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———————_—

CHARACTERIZATION OF A COMMON ANTIGEN LIPOPOLYSACCHARIDE

FROM PSEUDOMONAS AERUGINOSA AK1401

By

Mildred Rivera Betancourt

A DISSERTATION

Submitted to

Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1991

ABSTRACT

CHARACTERIZATION OF A COMMON ANTIGEN
LIPOPOLYSACCHARIDE FROM PSEUDOMONAS AERUGINOSA
AK1401
By

Mildred Rivera Betancourt

Lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa PAOl (05
serotype) was separated by gel filtration chromatography into two antigenically
distinct populations: the A-band and B-band LPS. The A-band population,
containing shorter polysaccharide chains (~30 repeat units), reacted with a
monoclonal antibody (MAb) to a P. aeruginosa common antigen but did not react
with antibodies specific tO OS-serotype LPS. In contrast, the LPS pOpulation
containing long polysaccharide chains (B-band) (>30 repeat units) reacted only
with the OS-specific MAbs. Chemical analysis of the A-band or common antigen
LPS indicated a lack of reactive amino sugar and phosphate, although low levels
of heptose and 2-ketO—3-deoxyoctulosonic acid were detected. Also, high levels
of rhamnose and stoichiometric amounts Of sulfate were detected in this LPS

isolate; the fatty acid composition was similar to that of the O-antigen-specific or

B band LPS. These results imply that PAOl strains synthesize two type of
molecules that are antigenically and chemically distinct.

To analyze the effect of various growth conditions on the size heterogeneity
of LPS, P. aeruginosa PAOl was grown in various media and at different
temperatures. The size distribution of the serotype-specific or B-band LPS and the
A-band or common antigen LPS were analyzed by both polyacrylamide gel
electrophoresis and immunoblots. Cells grown at high, near growth-limiting
temperatures, at low pH, in low concentrations Of phosphate, and in high osmotic
strength or salt concentrations, produced decreased amounts of very long chain
populations of O-antigen LPS molecules. Lower temperature and lower osmotic
strength, low sulfate, lower salt concentration, and elevated pH did not affect the
level of this LPS population. The size and amount Of common antigen LPS was
not significantly affected when the cells were grown under the above stress
conditions. Cells grown under normal, nonstressed conditions were agglutinated
only by serotype-specific MAbs. In contrast, cells grown under stress conditions,
in which the long-O-polymer LPS was absent, were agglutinated by both serotype
specific and common antigen-specific MAbs. The results indicate that specific
growth conditions limit the production of the long-O-polymer, allowing the
exposure and reactivity of the common antigen on the cell surface.

To corroborate that sulfur is incorporated into A-band LPS in stoichiometric
amounts, P. aeruginosa AK1401 was grown in 35S-labelled sulfate. The

advantage of using this PAC strain is that it is a convenient source of A-band LPS

since the synthesis Of O-antigen is defective. Gel filtration chromatography
separated the LPS into two major size populations: the A-band and B-band or
short chain (SC)-LPS. The elution profile, as well as the autoradiogram and the
inductively coupled plasma spectroscopy data showed that the A-band and B-band
LPS contained labelled sulfur. Thus, A-band LPS contains stoichiometric amounts
of covalently bound sulfur, perhaps as sulfate.

Finally, to further analyze the core oligosaccharide structure of A-band
LPS, a rhamnanase on bacteriophage A7 was used to specifically hydrolyze the
rhamnose polysaccharide chain. The chemical composition of the core components
Of the phage A7-digested A-band (Dig A-band) LPS was similar to that reported
previously for undigested A-band LPS. This Dig A-band isolate was also
incubated with MAbs against either inner core or outer core epitopes of P.
aeruginosa LPS, and the results were compared to that of the serotype SC-LPS.
The results from the immunoblots indicated that, even though the inner core region
Of all AK1401 LPS fractions share a common epitope, the outer core region of the

A-band LPS is different from that Of the B-band or SC-LPS.

TO my Lord Jesus Christ,
my daughter Tanya Y. Collazo,
my parents and

my friend Barbara Hamel

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my Lord Jesus Christ who
has given me strength and guided me throughout these years. I would also like
to express my gratitude to my mentor, Dr. Estelle J. McGroarty, for her guidance,
support and friendship. In addition, I am grateful to Dr. Alfred Haug, who
provided critical insight into my research, and to Dr. Jack Preiss, who believed
in my capability to achieve a Ph.D. degree.

I am in debt to my fellow students, Warren and Jill, for moments shared
in the laboratory, for the patience and moral support. Special thanks to Barbara
Hamel who has been a dear friend and given me a lot of support in the darkest

moments of "mental chaos. All Of you I will remember very dearly. Also I am
very grateful to the members Of my committee for their valuable comments and
suggestions during my dissertation.

I would like to acknowledge the Cystic Fibrosis Foundation for providing
funds which supported, in part, this research.

I am grateful tO Carol McCutcheon for her patience, excellent secretarial

skills, and for being a friend. Above all, I would like to acknowledge a special

professor in the Department of Chemistry, Dr. James F. Harrison, for being a

vi

source of spiritual strength and intellectual stimulus and an excellent friend. To
him and other good friends, Dr. Aileen Alvarado, Carmen Medina, Maria Suarez,
Dr. Pamela Fraker, Douglas Burdette, the biochemistry secretaries, Lydia, Joyce,
and the Puerto Rican crowd: thanks for the moral support and fi'iendship.
Finally, my deepest gratitude is to my beautiful and dear daughter Tanya
and my parents whose love, patience, encouragement and understanding at all

times were my inspiration in the realization of my goals.

vii

TABLE OF CONTENTS

List of Tables ..............................
List of Figure ..............................
List of Abbreviations .............................
Introduction ..............................
Chapter 1 Literature Review .........................
Characteristic, Ecology, and Pathogenicity Of Pseudomonas . .
Bacterial Cell Wall of Gram-negatives ...............

General Characterization of Outer-membrane
Lipopolysaccharide ...........................

Chemical Structure of Lipopolysaccharide .............

List of References ...........................
Chapter 2 Analysis of a Common-Antigen Lipopolysaccharide

from Pseudomonas aeruginosa .................

Abstract ..............................

Introduction ..............................

Results and Discussion .........................

List Of References ...........................

xvii

. 1

. 4

. 5

. 7

11

16

36

4O

Chapter 3 Growth Dependent Alterations in the Production
of Serotype and Common Antigen Lipopolysaccharides

in Pseudomonas aeruginosa PAOl .............. 58
Abstract .............................. 59
Introduction .............................. 61
Materials and Methods ......................... 63

Bacterial strains and culture conditions ............ 63

LPS isolation ............................ 64

Gel electrophoresis and Western blots (immunoblots) . . . 64

Slide agglutination assays .................... 65

Assays .............................. 66
Results and Discussion ......................... 67

LPS recovery and phosphate content of samples
from cells grown in different media .............. 67

Growth temperature effects on LPS composition ...... 69

Effect of salt concentrations in media on
LPS composition ......................... 76

Effects Of growth in high glycerol or high sucrose
on LPS composition ....................... 79

Effects of growth in low phosphate and low sulfate

on LPS composition ....................... 85
Effect of growth at low pH on LPS composition ...... 89
Whole—cell agglutination assays ................ 92
List of References ........................... 96

ix

List of References .......................... 165

Chapter 6 Summary and Perspectives .................. 168
Summary ............................. 169
Perspectives ............................. 175
List of References .......................... 179

Appendix A Heterogeneity of Lipopolysaccharide from
Pseudomonas aeruginosa: Analysis of

Lipopolysaccharide Chain Length ............. 182
Abstract ............................. 183
Introduction ............................. 185
Materials and Methods ........................ 187

Bacterial strains ......................... 187
Growth media .......................... 187
Isolation of LPS ......................... 188
Gel electrophoresis ....................... 188
Column chromatography .................... 189
Western blots .......................... 189
Assays ............................. 190
Results ............................. 191
Discussion ............................. 214
List of References .......................... 221

xi

LIST OF TABLES

Page
Chapter 1
1. Structure of O-repeating units in Pseudomonas aeruginosa
Lipopolysaccharide ........................ 26
Chapter 2
1. Analysis Of Column Fractions Of LPS from
P. aeruginosa PAOl ....................... 42
2. Fatty Acid Composition of LPS fractions from
P. aeruginosa PAOl. ....................... 45
Chapter 3
1. Level of LPS Recovered from Cultures of AK1401 and of
PAOl Smooth Strains H103 and PAOl716 Grown in
Different Media .......................... 68
2. Phosphate Content of LPS Isolates from Cells Grown
in Different Media ......................... 7O
3. Slide Agglutination Reactions for Cells of P. aeruginosa
Strains H103 and AK1401 Grown Under Different
Conditions .............................. 93

Chapter 4

1. Fatty acid composition of LPS fractions
from Pseudomonas aeruginosa AK1401. .......... 110

xii

2. Chemical analysis Of column fractions of LPS

from Pseudomonas aemginosa AK1401. .......... 112
Chapter 5
1. Chemical composition of LPS fractions
from Pseudomonas aeruginosa AK1401. .......... 157
Appendix A

1. Chemical Analysis of P. aeruginosa B-Band LPS
Fractions ............................. 203

2. Composition of the Pooled Column Fractions of LPS
from P. aeruginosa Strains 1715 and PAZl ........ 206

xiii

LIST OF FIGURES

Page
Chapter 1
1. Schematic molecular representation of the Gram-negative
envelope. ............................... 9
2. Proposed structure for the lipopolysaccharide molecule
of Pseudomonas aeruginosa PAOl. .............. 13

3. Chemical structure Of the lipid A component of Pseudomonas
aemginosa PAOl, Escherichia coli, and Salmonella
minnesota lipopolysaccharides. ................. 19

4. Proposed covalent structure Of the inner core of
Escherichia coli K-12. ...................... 23

5. Proposed structure for the O-polysaccharide repeat
unit of Pseudomonas aeruginosa A-band or common
antigen LPS. ............................ 28

Chapter 2

1. Western blots of LPS fractions (peaks 1, 2, 3, and X)
from P. aeruginosa reacted with monoclonal anti-503
or monoclonal E87 antibody and aligned with a
silver-stained SDS-polyacrylamide gel of the same
fractions .............................. 49

2. Western blots of four peak X isolates from P. aeruginosa
strains 1716 and PAZl ...................... 52

xiv

Chapter 3
1 .

2.

Chapter 4

1.

Chapter 5

Effect of growth temperature on LPS size. ........ 72,73

Effect of MgC12 concentration in the growth medium
on LPS size. ............................ 78

Effect of NaCl concentration in the growth medium on
LPS size. .............................. 81

Effect of 1 M glycerol in the growth medium on LPS size. 83

Effect of phosphate and sulfate concentrations in the growth
medium on LPS size. ....................... 87

Effect of pH of growth medium on LPS size. ........ 91

Gel filtration profile of Uf-LPS from P. aeruginosa
AK1401 grown in 35SO4-MBM media. ........... 107

Silver-stained SDS-polyacrylamide gel (A) and
autoradiograph (B) of P. aeruginosa AK1401. ....... 115

Fractionation profile of LPS from P. aeruginosa AK1401
separated on Sephadex G-200 .................. 136

Silver-stained SDS-polyacrylamide gels Of phage A7-digested
A-band LPS and PA01715 serotype-specific LPS from
P. aeruginosa. ....................... 140,141

Silver-stained SDS-polyacrylamide gel Of LPS fractions from

P. aeruginosa AK1401 and P. syringae pv. morsprunorum
after incubation with phage A7. ............. 144,145

XV

Western blot (A) and dot blot (B) of lipid A’s derived from
LPS fractions Of P. aeruginosa reacted with MAb 8A1. . 148

5. Western blot of lipid A’s derived from LPS fractions of

P. aeruginosa reacted with MAb 177. ............ 151
6. Western blot (A) and dot blot (B) of LPS fractions of

P. aeruginosa reacted with inner core—specific MAb 7-4. 153
7. Western blot (A) and dot blot (B) of LPS fractions of

P. aeruginosa reacted with outer core-specific MAb 101. 155

Appendix A

1. Silver-stained SDS-pOlyacrylamide gel (A) and Western

blots (B) of LPS from P. aeruginosa PAOl strains. . . . 193
2. Fractionation of LPS from P. aeruginosa strain 503

on Sephadex G-200. ....................... 197
3. Fractionation of LPS from P. aemginosa strain 1715 on

Sephadex G-200. ......................... 199
4. Fractionation of LPS from P. aeruginosa strain Z61 on

Sephadex G-200. ......................... 201
5. Silver-stained SDS-PAGE and Western blots of LPS

from P. aeruginosa fractions reacted with

monoclonal anti-503 LPS antibody .............. 209
6. Dot blots of pooled column fractions from P. aeruginosa

1715 LPS. ............................. 213

xvi

C10:0

C12:0

C14:0

CF

13C NMR

Dig A-band LPS
DNA

EDTA

EI

ELISA

FAB-MS

GC
GC/MS
HEPES

1H NMR

LIST OF ABBREVIATIONS

Decanoic Acid

Dodecanoic acid

Tetradecanoic acid

Cystic fibrosis

Carbon-l3 nuclear magnetic resonance

Phage A7-digested A-band lipopolysaccharide
Deoxyribonucleic acid

(Ethylenedinitrilo) tetraacetic acid

Electron ionization

Enzyme-lined immunosorbent assay

Fast atom bombardment-mass spectrometry
Fourier transformed infrared

Gas chromatography

Gas chromatography/mass spectrometry
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

Proton nuclear magnetic resonance

xvii

IATS

LAL

LOS

LPS

MAb

MBM

N aBD4

NB

NBA
3-OH—C10.0
2-OH-C12.0
3-OH-C12.0
3-0H—C14.0
p.f.u.

Rha
SC-LPS
SDS

SDS-PAGE

International Antigen Typing Scheme
2-keto-3-deoxyoctulosonic acid
Limulus amebocyte lycate
Lipooligosaccharides
Lipopolysaccharide
Monoclonal antibody
Modified basal medium
Sodium d4-borohydride
Nutrient broth

Nutrient broth agar
3-hydroxydecanoic acid
2-hydroxydodecanoic acid
3-hydroxydodecanoic acid
3-hydroxytetradecanoic acid
Plaque forming units
Rhamnose

Short chain-lipopolysaccharide
Sodium dodecyl sulfate

Sodium dodecyl sulfate-polyacrylamide gel electrophore

xviii

TB

TLC

Tris

TYE

Uf-LPS

Terrific broth

Thin layer chromatography
Tris(hydroxymethyl)aminomethane
Tryptone—yeast extract

Unfractionated-lipopolysaccharide

xix

INTRODUCTION

The outer membrane of Gram-negative bacteria is very important for
resistance to host defense factors and as a strong permeability barrier to many
antibiotics. One Of the membrane components that appears to be critical in
determining permeability is the lipopolysaccharide (LPS). It has been shown that
many strains of Pseudomonas aeruginosa produce two chemically and
immunologically distinct LPS molecules. These LPS isolates are known as A-band
and B-band LPS. B-band LPS is the O-antigen containing LPS and determines the
O-specificity of the bacterium, while A-band or common antigen LPS contains
shorter chains of predominantly neutral polysaccharide. The O-antigen and lipid
A region of the O-specific LPS is well characterized. Only the O-polysaccharide
chain structure of A-band LPS has been determined, but little is known about the
structure of the core-lipid A region of A-band LPS. Therefore, it is necessary to
chemically characterize the A-band core-lipid A region to be able to understand
the pathophysiological responses as well as any role that this component might
have in antibiotic resistance.

Two major goals of this thesis were 1) to isolate and characterize the LPS

from P. aeruginosa AK1401, defining the chemical differences between the A- and

2
B-band LPS, and determining the functional groups that might be replacing

phosphate in the A-band core-lipid A region; and 2) find a nondestructive method
to hydrolyze the O-polysaccharide chain from A-band LPS to further characterize
the core-lipid A.

Lipopolysaccharide from P. aeruginosa AK1401 and smooth-PAOl strains
were isolated and separated by gel filtration chromatography (Appendix A and
Chapter 2). The different LPS fractions were analyzed using chemical,
immunological, and gel electrophoretic techniques. Changes in the size
distribution of A-band and B-band LPS with variation in the growth conditions
including temperature, osmotic strength, and salt concentrations were studied
(Chapter 3). The size heterogeneity was characterized using sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblots.
Incorporation of sulfur into A-band LPS was further corroborated by growing the
cells in 35S-1abelled sulfate (Chapter 4). The elution profile from a gel filtration
column was monitored for total 358 counts, phosphate, and amino sugar. The
polyrhamnose chain of A-band LPS was digested with a rhamnanase associated
with bacteriophage A7 (Chapter 5). The phage A7-digested A-band LPS was
characterized using SDS-PAGE and chemical analysis to quantitate for heptose, 2-
ketO-3-deoxyoctulosonic acid, amino sugars, and phosphate. The inner core and
outer core region of A-band as well as B-band LPS was characterized using
monoclonal antibodies.

The first chapter provides general background on LPS from Gram-negative

3

bacteria. The final chapter summarizes the results found and proposes the
physiological importance of these results as they relate to the organism. The
chemical differences between the O-serotype specific or B-band and the common
antigen or A-band LPS are emphasized. An experimental approach to elucidate

the structure of A-band core-lipid A region is also given.

CHAPTER 1

Literature Review

5
Characteristics, Ecology, and Pathogenicity of Pseudomonads.

The family Pseodomonadaceae presently includes four genera, namely,
Pseudomonas, Xanthomonas, F rateuria, and Zoogloea (58). The type genus of the
family is Pseudomonas, one of the most complex groups of Gram-negative
bacteria. Members of this genus are characterized by their ability to grow in
simple media (58). Pseudomonads superficially resemble the enteric bacilli but
they differ in several fundamental respects. For example, they have polar flagella,
they are strongly oxidase-positive (except for P. maltophilia and some strains of
P. cepacia), and they are strict aerobes (58,77). Some strains produce water
soluble pigments; most P. aeruginosa strains produce a bluish green phenazine
pigment, pyocyanin, as well as fluorescein, a greenish yellow pteridine that
fluoresces (58). Pseudomonads have a considerably higher G+C content in their
DNA than Enterobacteriaceae, and most metabolize sugars via the 2-keto-3-
deoxygluconate (Entner-Doudoroff) pathway rather than via glycolysis (58,77).

Of all Pseudomonas species, by far the best studied genetically are P.
aeruginosa and P. putida (26,27,72). The genetically circular chromosome of P.
aeruginosa and P. putida allows a comparison of this species with the
Enterobacteriaceae (27,72). The gene arrangement and distribution in
pseudomonads are substantially different from that in the Enterobacteriaceae.
Three features of chromosomal gene arrangement have become apparent (27): (a)
the common noncontiguous arrangement of genes of biosynthetic pathways, which

contrasts with the contiguous arrangement commonly found in Enterobacteriaceae;

6

(b) the rarity of contiguous functionally related genes; and (c) the clustering of
genes with related functions into noncontiguous groups, described as supraoperonic
clustering.

Pseudomonads are found primarily in the soil, in water, or on plants, and
as a group are able to degrade a variety of organic compounds (58,77). Some
pseudomonads and other nonfermenters are found on skin or other body surfaces,
and in small numbers in the intestine (77). Pseudomanad strains can frequently
be isolated from assorted clinical materials, and they can be the cause of
nosocomial infections, particularly in patients in which the normal host defenses
are depressed (neoplasias, burns, cystic fibrosis, etc.) (16,28).

P. aeruginosa is among the Pseudomonas species that can be classified as
opportunistic human pathogens (10). Cystic fibrosis (CF) is an inherited disease
of children, adolescents, and young adults. Most patients with CF develop lung
infections and the dominant pulmonary pathogen is P. aeruginosa (3,18,24,64);
this organism is responsible for much of the morbidity and mortality associated
with chronic pulmonary infections in CF patients (8,15,24). The pathogenicity of
P. aeruginosa is associated with several virulence factors including extracellular
enzymes, lipopolysaccharides (LPS), and the production of exopolysaccharide,
known as alginate (7,9,19,39,53,61). Antibiotic therapy for such infections are
difficult since resistance in P. aeruginosa is comprehensive for many drug classes
due to low permeability of the cells’ outer membrane (21). Also, LPS appears to

be critical in determining permeability (54,74). To understand the nature of

7

antibiotic interaction with LPS as well as the role that this component has as a
virulence factor and in host response, it is important to characterize the chemical

structure and composition of the LPS molecule.

Bacterial Cell Wall of Gram-negatives.

The P. aeruginosa cell envelope is typical for gram-negative bacteria. It
includes an inner cytoplasmic membrane, a peptidoglycan layer, and an outer
membrane (Figure l). The cytoplasmic membrane is involved in cell division, in
the active transport Of materials across the bilayer, in synthesis of cell envelope
components, in the electron transport chain, and in oxidative phosphorylation (6).

The peptidoglycan layer, located between the inner and outer membranes,
is composed of a repeating disaccharide polymer crosslinked by peptides bridges,
and is important in maintaining mechanical rigidity (55). This peptidoglycan
structure is covalently linked to the outer membrane via a small lipoprotein (55).
Also found within the periplasm are a wide variety of enzymes which process
compounds into molecules capable of transport across the inner membrane (52).

The outer membrane is composed of proteins (structural proteins and
porins), phospholipids, various other amphiphiles, including the capsular-antigens,
the lipoproteins, and the endotoxins or lipopolysaccharides (LPS) (22,67). The
outer membrane has an asymmetric architecture, i.e., phospholipids (mostly
phosphoethanolamine) are present mainly in the inner monolayer; the high

molecular weight amphiphiles, including LPS, are located exclusively in the outer

Figure 1

Schematic molecular representation of the Gram-negative envelope.
Ovalsrand rectangles depict sugar residues. Circle represent the
polar headgroups of phospholipids. MDO are membrane-derived
oligosaccharides, and KDO is 3-deoxy-D—manno-octulosonic acid.

KDO and heptose make up the inner core of LPS.

 

    
     
  
  
 
 
 
   

 

g g g §<—O-Antigen
g g g Outer
Lipopoly- _ J . Core
saccharide L <— Heptose
2 ‘ j“: .‘ ‘F\KDL(i)pid A
Outer Membrane i
. . i ‘ ~' . , llil
Lipoproteln r‘ Peptidoglycan
Periplasm ‘— MDO
Ph h r 'd
Inner Membrane{ fig“ osp o‘IPI s

 

‘_ .
C ytoplasm Proteins

Figure 1

10

leaflet (22). It is through this outer membrane that Gram-negative bacteria
communicate with, interact with, and adapt to the natural environment. In this
interaction the outer membrane regulates the uptake of nutrients required for
growth (attributed to the porin proteins), prevents the penetration of toxic
molecules into the bacterial cell, and impedes microbial destruction by serum
components and phagocytic cells (22,52,55). As an integral component, LPS
participates in many of these active or passive membrane functions and is
indispensible for the proper assembly and architecture of the outer membrane.
The outer membrane Of Gram-negative bacteria forms a well regulated
permeability barrier due to the interactions of LPS and porins.

Like many other Gram-negative bacteria, extracellular polysaccharides with
the same structures as O—antigens have been found in cultural fluid or in the slime
Of different P. aeruginosa strains. Their production increases after cessation of
logarithmic growth. It is unclear whether these antigens are synthesized as pure
polysaccharides or if they are attached to a lipid moiety. Some P. aeruginosa
clinical isolates produce a mucoid alginate-like glycuronan. Such mucoid strains
are usually found in association with certain human pathological conditions,
especially with respiratory tract infections in CF patients (20,33). Kelly et a1 (32)
reported that there is an association between the development of mucoid or
alginate-producing variants and the loss of the long-chain or serotype LPS on the
cell surface. Alginate is an unbranched polysaccharide composed of 1,4-linked

residues of 6-D-mannuronic acid and a-L—guluronic acid which can be arranged

11
in homopolymeric or heteropolymeric blocks (2,75). Synthesis of alginate by

mucoid strains of P. aeruginosa is dependent on growth conditions and depends

on the temperature of growth (33).

General Characterization of Outer-membrane Lipopolysaccharide.

LPS from P. aeruginosa possesses the same general molecular architecture
as enterobacterial LPS. The high-molecular—weight LPS (S-form) molecule can
be divided into three parts: the hydrophobic lipid A component, and the two
hydrophilic parts composed of the O-antigenic polysaccharide attached to a core
oligosaccharide (Figure 2) (33). This structure is characteristic of wild-type
smooth strains. R-form or rough LPS is characterized by the absence of any 0—
antigenic side chain and is present in both smooth and rough strains (33,69).

Lipid A consists of a phosphorylated glucosaminyl disaccharide backbone,
to which several fatty acid chains are attached via ester and amide linkages
(33,81). Lipid A is the part of the molecule that by hydrophobic interaction
anchors the LPS in the outer membrane (Figure l) (67). It plays a significant role
in the organization and stability of the outer membrane. This component is
responsible for the LPS-induced endotoxic responses in mammals and participates
in the initiation Of a number of pathophysiological responses to infection (39).

The oligosaccharide core region is directly linked to the lipid A headgroup
via an unusual sugar present in most LPS molecules, 2-keto-3-deoxyoctulosonic

acid (KDO) (33,81). Within the core and lipid A headgroup region there is a

12

Figure 2 Proposed structure for the lipopolysaccharide molecule of

Pseudomonas aeruginosa PAOl (05 serotype according to the

IATS scheme).

 

T

O-ontigen

if

core

 

Lipid A

1

Figure 2

1

U

FucN Ac
Ac

 

 

 

WonImUAj
L.
Glc

—n

 

r ®e

 

Glc

-®9

 

rough

 

smooth

14

variety of ionic groups such as acidic phosphates and carboxyl moieties (33). Due
to the high level of phosphorylation in P. aeruginosa, compared to enterobacteria,
the core oligosaccharide has an exceptionally high metal-binding capacity (33,54).
This makes the outer membrane particularly dependent on divalent cations for
stability. Chelation of these cations by EDTA causes disordering of the membrane
with release of LPS (48,51). The disruption of outer membrane integrity and
increase in permeability induced by polycationic antibiotics results from binding
of these compounds to the anionic groups of LPS perhaps displacing the stabilizing
divalent cations (54,60). Therefore, the combination of the LPS’s negative charge
and the divalent cation cross-bridging of LPS provides the gram-negative cell
surface with a tight barrier, important for the cells’ resistance to hydrophobic
antibiotics, bile salts, detergents, protease, lipases, and lysozymes (54,55).

In smooth strains, O-polysaccharides are attached to the core region of LPS
and are made up of repeating units of identical oligosaccharides (33,81). The
portion of LPS molecules that have an attached polysaccharide is low for P.
aeruginosa isolates, usually less than 15% (69,81). The remainder of the
molecules contains only core and lipid A components. The structure of the
polysaccharide repeat unit defines the serotype of the strain (33,81). The O-side
chains from P. aeruginosa are rich in amino sugars, some of which are unique
among natural products (35,54). As mentioned previously, each of the at least 20
serologically distinguishable strains of P. aeruginosa produces a unique O-antigen

with a specific composition and structure (43,44). The presence and the amount

15

of O-antigen as well as the length of O-antigen chain influences various other P.
aeruginosa cell surface phenomena, including antibiotic susceptibility (1),
bacteriophage recognition (40), virulence and sensitivity to bactericidal action of
serum (7,11), and the capacity to induce protective antibodies (45).

Lipopolysaccharides of most bacteria exhibit structural heterogeneity in all
three regions of the molecule (56). In enterobacterial S-forms, as well as in
Pseudomonads, a collection Of LPS species is present which differ in the number
of repeating units, i.e., the length of the O-specific chain (56,69). As mentioned
before, a certain portion of molecules lacks the O—specific chain. In addition,
some of the charged substituents of the inner core and lipid A and the acyl groups
of lipid A, are not present in molar amounts (33,54,67,81).

The O-polymers from various P. aeruginosa strains have been resolved into
two chemically distinct sets, an amino sugar-rich fraction and a neutral sugar-rich
fraction (69,73,82,84). The shorter, neutral sugar-rich fraction has been shown
to contain a polysaccharide region composed of a three rhamnose repeat unit
(4,84). Rivera et al (69) have demonstrated that the P. aeruginosa strain PAD]
is capable of synthesizing more than one type of LPS. Analysis by gel filtration
chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) revealed that the PAOl LPS consisted of two antigenically and
chemically distinct molecules termed A- and B-band LPS (69). The B band,
usually composed of LPS with long O-antigen chain, is the LPS specie responsible

for the O-specificity of the organism, while a second LPS, the A band fraction,

16

is composed of molecules that only have intermediate size polysaccharide chains
(approx. 30 repeat units) (42; M. Rivera, T.J. Chivers, J.S. Lam, and E.J.
McGroarty, J. Bacteriol., submmited).

The isolation of P. aeruginosa rough strains from sputum in CF patients is
correlated with chronic and severe infection (12,29,47). The lack of O-antigen
expression accounts for the unusually high percentage (60-80%) of nontypeable
strains Of P. aeruginosa encountered in many studies of CF patients
(13,14,23,41,57,65). Lam and coworkers (41) have produced seven MAbs to the
A-band isolate. Using Western immunoblot analysis, they have shown that A band
molecules are present as a common antigen on many but not all serotypes of P.
aeruginosa. Furthermore, they found that A bands are present in a high
percentage Of clinical isolates and appeared to be the main antigen on nontypeable

strains deficient in high-molecular-weight B band-type LPS.

Chemical Structure of Lipopolysaccharide.

The primary structure of enterobacterial lipid A, as well as P. aeruginosa,
has been elucidated in great detail. In Figure 3 lipid A structures of Escherichia
coli (67,68), Salmonella minnesota (68), and P. aeruginosa (17,33) are shown.
In all of these cases, lipid A is composed of a 6-D-g1ucosaminyl-(1->6)—a-D—
glucosamine disaccharide phosphorylated at positions 1 and 4'. This hydrophilic
lipid A head group is acylated by four residues of (R)-3-hydroxy fatty acids at

positions 2, 3, 2’, and 3’. Structural heterogeneity in the lipid A (Figure 3)

17

includes variation in the fatty acid chain length and the location of ester-bound acyl
groups. For example, in E. coli the (R)-3-hydroxytetradecanoic acid (3-OH-
C14:0) is present in ester and amide linkage. The hydroxyl groups of the two 3-
OH-C14:0 residues bound to the nonreducing glucosaminyl residue at position 2’
and 3' carry dodecanoic (C 12:0) and tetradecanoic acid (Cl4:0), respectively. The
3-OH-C14:0 residues bound to the reducing glucosaminyl residue are not 3-O-
acylated. In P. aeruginosa lipid A, (R)—3-hydroxydodecanoic acid (3-OH-C12:0)
is amide-bound and (R)-3-hydroxydecanoic acid (3-OH—C10:0) is ester-linked to
the lipid A backbone. The latter are not substituted at their 3-hydroxyl groups
while the 3—OH-C12:0 residues at positions 2 and 2’ carry C12:0 and/or (S)-2-
hydroxydodecanoic acid (2-OH—C1220). As indicated in Figure 3, the hydroxyl
groups in positions 1 and 4’ may be substituted by phosphate or pyrophosphate,
phospho—D-glucosamine, and phosph04aminO-4—deoxy-L-arabinopyranose (17,68).
It is important to mention that the pathway for biosynthesis of lipid A in P.
aeruginosa is similar to, but not identical with that of enterobacterial lipid A
(17,66). The major precursor of the latter includes only amide-bound fatty acids
(66), while that of the former contains all fatty acids present in lipid A of the
mature LPS before addition of KDO (17).

Thus, while lipid A’s of different bacterial families share certain chemical
features they differ in others. The tetraacyl backbone is ubiquitous and highly
conserved and exhibits very low structural variability (66-68). However,

individual lipid A’s may differ from each other by the presence and nature of polar

Figure 3

18

Chemical structure of the lipid A component of (A)
Pseudomonas aeruginosa PAOl , (B) Escherichia coli, and (C)
Salmonella minnesota lipopolysaccharides. Dotted lines
indicate incomplete substitution. KDO is linked to the
primary hydroxyl group in position 6'. Numbers in circles

indicate the number of carbon atoms in the acyl chains.

 

19

one
@NH, 0
n 0
on - P
m '

.o m 0 on
"a
00.030 O'c'o
' I
.n 'R
R30H(2):H(1) (it). 2;”. ‘ Hi). i i.
C", C“,

Figure 3

20

and ionic head groups substituents, and the nature and chain lengths of fatty acids
acylating the 3-hydroxy fatty acids. Variations of these parameters create
structural diversity which is responsible for intrinsic heterogeneity of lipid A
(17,56,66).

It should be noted that certain Gram-negative bacteria synthesize LPS with
a lipid A structure that is radically different in architecture from that of
enterobacterial lipid A structure (25,49). As an example, lipid A of
Rhodopseudomonas viridis has been found to be devoided of glucosamine and
phosphate but to contain the rare sugar 2,3-diamino-2,3—dideoxy-D-glucose
monomer which carrys 3-OH-C14:0 residues (71). Hollingsworth and Lill-
Elghanian (25) isolated and characterized two major lipid A components of
Rhizobium mfolii ANU843. They demonstrated that the free lipid A component
contained a novel long-chain carboxylic acid and 2—amino-2-deoxy-gluco—hexuronic
acid and was totally devoided of phosphate.

The core region of enterobacterial and P. aeruginosa LPS consists of a
heterooligosaccharide which can be subdivided into the lipid A-proximal inner core
and the distal outer core region. The enterobacterial outer core region contains the
common sugars D-glucose, D-galactose, and N-acetyl-D-glucosamine (67),
whereas in P. aeruginosa D-glucose, D-galactosamine, L-rhamnose, and L-alanine
are the components found in this region (81). The inner core region of
Enterobacteriaceae as well as P. aeruginosa is composed of the unusual sugars

heptose, mainly in the L-glycero-D-manno and the D-glycero-D-manno

21

configuration, and 2-keto-3-deoxyoctulosonic acid (KDO, also termed 3—deoxy-D-
manno-Z-octulosonic acid, dOclA) (Figure 4) (33,66,67). 13C NMR spectroscopy
suggests that KDO is attached to lipid A by an a2-6' linkage (78,79). In general,
these residues are substituted by charged groups such as phosphate, pyrophosphate,
phosphoethanolamine, and pyrophosphoethanolamine, often in nonstoichiometric
amounts (33,66,67). The structural variability of the core within different bacterial
species is limited. Based on the sugar composition of the LPS isolated from
various enterobacterial R mutants, the core domain can be classified by
chemotypes, namely, Ra, Rb 1, sz, Rc, Rd 1, Rd2,and Re LPS (46,66). Type Ra
represent a complete core while type Rd has defects in adding any sugar unit of
the outer core region; type Re comprises only lipid A and the KDO units (probably
with branch substituents) of the core oligosaccharide. Chemotype variants of
P. aeruginosa rough mutants have been isolated which are analogous to those of
enterobacteria with the exception that Re mutants have not been reported
(5,30,38). Interestingly, a variety of nonenterobacterial wild-type strains of some
phototropic and pathogenic gram-negative bacteria such as Neisseria,
Acinetobacter, Campylobacter, Bordetella, Bacteroides, and Haemophilus
synthesizes LPS which consist only of core oligosaccharide and lack a long 0-
specific chain (67). These compounds have been termed lipooligosaccharides
(LOS).

The chemical analysis of the inner core is very difficult for a number of

reasons. (1) The ketosidic linkages of KDO are extremely acid-labile. (it) No

22

Figure 4 Proposed covalent structure of the inner core of Escherichia
coli K-12. Putative partial substitutions are indicated with

dashed bonds.

 

23

HO —-

HO

__ Heptose

 

 

 

R ion
O—Antigen eg

 

 

 

p-----—d

 

 

 

Outer Core

 

 

 

 

 

 

 

 

 

 

Figure 4

24

satisfactory procedure exists for the quantitative determination of KDO in
polysaccharides of unknown structure and substitution pattern. (iii) KDO
undergoes side reactions under the usual conditions of hydrolysis of
polysaccharides, leading to unknown or unstable products (80). Because of the
difficulty of analyzing intact LPS under nondestructive conditions, the proposed
structure on the enterobacterial inner core (Figure 4) cannot be considered
definitive. At least one KDO residue, or KDO-like sugar, has been found in
almost all gram-negative bacteria that have been studied (46,67), but in some
organisms, subtle modifications of KDO are observed. In Acinetobacter
calcoaceticus, an octulosonic acid isomer resembling KDO is attached to lipid A
which is resistant to acid hydrolysis (31). This isomer differs from KDO by the
presence of an additional hydroxyl group at C-3, and this group appears to play
a role in the acid stability of its ketosidic linkage. It is not clear whether LPS
lacking the KDO residue exists. However, Pask-Hughes and Williams (59)
reported that the LPS isolated from extreme thermophiles of the genus Thermus
lacked heptose, KDO, glucosamine, and phosphorus. Interestingly, hydroxylated
fatty acids were not reported. Also, Beconi and Hollingsworth (personal
communication) have isolated an LPS fraction from Rhizobium leguminosarum
viobar viacea grown in acidic conditions. The lipid A fraction contains the fatty
acids characteristic of this specie but the carbohydrate fraction lacks both heptose
and KDO.

The O-antigen is attached to a terminal sugar of the outer core (Figure 4),

25

and it is composed of a repeating oligosaccharide unit containing up to six sugar
residues (33,46,67). The nature, sequence, type of linkage, and type of
substitution of the individual monosaccharide residues within a repeating unit is
characteristic and unique for a given bacterial strain. Thus, the O-polysaccharide
chain is species-specific and determines the O-serological specificity of the
molecule and of the parent bacterial strain (33,46,67). The composition of
P. aeruginosa O-antigens has been found to differ in several ways from that of
other bacterial O-antigens studied (34). The P. aeruginosa antigens usually are
defficient in neutral sugars, and typically contain monoamino and diamino sugars,
many of which carry a carboxyl functional group. The structures of some of these
O-antigens are given in Table 1. As mention, the O-polysaccharide chain of P.
aeruginosa exhibits heterogeneity not only in the number of repeating units but
also modifications in the repeating unit (33,34). The most common is
nonstoichiometric O-acetylation; other variable modifications are nonstoichiometric
amidation and epimerization.

The majority of the P. aeruginosa strains contain a second LPS specie
whose polysaccharide chain differs serologically and structurally from O-antigen
chain (37,50,69,70,73). This LPS has been termed A-band or common antigen
LPS (Figure 5). In wild type strains, this common polysaccharide antigen is
shorter than the O-antigen chain; thus the common antigen is covered and not
exposed to the cell surface on smooth strains, but is accessible on the surface of

rough-type strains (41,50). The common antigen polysaccharide is a regular

A3 go.— th :83: 085 .3 BOO—BE Quote—:88 082% wins commie 3:092:25 05 2
wEEoooa 2588 05 o. macho.— ..xs mks .38 oEecseean:m_ocaEEm-N {3:582 we“ ”Boa emceeixoéxxoowme

-mdeEESoofi—YQN .<D~G<Zv 658883 .205 65830553 .280

”meowagofifi veneeflmcoz

 

 

-éaoda-_gagged?3220;qu : 033.
-aBo<zsod$EEZUEOG-EZDNGEEOOT3Vo<zaed€ _ o
228mb {2:22:32 .9-§<DE§2O€ CREAM
- Eizosmda- _ 3<D~o<zvso£s _ S<DEE§O€ oar».
-§o<zo=mdaa8,35%??qu§<D§§EO€ N 0.3m
-§o<zo=n_d8-§<D~o<zv§2O$§<DEE§OA4 2 gem
séézoéds-_S<a€<z§2d$§<35E§Oe m Ram
- _ casein- Sagas- _ 32230-93- §<DQ<zaoJ€ 3N
-_va§o<o-~£m-EaéogAJa-EVEZSOOR-§<DQ<zaoJ€ 2 new
35 wcueoqocé 09C. camp.
was ~23

 

outoseeombenemfi omeSMEee geesewaam E SE: win-«2.2-0 we anagram A mam—<8

27

Figure 5 Proposed structure for the O-polysaccharide repeat unit of

Pseudomonas aeruginosa A-band or common antigen LPS.

 

 

28

m eczmmm

OI
_ 0 Oil

 

 

:O 0 IO xu
IO 1 IO

29

homopolymer of rhamnose. On the basis of NMR and chemical analyses the
structure has been shown to consist of the repeating unit [— > 3)-oz-D-Rhap-(l- > 3)—
oz-D-Rhap-(l- >2)-oz-D-Rhap-(1- >] n (4,37,84). Surprisingly, the O-antigens of
many phytopathogenic Pseudomonas species, such as P. syringae, also contain D-
rhamnose as a main component, and some strains possess the same structure as the
P. aeruginosa common polysaccharide chain (36,76). Little is known about the
structure of the core oligosaccharide of P. aeruginosa A-band or common antigen
LPS. Yokota et al (84) reported that the oligosaccharide core contained 3-0—
methyl-6-deoxyhexose, xylose, and glucose. On the other hand, Arsenault and
coworkers (4) detected minor amounts of 3-0—methyl rhamnose, ribose, mannose,
glucose, and a 3- or 4-0—methylhexose in A-band LPS from P. aeruginosa strain.
Furthermore, Rivera et al (69), and Rivera and McGroarty (70) reported that the
A—band LPS isolated from smooth strains of P. aeruginosa contained low levels
of amino sugars, KDO, and no phosphate but instead stoichiometric amounts of
sulfate was detected. In this study, I have initiated a study of the chemical

structure of core-lipid A of A-band LPS.

10.

11.

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CHAPTER 2

Analysis of a Common-Antigen Lipopolysaccharide

from Pseudamanas aeruginasa

36

ABSTRACT

Lipopolysaccharide isolated from Pseudamanas aeruginasa PA01 (05
serotype) was separated into two anti genically distinct fractions. A minor fraction,
containing shorter polysaccharide chains, reacted with a monoclonal antibody to
a P. aeruginasa common antigen but did not react with antibodies specific to 05-
serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide
chains reacted only with the 05-specific monoclonal antibodies. The shorter,
common-antigen fraction lacked phosphate and contained stoichiometric amounts
of sulfate, and the fatty acid composition of this fraction was similar to that of the
O-antigen-specific fraction. The lipid A derived from the serotype-specific
lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from
Escherichia coli, while the lipid A derived from the common antigen did not react.
We propose that many serotypes of P. aeruginasa produce two chemically and
antigenically distinct lipopolysaccharide molecules, one of which is a common

antigen with a short polysaccharide and a unique core-lipid A structure.

37

INTRODUCTION

Lipopolysaccharide (LPS) isolated from Pseudamanas aeruginasa, like that
from enteric bacteria, is a heterogeneous mixture of molecules of different
polysaccharide chain lengths (16,26, Appendix A) and with different levels of
phosphate substitution (21,31). Structurally, the molecules can be divided into
three regions: the lipid A, the core oligosaccharide, and the O-antigen
polysaccharide. The lipid A from P. aeruginasa is similar to that of many gram
negative bacteria; it consists of a 4-phosphoglucosaminyl-(l—>6) glucosamine-1’—
phosphate head group to which saturated and hydroxy fatty acids are ester and
amide linked (21,31). The hydroxy fatty acids present in P. aeruginasa LPS are
different from that of enteric bacteria, lacking 3-OH-tetradecanoic acid but
containing 2-OH- and 3-OH-dodecanoic acids and 3-OH-decanoic acid (9,31,32).

The composition of the core oligosaccharide of P. aeruginasa is also
somewhat distinct from that of the core oligosaccharide of other gram-negative
species, containing D-glucose, D-galactosamine, L-rhamnose, and L-alanine as
well as the sugars commonly found in the inner core, L-glycero-D-mannoheptose
and 2-keto-3-deoxyoctulosonic acid (KDO) (26,28,31). Also, the core and lipid

A regions of P. aeruginasa isolates are especially high in phosphate (24,31). In

38

39

smooth strains, a long polysaccharide is attached to the core, but usually on only
a low proportion of the LPS molecules (26,31). The structure of the
polysaccharide repeat unit defines the serotype of the strain (6,15). Generally, O-
polysaccharides of the various P. aeruginasa serotypes are rich in amino sugars
(21,31). Neutral sugars are also found as components of many O-polymers
include L-rhamnose and D-glucose (17,31).

Characterization of the structures of various O-specific polysaccharide from
different serotypes of P. aeruginasa has been complicated in some cases by
chemical heterogeneity of the polysaccharide chains (5,6,32). Not only is the
length of the O-polysaccharide variable (26), but also the O-polymers have been
resolved into an amino-sugar-rich and a neutral-sugar-Iich fraction (29,32,34).
The shorter, neutral-sugar-rich fraction has been shown to be composed of a three-
rhamnose repeat unit (34). Monoclonal antibodies reactive against the
polyrhamnose isolate react with many different serotypes of P. aeruginasa,
suggesting that these molecules compose a common antigen for this organism (29).
We have recently reported the isolation and partial purification of an A-band LPS
fraction from PA01 strains which is low in phosphate and amino sugars and does
not react with serotype-specific monoclonal antibodies (26). In this study we

further characterized the structures of these two fractions of LPS.

RESULTS AND DISCUSSION

LPS was isolated from strains PAOl716 (ade-l36, lea-8, rif-l) (revertant),
PA01715 (ode-136, lea-8, rif-l , tal A 12) (an aminoglycoside-supersensitive mutant
[20]), and PAZl (met-28, trp-6, lysA12, his-4, ile-226, absA), a PA0222
derivative (2), by either the method of Darveau and Hancock (7) as previously
described (26) or the hot aqueous phenol method (33). The LPS isolates were
fractionated on a Sephadex G-200 (Pharmacia Fine Chemicals, Piscataway, NJ)
column at room temperature as previously described (23,26). Column fractions
were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) by using the buffer system of Laemmli (18); the gels were silver-
stained by the method of Dubray and Bezard (10). SDS-PAGE of the column
fractions revealed two distinct ladder patterns of apparently different sizes (results
not shown) which in the unfractionated sample were overlapping and thus
superimposed. Previously, we showed that LPS from P. aeruginasa PA01 strains
could be resolved into two chemically distinct sets of molecules, the shorter A
bands (later-eluting ladder) and the longer B bands (earlier-eluting ladder, [26,
Appendix A]). The main serotype-determining antigen, high in amino sugars, was

recovered primarily in peaks 1 and 2. The third peak, peak X, contained a

40

41

shorter, neutral-sugar polysaccharide. The bands in SDS gels that contained the
main O-antigen were termed the B bands, while the bands in peak X were termed
the A bands. The last peak recovered from the Sephadex column (peak 3)
contained the majority of the molecules, comprising core and lipid A with none
or only one or two O-repeat units. The fractions which made up the four peaks
were isolated, pooled, and dialyzed extensively (12,000 to 14,000 molecular
weight cutoff membranes) against column buffer without detergent at 37°C and
then against distilled water at 4°C. The dialyzed fractions were lyophilized and
suspended in distilled water to known weight concentrations for further analysis.

In an earlier study we showed that all four fractions contained heptose, a
component of the inner core of most LPS isolates (26). The level of this sugar per
weight of the four fractions reflected the molecular weight of the molecules,
suggesting that the heptose content is similar in all of the isolates (Table 1). In
contrast, the sugar 2-keto-3-deoxyoctulosonic acid (KDO), another component
common to the inner core of most LPS isolates, appeared to be very low in the
peak X isolate (Table 1). However, Caroff and coworkers have shown that this
sugar is not detected in the thiobarbiturate assay if the sugar is substituted at
specific positions, unless harsher hydrolysis conditions are used (4). When the
peak X isolate was assayed after hydrolysis with higher levels of acid and for
longer times, the KDO content increased ten-fold. Thus, the fraction contains
KDO, but this sugar may be substituted and thus not readily detected.

Presumably, the KDO residues in the other three peaks are not substituted; the

42

TABLE 1. Analysis of Column Fractions of LPS from P. aeruginasa PAOl

 

Amt (nmol/mg)a of:

 

 

LPS Heptoseb KDOb Phosphateb Sulfate LAL
Sample resultc
Peak 1 39 30 150 13 75
Peak 2 34 28 165 47 72
Peak 3 272 282 1,670 16 408
Peak X 83 2 8 190 57
Unfractionated NDd 162 1 ,200 37 N Dd

 

aAverage results from two or more isolates.

bData from reference 26.

cLAL, Limulus amebocyte lysate assay. Results are expressed as endotoxin units
per picogram.

dND, Not determined.

43
level of KDO detected in these fractions was not affected by the hydrolysis

conditions

Our earlier studies also indicated that the peak X isolate lacks phosphate
groups while the other fractions are highly substituted with phosphate (Table 1).
To determine whether another anionic group might replace the phosphate moieties,
we assayed the LPS fractions for sulfate by a barium chloranilate assay procedure
with K2804 as a standard (8). Briefly, 5 to 10 mg of LPS were hydrolyzed in 0.5
ml of 6 N HCl at 100°C for 1 h and extracted with 5 ml of chloroform-methanol
(2:1 [vol/vol]). The upper phase was washed with chloroform-methanol (17:3)
and dried in a boiling-water bath. The samples were then dissolved in 0.5 ml
water-methanol (1:1) and dried three times. This hydrolyzed sample was then
dissolved in H20, diluted into ethanol, and reacted with barium chloranilate.
Cross-reaction with KH2P04 was shown to be negligible. The levels of sulfate
detected in peaks 1, 2, and 3 were low (Table 1). In contrast, the molar amount
of sulfate detected in peak X was over twice the heptose content, indicating sulfate
levels greater than stoichiometric levels. The slightly elevated levels of sulfate in
peak 2 may reflect a small amount of A-band LPS contaminating this fraction (see
below).

Reactivity of the four pooled fractions in the Limulus amebocyte lysate assay
(Whittaker Bioproducts, Walkersville, MD) was also quantitated (Table 1). Levels
were calibrated with the Escherichia coli 0111:B4 LPS standard provided in the

kit. The reactivity for the high-molecular-weight LPS (peaks 1 and 2) compared

44
with that of low-molecular-weight LPS (peak 3) expressed on a per-weight basis

reflected the difference in sizes of the LPS molecules. The peak X sample did not
fit in this pattern, probably because of the chemical differences of the LPS
molecules. This A-band-containing fraction did not appear to be as reactive in this
assay as the B-band-containing fraction, at least when reactivity is expressed per
amount of heptose.

Peaks X and 3 and the unfractionated LPS were analyzed for fatty acid
composition (Table 2). LPS fractions (5 to 8 mg) were suspended in a 3-ml
solution of HCl-methanol (3:15 [vol/vol]) and hydrolyzed at 85°C for 18 h. The
fatty acid methyl esters were extracted into petroleum ether, dried, and suspended
into ethyl acetate. Samples of 3 to 4 pl were injected into an HP5890 gas
chromatograph (Hewlett-Packard Co., Palo Alto, CA) and separated on a 25-m
Ultra H column. The column was run at 150°C for 15 min; the temperature was
increased at 2°C/min up to 250°C and then increased at 25°C/min up to 350°C.
The retention times were compared to those of a bacteria fatty acid methyl ester
mixture (Supelco, Bellefonte, PA) for identification. The fatty acids detected were
very similar for all three samples and are similar to what has been reported
previously for LPS from P. aeruginasa (9,32). All three samples contained high
amounts of 2-OH- and 3-OH-dodecanoic acid and lacked 3-OH-tetradecanoic acid.
The content of the other fatty acids were similar for all three samples. These
results give further evidence that the A-band isolate is an LPS with a fatty acid

composition typical of this species.

45

TABLE 2. Fatty Acid Composition of LPS fractions from
P. aeruginasa PA01.

 

Recovery from samplea

 

 

Fatty Acid Peak 3 Peak X Unfractionated
C1010 3 0 4
C120 9 11 6
C16:0 6 6 2
3-OH-C10.0 5 5 16
2-OH-C12.o 33 32 33
3-OH-C12.0 37 43 40
Other 6 3 0

 

21Results are the average of two or more analyses and are expressed as a weight
percentage of the total.

46

To characterize the sugar components in the purified LPS fractions, the four
fractions were hydrolyzed in 2 N HCl for 2 h at 100°C and evaporated. The
samples were then spotted on cellulose F254 (Merck) thin-layer plates and
developed by using the solvent system described by Sawada et al. (29). The
chromatograms were reacted with alkaline silver nitrate to localize the sugars by
previously described procedures (29). This qualitative analysis revealed that
rhamnose was present in peak X, but it was not detected in the peak 1 and 2
samples (data not shown). As expected, rhamnose was also present in the peak
3 sample; rhamnose is a known component of the core oligosaccharide (31,32).

Recently Sawada and coworkers reported that acid hydrolysis of P.
aeruginasa LPS released a rhamnose-containing polysaccharide, shorter than the
main amino-sugar-containing polysaccharide, which reacted with a monoclonal
antibody E87 (29). We obtained a sample of this monoclonal antibody to see
whether it reacted with our A-band fractions. We also tested the fractions for
reactivity with the 05-specific monoclonal antibody anti-503 (12). Western
immunoblots of SDS-polyacrylamide gels were prepared as described by Towbin
et al. (30). The gels were electrotransferred with a model TE Transphor
Electrophoresis apparatus (Hoefer Scientific Instruments, San Francisco, CA) at
a constant current of either 150 or 300 mA for 18 h. The nitrocellulose blots were
visualized as described by Otten et al. (22) with either monoclonal anti-503
antibody specific for 05-serotype LPS (12) or monoclonal antibody E87 specific

for a common antigen of P. aeruginasa (29). In addition, dot blots were

47

performed by applying known quantities of LPS directly onto nitrocellulose and
reacting the blots with the monoclonal antibodies described above. The blots were
washed and visualized by using horseradish peroxidase-conjugated goat anti-mouse
immunoglobulin G antibody (Sigma Chemical Co., St. Louis, MO). Both the
Western immunoblot (Figure 1) as well as the dot blot (data not shown) indicated
that the E87 monoclonal antibody reacted with the X peak isolate while the
serotype-specific antibody reacted with peak 1 and 2 isolates. There was a weak
reactivity of the peak 2 fraction with the E87 antibody on the dot blot, suggesting
that this isolate had a minor amount of A-band LPS. Furthermore, the peak X
isolate showed some reactivity with the anti-503 antibody. To show that peak X
reactivity was due to B-band contamination of this fraction, four peak X isolates
were separated on SDS gels, transblotted, and reacted with the two monoclonal
antibodies (Figure 2). The results indicate that the E87 antibody reacted
exclusively with the more slowly moving bands in this gel while the anti-503
antibody reacted only with the faster-moving B-band molecules that were present
in low amounts in this sample. Thus, the A- and B-band LPS molecules appear
to be antigenically as well as chemically distinct.

To determine whether the lipid A components of these fractions were also
antigenically different, lipid A from peaks 3 and X as well as from
nonphosphorylated LPS from Chromatium vinasium were isolated and reacted on
dot blots with anti-lipid A monoclonal antibody 1D4 or 8A1 (27). The LPS from

C. vinasium was a gift from R. Hulbert (14). Purified LPS fractions were

48

Figure 1. Western blots Of LPS fractions (peaks 1, 2, 3, and X) from
P. aeruginasa reacted with monoclonal anti-503 (left) or
monoclonal E87 (right) antibody and aligned with a silver-
stained SDS-polyacrylamide gel (center; 15% acrylamide) of
the same fractions. A 5-pg portion of each sample was
applied to each gel. The gels were blotted as described in the

text.

H oeswfi

EWoB no

wmfl _

S u; z _ <5 58“.?
.. $35 8...-z.z<

49

    
  

._. Nn X.

 

0 fror
left) 11
silnr-
lidc) ti
la n
l in it

50
hydrolyzed to lipid A by treating the samples with 5 % acetic acid at 100°C for 2.5

h (14), with 0.1 N HCl at 100°C for 1 h (25), or with 20 mM sodium acetate (pH
4.5) at 100°C for 1 h (l). The samples were neutralized with NaOH, and the
polysaccharide was separated from the lipid A either by elution on a Sephadex G-
25 column with distilled water, or by sedimenting the lipid A at 10,000 x g for 30
min. Hydrolysis in 20 mM sodium acetate (pH 4.5) produced an insoluble lipid
A from the peak 3 fraction, but no lipid A could be recovered from peak X or the
C. vinasium sample by using this hydrolysis protocol. Thus, these two samples,
as well as peak 3, were hydrolyzed for 1 h at 100°C in either 0.1 N HCl or 5%
acetic acid. The isolated lipid A samples were lyophilized and suspended in
distilled water. Samples were spotted onto nitrocellulose at different
concentrations and reacted with either 1D4 or 8A1 monoclonal antibody specific
for lipid A of Escherichia coli (27). On all the blots studied, only lipid A from
peak 3 reacted, but all of the peak 3 lipid A isolates reacted with both antibodies
(data not shown). Perhaps phosphate on the lipid A is a part of the epitope for
these antibodies. The results indicate that the lipid A from peak X is antigenically
distinct from that of peak 3 and presumably from that of the other B-band
fractions.

In conclusion, the differences between the A- and B-band LPS fractions of
P. aeruginasa are significant. First, the B-band molecules contain much longer
polysaccharide chains, as determined by SDS-PAGE and by separation on

Sephadex G-200. Second, the B bands are high in amino sugars and low in

Figure 2.

51

Western blots of four peak X isolates. Isolates I, II and III
were from strain 1716, and isolate IV was from strain PAZl.
The blot on the left was reacted with monoclonal antibody
E87 and the blot on the right was reacted with monoclonal
anti-503 antibody; the gels were aligned with a silver-stained
SDS-polyacrylamide gel (center; 15 % acrylamide) of the same
isolates. A 5-pg sample of each isolate was applied, and after
electrophoresis, the gels were blotted or stained as described

in the text.

52

N Rama

3:. S...
Eoumo>> 533;
8322 22.5 535 Bu

.liillul Ii lrlt
. . . . -

i 1, 11114.

r

\.

.r-

, 1,711- ; 3".32‘

  

 

firms-1‘, A,

:37 ‘ 2‘"

 

rv

3(IIlslIl‘ltlI

>_ _= = _ >_ _= __ _ “83.8.
9.8%

 

53

rhamnose, while the A bands contain rhamnose and are low in amino sugars.
These differences in O-polymer structure are also reflected in the reactivities of
antibodies to these polymers. The O-specific antibodies reacted only with the
amino-sugar-containing polymers, while the A bands reacted only with the
polyrhamnose-specific antibody. Third, the B bands are high in phosphate content
but low in sulfate, while the A bands lack phosphate but contain sulfate groups.
This is the first report that we know of which indicates sulfate as a component of
LPS. However, nonphosphorylated lipid As have been reported for a number of
bacteria (19); in such isolates, sulfate may replace the phosphate on lipid A. In
preliminary studies we have detected stoichiometric levels of sulfate on the lipid
A of the nonphosphorylated LPS from C. vinasium (14; M. Rivera, A.A.
Peterson, R.T. Coughlin, and E.J. McGroarty, manuscript in preparation).

A fourth difference between the A- and B-band LPS is the reactivity of the
lipid As with monoclonal antibodies to lipid A from E. coli. These antibodies
reacted with the B-band lipid A but not with lipid A derived from the A bands.
The anti-lipid A antibodies also did not react with lipid A from C. vinasium,
suggesting that phosphates in the lipid A head group are critical in binding these
antibodies; sulfate may not serve as a replacement at the binding site.

A final difference between the A- and B-band LPS isolates is their
distribution among the serotypes of P. aeruginasa. Each serotype class has a
unique B-band type of O-polymer with a different chemical structure (15). In

contrast, many of the serotype strains may contain an A-band type of LPS with

54

similar structure, since reactivity with the E87 monoclonal antibody is found in a
large number of serotype strains (29). Recently, Lam and coworkers (M.C. Lam,
E.J. McGroarty, and J .S. Lam, unpublished results) have produced seven
monoclonal antibodies to the A-band isolate. Using Western immunoblot analysis,
they have shown that A-band molecules are present as a common antigen on
strains of many, but not all, serotypes. Interestingly, they found that A bands
were present in a high percentage of clinical isolates and appeared to be a main
antigen on nontypeable strains deficient in high-molecular-weight B-band-type
LPS.

We propose that, for strains containing both A- and B-band-type molecules,
the longer B-band polymers extend from the surface and constitute the main
antigenic structure exposed on the cell. The shorter A bands may be covered and
masked by the B-band O-polymers. However, under certain conditions, such as
prolonged antibiotic therapy, clinical isolates are found to be nontypeable and
appear to lose the O-polymer-containing B-bands (3,11,13). For such clinical
isolates the A bands may become exposed and serve as an important antigenic

determinant.

10.

11.

12.

LIST OF REFERENCES

Amano, K., E. Ribi, and J.L. Cantrell. 1983. J. Biochem. 93,
1391—1399.

Angus, B.L., J.A.M. Fyfe, and R.E.W. Hancock. 1987. J. Gen.
Microbial. 133, 2905-2914.

Bryan, L.E., K. O’Hara, and S. Wong. 1984. Antimicrob. Agents
Chemother. 26, 250-255.

Caroff, M., S. Lebbar, and L. Szabo. 1987. Carbohydr. Res. 161,
C4-C-7.

Chester, LR, and RM. Meadow. 1975. Eur. J. Biochem. 58, 273-282.

Chester, I.R., P.M. Meadow, and T .L. Pitt. 1973. J. Gen. Microbial. 78,
305-318.

Darveau, R.P., and R.E.W. Hancock. 1983. J. Bacterial. 155, 831-838.
Dittmer, J .C., and M.A. Wells. 1969. Methods in Enz. 14, 482-530.

Drewry, D.T., L.A. Lamax, G.W. Gray, and SP. Wilkinson. 1973.
Biochem. J. 133, 563-577.

Dubray, G., and G. Bezard. 1982. Anal. Biochem. 119, 325—329.

Godfrey, A.J., L. Hatlelid, and LE. Byran. 1984. Antimicrob. Agents
Chemother. 26, 181—186.

Godfrey, A.J., M.S. Shahrabadi, and LE. Bryan. 1986. Antimicrob.
Agents Chemother. 30, 802—805.

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l3.

14.

15.

16.

17.

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24.

25.

26.

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56

Hancock, R.E.W., L.M. Matharia, L. Chan, R.P. Darveau, D.P. Speert,
and GB. Pier. 1983. Infect. Immun. 42, 170-177.

Hurlbert, R.E., J. Weckesser, H. Mayer, and I. Fromme. 1976. Eur. J.
Biochem. 68, 365—371.

Knirel, Y.A., E.V. Vinogradov, N .A. Kocharova, N.A. Paramonov, N .K.
Kochetkov, B.A. Dmitriev, E.S. Stanislavsky, and B. Lanyi. 1988. Acta
Microbial. Hung. 35, 3—24.

Koval, S.F., and P.M. Meadow. 1977. J. Gen. Microbial. 98, 387-398.

Kropinski, A.M., B. Jewell, J. Kuzio, F. Milazzo, and D. Berry. 1985.
Antibiat. Chemother. 36, 58—73.

Laemmli, U.K. 1970. Nature (London) 227, 680—685.
Mayer, H., and J. Weckesser. 1984. In Handbook of Endatoxin, Vol. 1:
Chemistry of Endotaxin (E.Th. Rietchel, ed.), pp. 221-247. Elsevier

Science Pub. B.V., Amsterdam.

Mills, B.J., and R.G. Holloway. 1976. Antimicrob. Agents Chemother.
10, 411—416.

Nikaido, H., and R.E.W. Hancock. 1986. In The Bacteria, Val. X (J .R.
Sokatch, ed.), pp. 145—193. Academic Press, Inc., New York.

Otten, S., S. Iyer, W. Johnson, and R. Montgomery. 1986. J. Bacterial.
167, 893—904.

Peterson, A.A., and E.J. McGroarty. 1985. J. Bacterial. 162, 738—745.

Peterson, A.A., R.E.W. Hancock, and E.J. McGroarty. 1985. J.
Bacterial. 164, 1256—1261.

Qureshi, N., K. Takayama, and E. Ribi. 1982. J. Biol. Chem. 257.
11808-11815.

Rivera, M., L.E. Bryan, R.E.W. Hancock, and E.J. McGroarty. 1988.
J. Bacterial. 170, 512-521.

Rocque, W.J., R.T. Coughlin, and E.J. McGroarty. 1987. J. Bacterial.
169, 4003—4010.

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57

Rowe, P.S.N., and P.M. Meadow. 1983. Eur. J. Biochem.. 132,
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Sawada, S., T. Kawamura, Y. Masaho, and K. Tomibe. 1985. J. Infect.
Dis. 152, 1290—1299.

Towbin, H., T. Staehelin, and J. Gorden. 1979. Prac. Natl. Acad. Sci.
USA 76, 4350—4354.

Wilkinson, S.G. 1983. Rev. Inf. Dis. 5, $941—$949.
Wilkinson, S.G., and L. Galbraith. 1975. Eur. J. Biochem. 52, 331—343.

Westphal, O., O. Liideritz, and F. Bister. 1952. Z. Naturfarsch. Teil B
7, 148-155.

Yokota, S., S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito.
1987. Eur. J. Biochem. 167, 203-209.

CHAPTER 3

Growth Dependent Alterations in the Production
of Serotype and Common Antigen Lipopolysaccharides

in Pseudamanas aeruginasa PAOl

58

ABSTRACT

Pseudamanas aeruginasa PAOl is grown in various media and at different
temperatures, and the heterogeneity of the extracted lipopolysaccharide (LPS) was
characterized by polyacrylamide gel electrophoresis. The size distributions of the
serotype-specific LPS and the common antigen LPS were analyzed on Western
blots (immunoblots). Cells grown at high, near-growth-limiting temperatures, at
low pH, in low concentrations of phosphate, or in high concentrations of NaCl,
MgClz, glycerol, or sucrose produced decreased amounts of the very long-chain
population of O-antigen LPS molecules. Lower temperatures and lowered
glycerol, lowered sucrose, low sulfate, lower salt concentrations, and elevated pH
did not significantly affect the level of this LPS population. The size and amount
of common antigen LPS was either unaffected or increased slightly when the cells
were grown under the above stress conditions. Cells grown under normal,
nonstressed conditions were agglutinated only by serotype-specific antibodies. In
contrast, cells grown under stress conditions, in which the long-O-polymer LPS
was absent, were agglutinated by both serotype-specific and common antigen-
specific antibodies. The results indicate that the long 0 polymers cover and mask

the shorter common antigen. However, specific growth conditions limit the

59

60

production of the long 0 polymer, allowing the exposure and reactivity of the

common antigen on the cell surface.

INTRODUCTION

The lipopolysaccharide (LPS) from Pseudamanas aeruginasa, like that from
other gram-negative bacteria, is a heterogenous mixture of molecules of different
polysaccharide lengths (19,32) and with variable levels of substitutions at specific
sites (27,38). In addition, LPS isolates from many serotypes of P. aeruginasa
contain two chemically and anti genically distinct fractions, a serotype-specific LPS
and a common antigen LPS (22,31,32,34). The size heterogeneity of LPS from
P. aeruginasa, Serratia marcesens and Salmonella anatum is reported to be altered
by growth temperature (1,25,30). These temperature-induced changes in LPS
heterogeneity reportedly affected various cell surface properties including
bacteriophage-inactivating capacity (25) and efficiency in plasmid transformation
(1). Altering growth conditions, such as medium composition, is also reported to
modulate the LPS chain length of Escherichia coli and to alter the sensitivity of
the cells to a neutrophil bactericidal protein (36). Further, growth of P.
aeruginasa in low magnesium or adaptive growth in polymyxin or aminoglycosides
alters the sensitivity of the cells to EDTA and polymyxin (12,17,33). In a similar
fashion, polymyxin resistance can be induced in Pseudamanas fluorescence by

growth in limiting phosphate (8). Such alterations in antibiotic sensitivity may

61

62

result from changes in the outer membrane which affect the permeation of these
drugs. Alterations in outer membrane structure induced by specific growth
conditions may change cation binding sites (12). Since a major site of cation
binding in the outer membrane is with LPS, we have initiated an analysis of the
influence of various growth conditions on the size heterogeneity of LPS in P.
aeruginasa. The results reported here indicate that near-growth-limiting conditions
including high temperature, high concentration of salt, sucrose, or glycerol, low
phosphate concentration, and low pH altered the size heterogeneity of the serotype-
specific LPS produced and allowed exposure of the common antigen LPS on the
cell surface. In addition, cells grown in limiting phosphate produced LPS that was
recovered mainly in the phenol phase, similar to what was observed for LPS from

a rough mutant strain.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Smooth strains of P. aeruginasa PA01 used in this study were H103 (15),
PAOl716 (24) and the polymyxin-resistant strain H185 (26). Strain AK1401, an
LPS—defective rough mutant of PA01, was also studied (1). Cultures of 1 liter
were grown in 2.8-liter Fernbach flasks on a rotary shaker operating at 230 rpm.
Cultures were grown at temperatures between 17 and 42°C in either tryptone (1% ,
wt/vol)-yeast extract (0.2%, wt/vol) broth (1' YE) or in terrific broth (TB)
consisting of 24 g of yeast extract, 4 ml of glycerol, and 12 g of tryptone per liter
of 0.017 M KH2P04 - 0.072 M KZHPO4. Cultures of H103 and AK1401 were
also grown in a modified basal medium (MBM) composed of 5 mM NaCl, 5 mM
KCl, 40 mM glucose, 30 mM (NH4)2HPO4, 30 mM (NH4)ZSO4, and 0.5 mM
MgC12. For H103 cultures, 0.05 mg/ml of adenine sulfate, and 0.1 mg/ml of L-
leucine (pH 7.0) were added; for cultures of AK1401, 20 pg of L-leucine, L-lysine
and L-threonine per liter and 5 pg of thiamine per ml were added. For cells
grown in low sulfate, MBM was modified; the final (NH4)ZSO4 concentration was
reduced to 0.1 mM and the concentration of (NH4)2HPO4 was increased to 60

mM. For cells grown in low phosphate the (NH4)2HPO4 in MBM was decreased

63

64
to 3 mM and the (NH4)ZSO4 was increased to 60 mM. To analyze the effect of

medium pH, we modified MBM; glucose was replaced with 40 mM citrate and the
pH was adjusted with NaOH or HCl. Cells were grown in the various media at
defined temperatures for at least two transfers before the culture was harvested late

in logarithmic growth phase.

LPS isolation

Cells form 1 liter of medium were washed with distilled water and extracted
with hot aqueous phenol (37). The two phases were separated at room
temperature by spinning at 5,000 x g for 5 min. The phenol and aqueous phases
were dialyzed extensively against 5 mM HEPES (N-2-hydroxyethylpiperazine-N'-
2—ethanesulfonic acid), pH 7.5, and then with distilled water. The dialyzed
samples were spun at low speeds, and the supernatant solutions were treated with
15 ug/ml DNase I (Sigma Chemical Co., St. Louis, MO) and 5 pg/ml RNase A
(Sigma) in 10 mM MgC12 for 30 min at 4°C. The samples were washed once
with 0.1 mM MgC12 and once with distilled water at 76,000 x g for 2 h and then
lyophylized. Unless otherwise noted, greater than 85 % of the LPS was recovered

in the aqueous phase, and only the aqueous phase-fractions were analyzed.

Gel electrophoresis and Western blots (immunoblots).
LPS samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) as previously described (31,32). The gels were

65
silver stained by the method of Dubray and Bezard (9). Western immunoblots of

SDS-polyacrylamide gels were prepared as described previously (4,31,32,35). The
0-5 serotype-specific LPS was detected with the monoclonal antibody anti-503
(14,31,32) and the common antigen LPS was detected with the monoclonal
antibody E87 (31,34). The blots were washed and visualized by using horseradish

peroxidase-conjugated goat anti-mouse immunoglobulin G antibody (Sigma).

Slide agglutination assays.

Cells grown under specific test conditions were harvested late in the
logarithmic growth phase. The cells were sedimented and suspended in saline
containing 0, 0.3 or 1.5% Formalin. After the cells were incubated for 30 min
at room temperature, the treated cells were washed twice in saline and resuspended
in saline at high concentrations (optical density at 560 nm of ~ 20). The cells
were mixed with an equal volume of either 0-5 specific monoclonal antibody
MF15-4 (21) or common antigen-specific monoclonal antibody E87 (34). Cells
were also mixed with saline or with control antiserum. After 5 min, agglutination
was assessed by light microscopy. The level of agglutination was quantitated by
measuring the relative number of cells which had clumped compared with the
number that were dispersed. The agglutination tests were performed at least twice
for each culture condition with independently isolated cells. Cells were used in

the agglutination studies immediate after isolation and treatment in all cases.

66
Assays.

Assays for 2-keto-3-deoxyoctulosonic acid (KDO) and protein were done as
described previously (32). Phosphate levels were quantitated by inductively

coupled plasma emission spectroscopy (6).

RESULTS AND DISCUSSION

LPS recovery and phosphate content of samples from cells grown in different
media.

Lipopolysaccharide was isolated from cells grown in several different media
by using hot aqueous phenol (37), and the recovery of LPS in the aqueous and
phenol phases was quantitated by assaying for 2-keto-3-deoxyoctulosonic acid.
Except for the rough mutant AK1401 and the smooth strain H103 grown in low
phosphate, greater than 85 % of the LPS was recovered in the aqueous phase. For
LPS from strain AK1401, between 25 and 80% of the LPS was recovered in the
aqueous phase, depending on the growth medium used. Also, for cells of H103
grown in MBM low in phosphate, only 25% of the LPS was recovered in the
aqueous phase with the remainder isolated from the phenol phase. The low
recovery of LPS in the aqueous phase for these cultures may result from the lack
of hydrophilic long O-polymer-containing LPS in the isolates (see below). The
amount of LPS recovered in the aqueous phase for both the smooth and rough
strains varied depending on the growth medium (Table 1). Generally, the LPS
recovered constituted between 2 to 6% of the cell dry weight. This level is similar

to that reported for LPS levels from P. aeruginasa (7). Modification of the media

67

68

TABLE 1. Level of LPS Recovered from Cultures of AK1401 and of PA01
Smooth Strains H103 and PAOl716 Grown in Different Media.a

 

 

 

 

 

Medium PA01 Smooth Strainsb AK1401
TB 2.5 i 0.6 1.0 i 0.1
TYE 5.0 :t 1.0 3.9 i 0.6
MBM 6.1 i 1.1 4.9 i 0.4
MBM (low Pofi‘)c 2.0 i 1.0 3.1 :1: 0.7
MBM-citrated 6.0 :t 0.5 5.3 :I: 0.9

 

 

aThe recovery is expressed as a percentage of the dry weight of the cells (mean
:1; standard deviation). Only recovery from the aqueous phase after phenol
extraction is reported.

1’Recovery for the two smooth strains was not different, and the average recovery
is reported.

‘7ic MBM medium contained lower levels of (NH4)2PO4 as indicated in Materials
and Methods.

dGlucose was replaced by citrate in the MBM (see Materials and Methods).

69

or other growth conditions as described below did not significantly alter the level
of LPS recovery (data not shown).

The phosphate contents of the various LPS isolates also did not vary
appreciably with the growth medium used (Table 2). LPS from the smooth strains
grown in TB, which is high in phosphate, had higher phosphate levels, while LPS
from cells grown in minimal medium low in phosphate contained lower levels of
phosphate. Similar trends were detected in the isolates from strain AK1401.
Other modifications of the growth media or growth conditions as described below
did not significantly affect the LPS phosphate content (data not shown).

Using these different media we tested the influence of specific growth
conditions on the size and heterogeneity of the LPS produced by the smooth and
rough strains. The structure of the gram—negative envelope is known to change in
response to growth temperature, medium composition, and medium osmolarity (2).
Furthermore, the outer membrane of P. aeruginasa is exceptionally sensitive to
rapid changes in temperature, pH, and toxicity (5). Thus, these growth conditions

were examined for their influence on LPS composition.

Growth temperature effects on LPS composition.

Cultures of PAOl7l6, H103, and AK1401 were grown in TB and TYE at
temperatures between 17 and 42°C for two transfers and then harvested. The size
heterogeneity of the various isolates from the aqueous-phase fraction was defined

by SDS-PAGE (Figure 1; data not shown). When large amounts of the samples

70

TABLE 2. Phosphate Content of LPS Isolates from Cells Grown in Different

 

 

 

Mediaa
pmol/mg LPS
Medium PA01 Smooth Strainsb AK1401
TB 21:01 L8i01
TYE 1.7 i 0.1 1.7 i 0.1
MBM 1.6 i 0.1 1.4 i 0.2
MBM (low P02“) 1.4 i 0.1 1.1 i 0.1

 

aPhosphate contents are given as the weighted average :I: standard deviation of the
levels recovered from the phenol phase and aqueous phase.

bThe levels represent the average amount detected in LPS from strain H103, H185
or PAOl7l6.

Figure 1.

71

Effect of growth temperature on LPS size. Silver-stained gels (A
and B) and Western blot (C) of LPS from strains PAOl7l6 or
AK1401 grown in TB at the temperatures indicated. Samples of 10
(A), 0.25 (B), or 30 (C) pg were applied. The Western blot (C) was
visualized with the monoclonal antibody E87. Numbers to the left

of panel A denote the major size populations.

72

     

A
PAO I7l6 AK ,40'
17° 22° 27° 32° 37°42° 17° 37o
' 92?, . :2!

Figure 1

 

      

B
PAO I7I6 AKI40|
|7° 22° 27° 32°37° 2o 37.

 

73

C
AK I40| PAO l7|6

 

 

 

Figure 1

74

were applied to the gel, the ladder pattern of the high-molecular-weight O-
polymer-containing molecules could be detected (Figure 1A). For the isolates
from the smooth strain, three to four regions of intense staining could be detected
which represented three to four populations of molecules with O-polymers of
different lengths. LPS from cells grown at low temperatures had higher levels of
the very long O-polymer-containing molecules (population 1) and lacked the
shorter-chained population, denoted 2a. As the growth temperatures increased, the
level of molecules of the highest molecular weight dropped and the amount of
short-chained molecules increased. The level of the intermediate-sized molecules
(population 2) showed only marginal changes. These results are similar to those
of a previous study which showed a decrease in high-molecular-weight LPS in P.
aeruginasa with increasing growth temperature (20). The LPS from strain
AK1401 grown in TB appeared rough and did not contain very high levels of O-
polymer-containing molecules. Only very weak staining in the region of the
common antigen bands was detected in the LPS from cells of AK1401 grown in
TB at 17 and 37°C (see below). This reflected a general inhibition in common
antigen LPS production in all strains grown in TB (data not shown).

When these same samples were applied at low concentrations, the
heterogeneity of the predominant, smaller-sized molecules could be resolved
(Figure 1B). For the PAOl7l6 samples, as the growth temperature increased,
there was an increase in the band immediately above the fastest-moving band. The

band of higher mobility presumably corresponds to molecules containing core-lipid

75

A, while the slower moving band likely contains molecules capped with one 0
repeat. This increased capping with increasing growth temperature has been noted
by others (20). The LPS from the rough mutant also revealed the same two
bands, confirming a previous study which showed that AK1401 is an SR mutant
(22). For both strains grown at temperatures above 32°C, the LPS contained a
second, slower-migrating band which could be resolved above each of the two
fastest-migrating bands; these secondary bands increased in relative amounts with
increasing growth temperature.

Since the LPS isolates contain a common antigen LPS in addition to the
main, serotype-specific LPS, the change in the size distribution of this population
was analyzed on immunoblots. The LPS isolates from the two strains grown at
different temperatures were separated by SDS-PAGE and then electrotransferred
to nitrocellulose. The common antigen LPS bands were visualized with
monoclonal antibody E87, which is specific for the P. aeruginasa common antigen
(31,34). The transblots indicated that for the PAOl7l6 strain, the level and size
distribution of the common antigen molecules was unaffected by growth
temperature (Figure 1C). In addition, the AK1401 strain contained common
antigen LPS, and the level and size distribution also did not change significantly
with growth temperature. However, the size distribution of the common antigen

LPS from strain AK1401 was somewhat different from that of the smooth strain.

76

Effect of salt concentrations in media on LPS composition.

Cells of H103 and H185 were grown at 37°C in TYE broth containing 0,
3, 10, 30 or 100 mM MgC12. Growth was completely inhibited for both strains
in medium containing 300 mM MgC12. Cultures of H103 were also grown at
37°C in TYE containing 0, 50, 150 and 500 mM NaCl. Growth was inhibited by
1.5 M NaCl in the medium.

The size distribution of the isolates from H103 and H185 grown in various
concentrations of MgC12 were analyzed by SDS-PAGE. The gels of the H103
isolates (Figure 2) and H185 (not shown) were either silver stained or were
transblotted and reacted with monoclonal antibody E87 or anti-503. When these
samples were applied to gels at high concentrations, the long-chain LPS could be
detected. Silver-stained gels of the H103 samples indicated that there was little
change in the O-antigen length with Mg2+ concentration except at 100 mM
(Figure 2B). At this highest concentration, there was a dramatic decrease in the
level of the highest-molecular-weight population of LPS. This population
(population 1) represents the longest O-specific LPS molecules as detected on
Western blots with the O-specific monoclonal antibody (Figure 2A). The size
distribution of the common antigen LPS, measured in blots with monoclonal
antibody E87 (Figure 2C), also did not change appreciable with the concentration
of MgC12 in the growth medium, except at the highest concentrations. The
samples from cells grown in 100 mM MgC12 had a slight increase in the longer

common antigen molecules. When the samples were applied to the gel at low

Figure 2.

77

Effect of MgC12 concentration in the growth medium on LPS size.
Silver-stained gels (B and D) and Western blots (A and C) of LPS
from strain H103 grown in TYE nutrient medium containing 1 (lane
1), 3 (lane 2), 10 (lane 3), 30 (lane 4) or 100 (lane 5) mM MgC12.
Samples of'10 (B), 5 (A and C), or 0.05 (D) pg of LPS were applied
to the wells. After electrophoresis, the gels were either silver-
stained (B and D) or transblotted onto nitrocellulose and reacted with

either anti-503 (A) or E87 (C) monoclonal antibody.

78

A B C D
5432| |2345 5432| l2345

 

J

 

Figure 2

79

concentrations, the heterogeneity of the predominant, short-chained LPS could be
measured. The results showed that the level of LPS containing one 0 repeat was
not significantly altered when the M g2+ concentration in the medium was
increased (Figure 2D). Essentially identical results were obtained with the LPS
samples from strain H185.

When the H103 cells were grown in TYE containing high concentration of
NaCl, the LPS composition changed in a manner similar to that for cells grown
in high MgClz. The levels of the very-long-chain O-antigen molecules decreased
on cells grown in 500 mM NaCl but were unaffected by lower NaCl
concentrations (Figure 3A). Furthermore, the amount and length of the common
antigen also increased at the highest NaCl concentration but was unaffected by
lower levels of salt (Figure 3B). The heterogeneity of the short-chain LPS was

also unaffected by the NaCl concentrations (data not shown).

Effects of growth in high glycerol or high sucrose on LPS composition.
Since near-growth-limiting concentrations of salt appeared to inhibit the
synthesis of the long-O-antigen molecules and to induce an increase in common
antigen LPS amount and length, we asked whether this phenomenon might depend
on the osmotic strength of the media. Thus, cultures of H103 were grown in TYE
containing either 1.0 M glycerol or 1.0 M sucrose, which are both near growth
limiting. After two transfers in these media, the cells were harvested and the LPS

was isolated. When the aqueous-phase isolates were analyzed by SDS-PAGE, both

Figure 3.

80

Effect of NaCl concentration in the growth medium on LPS size.
Western blots of LPS from strain H103 grown in TYE nutrient
medium containing 0 (lane 1), 50 (lane 2), 150 (lane 3), or 500 (lane
4) mM NaCl. A 10-pg sample of the LPS was applied to the wells.
After electrophoresis, the gels were transblotted into nitrocellulose
and reacted with either anti-503 (A) or E87 (B) monoclonal

antibody.

81

A B
|234 l234

 

Figure 3

82

samples appeared to have a very long chain population that was shorter (population
1) than the LPS from cells grown in the absence of glycerol (Figure 4) or sucrose
(not shown). However, the population 1 set of molecules was not completely
missing in either of these isolates.

Thus, the LPS changes induced by growth in high salt are likely not
triggered by high osmolarity since growth in high glycerol and high sucrose had
much less of an effect on O-polymer synthesis. That high salt may induce an
alteration in the LPS composition and antigen reactivity may be critical for cystic
fibrosis (CF) patients infected with P. aeruginasa. Lam et al. (22) have shown
that a high percentage of clinical isolates produced common antigen LPS. Of the
strains which produced the A-band (common antigen) LPS, 68% could be
agglutinated by anti-common antigen monoclonal antibodies. This study also
indicated that during infection of a CF patient with P. aeruginasa, the initial
isolates were serotypeable and produced long-chain LPS. However, during
infection the strain became nontypeable and the cells were agglutinated with
common antigen monoclonal antibody. Also, the patient produced antibody to the
A-band LPS during the infection. Presumably, the environment of the lung of
these patients selects for cells which produce lower amounts of long-chain,
serotype-specific LPS. Others have reported that during infection of CF patients,
P. aeruginasa strains lose their O-antigenic determinants (28,29), become
nontypeable (16), and serum sensitive (16,29), and produce a new,

polyagglutinable antigen (l6,23,28,29). It has been suggested that the

Figure 4.

83

Effect of 1 M glycerol in the growth medium on LPS size. Silver-
stained gel (A) and Western blot (B) of LPS from strain H103 grown
in TYE nutrient medium containing 0 (lane 1) or 1 (lane 2) M
glycerol. A 20-pg sample of LPS was applied to each well. The

Western blot was reacted with anti—503 monoclonal antibody.

84

A B
1212

1.19:; 'WI

 

Figure 4

85
P°\Yagglutinable antigen is in the core-lipid A which becomes exposed when the

0 polysaccharide is diminished (11). However, recent evidence indicates that the
common antigen polysaccharide is present and exposed on many nontypeable
strains (22). The presence of altered or elevated levels of specific ions in the
sputum of CF patients (10,18) may induce a decrease in long-O-polymer LPS
synthesized by the infecting P. aeruginasa just as we have observed for cells in
culture. In addition, in the CF patient, extensive antibiotic therapy may select for
B-lactam (l3) - and aminoglycoside (3) - resistant strains, some of which appear
rough or have decreased amounts of O polymer. Thus, there may be several
selective pressures that induce P. aeruginasa strains infecting CF patients to lose

their long 0 antigen.

Effects of growth in low phosphate and low sulfate on LPS composition.
The serotype-specific LPS has been shown to be high in phosphate and low
in sulfate, while the common antigen LPS appears to lack phosphate but contain
sulfate (31). To analyze the influence of these anions on the production of the two
types of LPS, we grew cells of strain H103 and AK1401 in MBM, in MBM low
in sulfate, and in MBM low in phosphate. The LPS isolates were separated by
SDS-PAGE, and the gels stained with silver. When the H103 samples were
loaded onto the gel at high concentration, the ladder pattern of the high-molecular-
weight LPS could be detected (Figure 5C). The samples from cells grown in

minimal medium and minimal medium low in sulfate appeared similar. In

Figure 5.

86

Effect of phosphate and sulfate concentrations in the growth medium
on LPS size. Silver-stained gels (C and D) and Western blots (A
and B) of LPS isolated from strain H103 grown in MBM (lane 1),
MBM low in sulfate (lane 2), or MBM low in phosphate (lanes 3
and 4). Samples in lane 3 were recovered from the aqueous phase,
while samples applied to lane 4 were from the phenol phase.
Samples of either 10 pg (A, B and C) or 0.1 pg (panel D) were
applied to the gels. Western blots were reacted with either anti-503

monoclonal antibody (A) or E87 monoclonal (B).

87

m 83E

 

mm
m

 

mm_
<

88

contrast, the LPS isolated in the aqueous phase from cells grown in low phosphate
appeared to contain fewer of the molecules with long 0 polymers. This difference
in the amounts of O-polymer-containing LPS was also detected on Western blots
of these same samples reacted with monoclonal antibody anti-503 (Figure 5A).
The cells grown in MBM and in MBM low in sulfate appeared to have similar
amounts of the very long O-specific LPS, while the LPS from the aqueous-phase
isolates of cells grown in low phosphate had a reduced amount of these long 0
polymers. Western blots of these samples stained with the common-antigen-
specific monoclonal antibody E87 indicated little difference in the levels and size
of common antigen LPS from cells grown in MBM and MBM low in sulfate
(Figure 5B). In contrast, the aqueous-phase isolates of cells grown in low
phosphate had a significantly greater level of common antigen LPS. Similar to the
analyses described above, the LPS isolated from the phenol phase from the cells
grown in low phosphate contained very little long-O-polymer LPS and was
composed mainly of core-lipid A molecules (population 3) and common antigen
LPS (Figure 5C). Analysis of the heterogeneity of the predominant short-chained
molecules (Figure 5D) indicated that the level of capping of the core-lipid A by
one or two 0 repeats was similar for the isolates from cells grown in MBM and
in low-sulfate MBM and from the phenol-phase isolates of cells grown in low
phosphate. However, the aqueous-phase isolates of cells grown in low phosphate

had a lower proportion of molecules capped with one 0 repeat. Surprisingly, the

89
LPS isolated from AK1401 grown in MBM and MBM low in phosphate was

essentially identical on SDS gels (data not shown).

Effect of growth at low pH on LPS composition.

To analyze the effects of medium pH on the LPS produced, we grew
cultures of strains H103 and AK1401 in MBM containing citrate instead of glucose
as the carbon source. The citrate buffered the medium in the acid range to within
0.2 pH units of the value set before inoculation. The growth rate of the two
strains at neutral pH in minimal media did not change with the change in carbon
source.

The heterogeneity of the LPS isolated from cells grown at different pHs was
detected on SDS gels (Figure 6). When the samples were loaded at high
concentrations, the size heterogeneity of the H103 isolates did not vary between
pH 6.9 and 7.8 and the size distribution of the samples isolated at neutral pH was
the same for cells grown on both carbon sources. However, the production of
long-chain LPS from cells grown at pH 5.5 was severely restricted. This pH is
near the lower limit for growth of this organism and appears to induce a stress-
related response in the smooth strain similar to that seen with high temperature and
high salt. Surprisingly, this low pH had little effect on the production of common
antigen LPS by AK1401 (Figure 6A). Analysis of the heterogeneity of the
dominant short-chain components (Figure 6B) showed that, for both strains, low

medium pH decreased the level of capping of the core with one 0 repeat and

Figure 6.

90

Effect of pH of growth medium on LPS size. Silver-stained gel of
LPS from strains AK1401 and H103 grown in MBM containing 40
mM glucose or citrate. The pH of the medium was adjusted as
indicated. Samples of either 12.5 pg (A) or 0.125 pg (B) were

applied.

91

H103

AK1401

6.6.820

35:0

8.25“

302.5

5.5 6.9 7.1 7.8 7.0

7.0 5.6 7.1 7.6 8.3

A

pH=

2.5:

mag: H.,

§==: .
gt ..

6
e
r
u

.We

F

 

       

- 'M‘" B

"an!

92

decreased the level of the slower-moving band in the doublet pattern; these
changes are similar to what was seen in the short-chain LPS population with

growth at lower temperatures (Figure l).

Whole-cell agglutination assays.

In the studies described above, we found several growth conditions
including high temperature, high salt concentrations, high glycerol and sucrose
concentrations, low phosphate, and low pH which induced a change in the size
heterogeneity of the serotype-specific LPS, resulting in a decrease in the number
of molecules with a long 0 polymer. These same growth conditions either had no
effect on the common antigen LPS produced or they induced an increase in the
amount and size of the common antigen molecules. To determine if the
reactivities of these LPS antigens on the cell surface were altered by such changes
in LPS composition, we used monoclonal antibodies MF15-4 and E87 to
agglutinate whole cells. Cultures of H103 were grown in the various test media
at specific temperatures for two transfers. A culture of AK1401 was also grown
in TYE broth as a control since it lacks serotype-specific LPS (Figure 1). The
cultures were treated as described in Materials and Methods and then mixed with
one of the monoclonal antibodies. Results of the agglutination assays are shown
in Table 3. The H103 strain grown at 37°C in TYE broth showed no
agglutination with the monoclonal antibody to common antigen even when the cells

were Formalin treated, while the serotype-specific antibody, MF15-4, readily

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agglutinated these cells. The agglutination with MF15-4 was strongest when the
cells were pretreated with Formalin, suggesting that formaldehyde alters the O
polymer to make it more accessible to antibody interaction. The lack of
agglutination by common antigen-specific E87 may be due to the longer serotype
molecules masking the shorter common antigen polymers. Thus, when these same
cells were grown in TYE broth at 42°C or in 0.5 M NaCl or when they were
grown in low-phosphate-containing minimal medium, conditions which permitted
synthesis of only shorter O polymers, the cells were agglutinated with E87
antibody. The agglutination of these cells with E87 was strongest if the cells had
been pretreated with Formalin, again indicating that formaldehyde alters the
polysaccharide to allow for better reactivity. These same cells were also strongly
agglutinated by the serotype-specific MF15-4 antibody, indicating that both
antigens were present on the surface of the cells. Since greater than 90% of the
H103 cells were agglutinated with serotype-specific antibodies, it appears that most
of the H103 cells in the cultures grown under any of these conditions contained
both types of LPS in their outer membrane.

The cells grown in TYE containing 1 M glycerol showed only weak
agglutination with E87. Thus, the loss of only the longest of the very-long-chain
O polymers (Figure 4) did not cause a significant increase in the exposure of the

common antigen molecules on the cells. Cells grown in minimal medium also

m

 

95
showed only weak agglutination with E87; these cultures also produced the long

0 polymers which masked the common antigen. As expected, agglutination of the
AK1401 strain was detected only with E87. This agglutination was very strong,
presumably because these cells lack a masking O polymer.

In conclusion, analysis of LPS isolates from cells grown under different
conditions showed that several stress conditions including high, near-growth-
limiting salt concentrations, high, near-growth-limiting temperatures, and low,
near-growth-limiting pH induce a dramatic decrease in the length of the O-specific
LPS. Furthermore, for cells grown at high temperature, there was an increase in
capping of the predominate short-chained population, as has been reported
previously (20). Growth in high salt, either MgC12 or NaCl, also induced an
increase in the amount or length of the common antigen. In contrast, growth at
high temperatures did not alter the common antigen. Loss of the long 0 polymer
allowed for reactivity of the common antigen on the cell surface, which normally
is not accessible to antibody. Since > 90% of all smooth cells tested were
agglutinated by serotype-specific antibody and since > 80% of cells from cultures
grown at high temperatures or in high salt were agglutinated by common antigen-
specific antibody, it appears that most cells in these cultures contain both antigens
on their surface. Thus, these two LPS antigens do not appear to represent
classical antigenic variation. Presumably, when high levels of the long 0 polymer
are synthesized, the shorter common antigen is present on most cells but is

covered and inaccessible to antibody.

10.

11.

12.

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Brown, M.R.W., and P. Williams. 1985. J. Antimicrob. Chemother.
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Bryan, L.E., K. O’Hara, and S. Wong. 1984. Antimicrob. Agents
Chemother. 26, 250-255.

Burnette, W.N. 1981. Anal. Biochem. 112, 195-203.
Costerton, J.W., M.R. Brown, and J.M. Sturgess. 1979. In
Pseudamanas aeruginasa: Clinical Manifestation and Current Therapy

(R.G. Doggett, ed.) pp 63-88. Academic Press, London.

Coughlin, R.T., S. Tonsager, and E.J. McGroarty. 1983. Biochemistry
22, 2002-2007.

Darveau, R.P., and R.E.W. Hancock. 1983. J. Bacterial. 155,
831-838.

Dorrer, E., and M. Teuber. 1977. Arch. Microbial. 114, 87-89.
Dubray, G., and G. Bezard. 1982. Anal. Biochem. 119, 325—329.

Filliat, M., C. Galabert, J.P. Chazalette, and A. Dauplan. 1981.
Managr. Pediatr. 14, 142-145.

Fomsgaard, A., R.S. Conrad, C. Galanos, G.H. Shand, and N. Hoiby.
1988. J. Clin. Microbial. 26, 821-826.

Gilleland, HE. 1988. Can. J. Microbial. 34, 499—502.

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13.

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97

Godfrey, A.J., L. Hatfield, and LE. Byran. 1984. Antimicrob. Agents
Chemother. 26, 181-186.

Godfrey, A.J., M.S. Shahrabadi, and LE. Bryan. 1986. Antimicrob.
Agents Chemother. 30, 802—805.

Hancock, R.E.W., V.J. Raffle, and T.I. Nicas. 1981. Antimicrob.
Agents Chemother. 19, 777-785.

Hancock, R.E.W., L.M. Mutharia, L. Chan, R.P. Darveau, D.P.
Speert, and GB. Pier. 1983. Infect. Immun. 42, 170—177.

Kenward, M.H., M.R.W. Brown, and J.J. Fryer. 1979. J. Appl.
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Kilbourn, JP. 1984. Curr. Microbial. 11,19—22.

Koval, S.F., and P.M. Meadow. 1977. J. Gen. Microbial. 98,
387—398.

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1960-1966.

Lam, J.S., L.A. MacDonald, M.Y.C. Lam, L.G.M. Duchesne, and
G.G. Southam. 1987. Infect. Immun. 55, 1051-1057.

Lam, M.Y.C., E.J. McGroarty, A.M. Kropinski, L.A. MacDonald,
S.S. Pedersen, N. Hoiby, and J .S. Lam. 1989. J. Clin. Microbial. 27,
962-967.

Luzar, M.A., and TC. Montie. 1985. Infect. Immun. 50, 572-576.

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McConell, M., and A. Wright. 1979. J. Bacterial. 137, 746—751.
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(J .R. Sokatch, ed.) pp. 145-193. Academic Press, New York.

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Ojenigi, B., L. Back, and N. Hoiby. 1985. Acta Pathol. Microbial.
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CHAPTER 4

Common Antigen Lipopolysaccharide of

Pseudamanas aeruginasa Contains Sulfur.

99

ABSTRACT

Lipopolysaccharide (LPS) was isolated from Pseudamanas aeruginasa strain
AK1401 which lacks O-antigen. The cells were grown in modified basal medium
containing 350 pCi of 35S-labelled sulfate, and the isolated LPS was separated by
gel filtration chromatography into two major size populations of LPS. These
fractions were characterized as a common antigen LPS (A-band) and a serotype—
specific short-chain LPS (SC or B-band). The elution profile showed that the A-
band and B-band LPS contained labelled-sulfur. This is in agreement with the
results of an autoradiogram of the polyacrylamide gel separation of unfractionated
(Ut) LPS sample. Chemical analysis of the A-band LPS showed low levels of
detectable 2-keto-3-deoxyoctulosonic acid (KDO) and no phosphate. In addition,
A-band LPS showed a 1:1 molar amount of sulfur to heptose suggesting that two
to three sulfur atoms are present in the A-band molecule probably as sulfate. The
molar amounts of sulfur detected in Uf- and SC-LPS isolates were one half the
heptose content. We propose that this A-band LPS possesses an ’unusual’ lipid
A with the distinctive feature that the phosphate groups in the 1- and 4’-position

of the diglucosamine head group are missing and may be replaced by sulfate.

100

INTRODUCTION

Lipopolysaccharide (LPS) is a major constituent on the surface of gram-
negative bacteria and is essential for the assembly, organization (18,25), and
functioning of the outer membrane (18,24). The LPS from Pseudamanas
aeruginasa, as well as that from Enterabacteriaceae, can be divided into three
structural regions: the lipid A, the core oligosaccharide, and the O-antigen
polysaccharide (16,29). Structural similarities between LPS from P. aeruginasa
and enterobacterial LPS include a bisphosphorylated D-glucosamine disaccharide
lipid A backbone and the inner core sugars L-glycera-D-manna-heptose and
2-keto-3-deoxyoctulosonic acid (KDO) (16,18). However, LPS from P.
aeruginasa is unique in its large number of phosphate residues, the presence of L-
alanine in the core, and the presence of 2-OH- and 3-OH-dodecanoic acids and 3-
OH-decanoic acid instead of 3-OH-tetradecanoic acid characteristic of enterics
(9,16,29,35,36).

In smooth strains, a long polysaccharide is attached to the core, but in P.
aeruginasa a low proportion of the LPS molecules carry the polysaccharide
(30,35). Chemical differences in the O-polysaccharide (O-antigen) of different

strains distinguish P. aeruginasa into 20 different O-serotypes (22,23).

101

102

Characterization of LPS structure has been complicated not only by chemical
heterogeneity in the lipid A (2,4,35) and incomplete substitution in the core
(16,24,29,35) but also by chemical heterogeneity of the O-antigen side chain
(16,24,35). Beside the variability in the O-polysaccharide length (16,30), the LPS
isolates of P. aeruginasa have been resolved into amino-sugar-rich and neutral-
sugar-rich fractions (30,32,36,39).

Recently, it has been shown that strains of P. aeruginasa synthesize two
immunologically and chemically distinct forms of LPS known as A-band and B-
band LPS (27,30,31). B-band LPS is the O-antigen-containing LPS and
determines the O-specificity of the bacterium, while A-band LPS or common
antigen contains shorter chains of predominantly neutral polysaccharide. Others
have observed a common lipopolysaccharide antigen in standard and clinical strains
of P. aeruginasa (20,32). Arsenault et al. (T .L. Arsenault, D.W. Hughes, D.B.
MacLean, W.A. Szarek, A.M.B. Kropinski, and J.S. Lam, Can. J. Chem., in
press) demonstrated that the high neutral sugar content of the A-band LPS is
attributed to an unusually high level of D-rhamnose. This is similar to the data
reported by Yokota et al. (38,39) and Kocharova et al. (17) who also isolated a
neutral polysaccharide from P. aeruginasa containing predominantly D-rhamnose.
The main polymer chain of the rhamnose-rich polysaccharide is reported to consist
of the repeating unit ->3)Rha-(a1->3)Rha-(oz1->2)Rha-(a1- (17,38,39; T.L.
Arsenault et al., Can. J. Chem., in press).

In a recent study, Rivera and McGroarty (31) showed that the fatty acid

103

composition of the A-band LPS from P. aeruginasa PAOl strains was similar to
the serotype-specific B-band LPS, but that the A-band fraction did not contain
KDO or phosphate. Instead, sulfate was detected in this LPS isolate. As
previously suggested (31), sulfate may replace the phosphate on lipid A of A-band
LPS. It is interesting to note that sulfolipids have been identified in various
bacteria (10—12,15,21); they are highly hygrosc0pic, and occur mainly in
membranes which have predominantly acidic lipids (11,12). In aqueous solutions
between pH 7.5 and 9.0 there is negligible hydrolysis of the sulfate from
sulfolipids (1 1). In this study we have demonstrated that sulfur is incorporated

into A-band LPS in stoichiometric amounts.

RESULTS AND DISCUSSION

The bacterial strain used in this study was P. aeruginasa AK1401, an LPS
defective rough mutant of the restrictionless mutant isolate OT684 (leu-l Iys-l res-
4, 3). This strain produces A-band LPS but is defective in the synthesis of 0-
antigen. Cultures were grown at 37°C in modified basal medium (MBM) (27)
with the following modification: the final (NH4)ZSO4 concentration was reduced
to 0.9 mM, and 0.1 mg/ml of L-leucine, L-lysine, and L-threonine, 2 pg/ml of
thiamine, and 350 pCi of 35S-labelled calcium sulfate were added per liter of
culture. Cells were adapted to MBM for at least two transfers before growing in
labelled sulfate. The cells were harvested late in the logarithmic phase. Labelled
cells from 3 liters of medium were washed with distilled water and extracted with
hot aqueous phenol (33). The aqueous phase was extensively dialyzed and
lyophilized as described previously (27) except that 50 mM K2S04 was added to
the 5 mM HEPES (N -2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.5,
to insure that the 35S-labelled sulfate remained covalently bound.

The LPS isolate was fractionated on Sephadex G-200 (Pharmacia Fine
Chemicals, Piscataway, NJ) column (69 cm by 25 mm) at room temperature by

using the buffer system (pH 8.0) of Peterson and McGroarty (28). Approximately

104

105
5 .9x104 cpm of 35S-labelled LPS was applied to the column, and 4-ml fractions

were collected at a flow rate of 6.4 ml/h. Column fractions were analyzed for
amino sugar content (8) and total phosphate (1). 35S counts (cpm/m1) were
measured using a Beckman LS7000 Microprocessor Controlled Scintillation
Counter. The column fractions were also characterized by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) using the system of Laemmli
(19), and the gels were silver-stained by the method of Dubray and Bezard (7).
The elution profile showed three 358 peaks (Figure 1, peaks 1,2, and 3) and two
amino sugar peaks (Figure 1, peaks 1 and 2), while phosphate could be detected
only in peak 2. Note that peak 3 contains only 35S and elutes as a broad peak
(fraction number 75 to 90). Presumably this peak contains either free sulfate that
has been released from the LPS during the column fractionation or sulfur bound
to small oligosaccharides. The dashed line indicates the position of elution of free
35S-labelled sulfate suggesting the second possibility is more likely (Figure l,
arrow). The SDS-PAGE of the column fractions, when applied in order of
elution, revealed a diagonal ladder pattern representing molecules of different sizes
(Figure 1, A— and B-bands). The A-band LPS corresponds to the slower migrating
set of bands (Figure 1, peak 1) while the second population of molecules,
designated the B-band or SC-LPS (Figure 1, peak 2), corresponds to the faster
migrating set of bands. As indicated in earlier studies (30,31), the SC-LPS
fraction is composed of molecules containing a core-lipid A with none or only one

O-repeat unit similar to that of the main serotype-determining antigen. The

Figure 1.

106

Gel filtration profile of Uf-LPS from P. aeruginasa AK1401
grown in 3SSO4-MBM media. Fractions were analyzed for
amino sugar (I), total phosphate (A), and 353 counts (0).
Silver-stained SDS-polyacrylamide gels of column fractions
are aligned under their appropriate fraction number. A
represents the slow-moving set of bands, and B represents the
faster moving set. The dashed line is free 35803; eluting

from the column at the inclusion volume (arrow).

107

H (nu/rude) stunoo s“

 

3000~

2000‘

IOOOq

 

1
R0

H0

100

 

90
Fraction Number

80

7O

 

60

 

 

I
9

o o'
H (“‘V) aroudsoud 10:01

I
N

OJ~

 

 

0304

020~

H (“‘V) J0598 09in

'010-

Figure l

 

108
fractions corresponding to A- and B-bands LPS (Figure 1, peaks 1 and 2,

respectively) were pooled and extensively dialyzed (12,000- to 14,000-molecular-
weight—cutoff membranes), at room temperature, against a buffer composed of 50
mM Tris, 10 mM EDTA, 50mM triethylamine, and 0.02% sodium azide, pH 8.5.
The pooled fractions were dialyzed further against 10 mM MgC12 and then
against distilled water. The dialyzed fractions were lyophilized and stored at 4°C
for further analysis.

Since the A-band LPS analyzed in previous studies was isolated from
smooth strains of P. aeruginasa, the isolates were always contaminated with small
amounts of an intermediate chain-length serotype-specific LPS (30,31). By using
the AK1401 strain we obtained purified A-band LPS devoid of any O-serotype
LPS contaminants. To verify that the fatty acid composition of this A-band LPS
isolate corresponds to what has been reported previously (31), the fatty acid
analysis of this A-band LPS, as well as SC-LPS and Uf-LPS, was performed as
described elsewhere (13). For total membrane fatty acid composition, 0.2 ml of
resuspended total membrane pellet was used. The identity of the fatty acids was
confirmed by gas chromatography/mass spectrometry (GC/MS). GC/mass
spectrometry was performed on a J EOL AXSOS double focusing mass spectrometer
equipped with a Hewlett-Packard 5 890 GC and a capillary column (Supelco SPBI;
using helium as carrier at a flow rate of l-ml/min) interfaced directly into the ion
source. The GC was programmed at an initial temperature of 150°C, and heated

at a rate of 3.0 deg/min to a final temperature of 300°C, with a final hold of 10.0

109

min; the total run length was 60.0 min. Mass spectra were acquired at a rate of
approximately 1 scan per second. Compounds were ionized by electron ionization
(EI) at 70 eV. A bacterial acid methyl esters mixture (Supelco, Bellefonte, PA)
was used to determine retention times as well as fragmentation patterns for the
fatty acid methyl esters. The fatty acid compositions for the various LPS fractions
are presented in Table l. The fatty acids of P. aeruginasa strain AK1401 LPS
consisted of C12:0 and the hydroxy acids characteristic of the Pseudamanas, 3-
OH-C10:0, 2-OH-C12:0, and 3-OH-C12:0 (9). All three samples contained high
amounts of 2-OH- and 3-OH-dodecanoic acid and lacked the 3-OH-tetradecanoic
acid. As previously reported (31), these results show that the A-band isolate of
strain AK1401 is similar to the A-band LPS isolated from smooth strains and
confirm that it has the fatty acid composition typical of this species. In addition,
the total cell fatty acid composition of P. aeruginasa AK1401 is in agreement with
that of an earlier report (14).

Also, our previous studies indicated that the A-band LPS (peak X) isolated
from smooth PA01 strains of P. aeruginasa lacked phosphate but contained sulfate
(30,31). To establish that A-band LPS from AK1401 follows the same trend,
composition of the LPS isolates was analyzed for heptose (37) and phosphate (1).
KDO (6) levels were determined after hydrolyzing the LPS samples in 0.5 N
H2804 at 100°C for 30 min. Sulfur levels were quantitated by inductively coupled
plasma emission spectrometry carried out by the Soil and Plant Analysis Lab,

Madison, WI. Protein concentrations were estimated by the Pierce BCA protein

110

Table 1. Fatty acid composition of LPS fractions from Pseudamanas
aeruginasa AK1401.

Recovery from Samplea

 

 

Fatty Acid Uf-LPS SC-LPS A—band LPS Totalb
3-OH-C10.0 21.3 7.9 11.9 2.1
912:0 8.8 8.6 3.2 1.3
2-C)I—1.C12.0 36.1 37.0 47.6 4.6
3-OH-C1M 33.9 46.6 37.4 1.2

CI 6: 19 _c _. _ 6.5
C; 6 = o _ — — 43.9
C1 8 : 19 —- — — 36.1
C1 8 : 0 — — — 1.7
C 1 9 : 0A -— — — 2.0
\

 

aR
eslllts are the averages of two or more analyses and are expressed as a weight
I I‘Qentage of the total.

1’1‘

C Qtal membrane fatty acid composition. There was less than 1.0% of C141),
1 5 :0, i'C17:0a C17:0A’ C17:0, C190 and C20:0 i" this sample-

CN—
Qt detected.

111

assay (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as the
standard. Analysis for protein in the pooled fractions after gel filtration indicated
no detectable levels of protein, while the Uf-LPS isolate contained less than 14%
(wt/wt) protein. The chemical composition of Uf-LPS and SC-LPS isolates were
similar (I‘ able 2). In previous studies we showed that all the size fractions of LPS
from P. aeruginasa PAO strains contained heptose, a component of the inner core
of most LPS isolates (30,31). The level of this sugar per weight in the isolates
reflected the molecular weight of the molecules, suggesting that the heptose
content is similar in the different size fractions. In contrast, the sugar KDO,
another component common to the inner core of most LPS isolates, appeared to
be very low in the A-band LPS isolate (Table 2). Even though the KDO levels are
low in this fraction we cannot rule out the possibility that this sugar is substituted
and thus not reactive in the assay (5). Furthermore, A-band LPS isolated from
AKl 401 had no detectable phosphate levels (Table 2), substantiating our earlier
findings (31). Interestingly the three LPS isolates showed similar molar amounts
Of SUlfur. The molar amounts detected in Uf-LPS and SC-LPS samples were
a'QQYOximately one-half the heptose content. Assuming that there are two to three
heptose in the core region of LPS (35), then there is at least one sulfur, perhaps
as sulfate, present in the SC-LPS. On the other hand, A-band LPS showed
approximately a 1:1 molar ratio relative to heptose suggesting that two to three
sulfurs may be bound to these molecules. This is consistent with the gel filtration

profile. A possible explanation for why we are observing sulfur in the SC-LPS

112

Table 2. Chemical analysis of column fractions of LPS from Pseudamanas
aeruginasa AK1401.

 

Amount (nmol/mg)“ of:

 

 

Heptose KDO Phosphate Sulfur
LPS Sample
Uf-LPS 304 200 2,538 102
SC-LPS 306 250 1,934 155
A-band LPS 163 8 0 129

 

“Average result from two or more isolates.

113

pOpulation may be that beside phosphate being incorporated in the core-lipid A,
stoichiometric amounts of sulfur may also be attached. The probability of having
an A—band type of LPS that is devoid of polysaccharide polymer that comigrates
on the SDS-gel and coelutes in gel filtration with the serotype-specific SC-LPS also
cannot be ruled out. Nonphosphorylated lipid As (13) and LPS lacking both
phosphate and KDO (26) have been reported for a number of bacteria. As
previously suggested, the A-band LPS isolate may have sulfate replacing phosphate
on lipid A (31).

Finally, the common antigen LPS from smooth strains of P. aeruginasa has
been shown to possess a regular banding pattern of intermediate polysaccharide
length according to the SDS-PAGE (27,31). The AK1401 pooled A- and B-band
LPS together with Uf-LPS were separated in SDS-PAGE to define their size
heterogeneity and to show the presence of labelled sulfate. The lyophilized
fractions were suspended in distilled water to a known weight concentration and
“PPlied to SDS-gels. Silver staining of Uf-LPS revealed two sets of bands (Figure
2A, lane 1) representing two populations of molecules differing in polysaccharide
length- The slower migrating A-bands showed a ladder pattern with regular
Spaci “g. When the separated fractions were reapplied to SDS-gels, only one set
Of bands was observed for SC-LPS (B-band) (Figure 2A, lane 3). The A-bands
Showed the ladder pattern with the regular spacing of the common antigen LPS
(Figul‘e 2A, lane 2; 27) with a slight contamination of low-molecular-weight LPS.

T .
0 fIlr’ther demonstrate that sulfur is covalently bound to thrs LPS, an

Figure 2.

114

Silver-stained SDS-polyacrylamide gel (A) and autoradiograph (B)
of P. aeruginasa AK1401. (A) Samples of 5 pg of Uf-LPS (lane 1),
A-band LPS (lane 2), and SC-LPS (lane 3) were applied to a 15%
acrylamide gel which had been polymerized overnight with a butanol
overlay. Band sets A and B denote LPS major size populations.

(B) Autoradiograph of SDS-polyacrylamide gel of Uf-LPS isolate.
Approximately 5.9 x 104 counts (~5 mg) was applied to a 15 %
acrylamide gel (1.5 mm thick) and was exposed in 3 X-ray film for

6 weeks.

115

 

o5:
. 333:.

[I‘ll

A

 

 

. ._

I‘ll
B

._-_

Figure 2

116
autoradiogram of the 35S-labelled Uf-LPS separated by SDS-PAGE was analyzed.

Approximately 5.9x104 cpm of the sample (approx. 5 mg) was applied to a 15%
SDS—gel (1.5 mm thick), with a 7.5% acrylamide stacking gel. Electrophoresis
was performed as previously described. Following electrophoresis the gel was
soaked in intensify-enhancer solutions (Intensify-Universal Autoradiography
Enhancer, Dupont-Biotechnology Systems, Boston, MA) according to the
manufacturer’s instructions. A high speed X-ray film was exposed to the dried gel
at -76 °C for 6 weeks. The autoradiogram indicated that the Uf-LPS sample had
a high amount of labelled sulfur (Figure 2B). The regions developed in the
autoradiogram correspond to the components detected by silver staining in the
SDS- gel (Figure 2A, lane 1). Free 3SS-labelled sulfate was run along with the
LPS sample and no band corresponding to the free isotope was detected on the
alltoradiogram (results not shown). Presumably the labelled-sulfate diffused from
the gel into the enhancer solution. These data are consistent with 35S-labelled
“Ulfilr being covalently bound to LPS. Preliminary studies have also shown that
RhiZObium melilati incorporates sulfate into LPS (Hollingsworth, personal
com t'nunication). Other microorganisms have also been reported to incorporate
sulfate in their lipid-linked oligo- and polysaccharides (10-12,15,21); thus the
p reselice of sulfur in LPS may not be confined to P. aeruginasa.
We have presented several pieces of evidence which indicate that A-band
LPS isolated from P. aeruginasa AK1401 has sulfur covalently bound to LPS,

perhaps as sulfate. The gel filtration column separated the two major size

117
populations (A- and B-band) and the A-band LPS as well as the short chain (SC)

B-band LPS molecules appeared to have covalently bound sulfur. This is in
agreement with the autoradiogram of the unfractionated LPS sample. Chemical
analysis of the A-band LPS substantiated our earlier results which indicated low
levels of detectable KDO and no phosphate (31). Furthermore, this isolate showed
a 1:1 molar ratio of sulfur to heptose suggesting that two to three sulfur atoms are
present in the A-band LPS molecule probably as sulfate. Given the findings
reported here, we reconfirm that A-band LPS of P. aeruginasa contains sulfur.
The data presented in this study as well as previous reports (17,20,27,30-
32,39) indicate that P. aeruginasa synthesizes more than one type of LPS.
Furthermore, these results confirm that sulfur is incorporated into the LPS. Our
results indicate that the chemical structure of the common antigen LPS differs in
several respects from that of the O-serotype-specific LPS. It is reasonable to
Propose that the biosynthetic pathways of these two chemically distinct LPS
mOIeCules diverge at some point.

The biosynthesis of LPS has been studied in detail for Salmonella
O’Phihzurium and Escherichia coli. Several studies have indicated significant
differences in lipid A synthesis among the different gram-negative bacteria (9,34;
1' Lightfoot, T. Dasgupta, and J.S. Lam, Abstr. Annu. Meet. Am. Soc.
1Microbiol. 1991, D69, p. 90). For example, Goldman et al. (9) has shown that
the major lipid A precursor species from P. aeruginasa are completely acylated

prior to addition of KDO, while enteric lipid A precursors just prior to KDO

118

attachment contains only 3-OH-C14:0 and lacks the other nonhydroxy fatty acids
characteristic of mature LPS. It has been shown that the synthesis or expression
of P. aeruginasa A- and B-band LPS appear to be partially independent (J.
Lightfoot et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1991, D-69, p. 90).
Genes for A-band synthesis have been shown to map at a location different from
the B—band genes. P. aeruginasa PAD is not the only bacterium reported to
simultaneously express two LPS polysaccharide antigens. Klebsiella pneumoniae
01 is reported to synthesize two structurally distinct D-galactan polymers (34) and
the genes involved in the expression of the two galactans are not closely linked.

Studies are now under way to chemically analyze the core-lipid A region
of the A-band LPS. The characterization of the structure will give a better

understanding of the function of this molecule in the mechanism of virulence and

resrstance to antibiotics.

 

10.

11.
12.

13

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Laemmli, U.K. 1970. Nature (London) 227, 680—685.

Lam, M.Y.C, E.J. McGroarty, A.M. Kropinski, L.A. MacDonald, S.S.
Pedersen, N. Hoiby, and J.S. Lam. 1989. J. Clin. Microbial. 27,
962-967.

Lerouge, P., P. Roche, C. Faucher, F. Maillet, G. Truchet, J.C. Promé,
and J. Dénarié. 1990. Nature 344, 781—784.

Liu, P.V., H. Matsumoto, H. Kusama, and T. Bergan. 1983. Int. Syst.
Bacterial. 33, 256—264.

Liu, P.V., and S. Wang. 1990. J. Clin. Microbial. 28, 922-925.

Liideritz, O., M.A. Freudenberg, C. Galanos, V. Lehmann, E.T.
Rietschel, and DH. Shaw. 1982. Curr. Top. Membr. Transp. 17, 79-151.

Makela, RH, and B.A.D. Stocker. 1984. In Handbook of Endataxin,
Vol. I . Chemistry of Endataxin. E.T. Rietschel (ed.), p. 59—137. Elsevier
Science Publisher B.V., Amsterdam.

Mayer, H., and Weckesser. 1984. In Handbook of Endataxin, Vol. 1:
Chemistry of Endataxin. E.Th. Rietschel (ed.), p. 221—247. Elsevier
Science Publishers B.V., Amsterdam.

McGroarty, E.J., and M. Rivera. 1990. Infect. Immun. 58, 1030-1037.
Peterson, A.A, and E.J. McGroarty. 1985 . J. Bacterial. 162, 738-745.

Raetz, C.R.H. 1990. Annu. Rev. Biochem. 59, 129-170.

30.

31.

32.

33.

34.

35.

36.
37.

38.

39.

121

Rivera, M., L.E. Bryan, R.E.W. Hancock, and E.J. McGroarty. 1988.
J. Bacterial. 170, 512—521.

Rivera, M., and E.J. McGroarty. 1989. J. Bacterial. 171, 2244-2248.

Sawada, S., T. Kawamura, Y. Masuho, and K. Tomibe. 1985. J. Infect.
Dis. 152, 1290—1299.

Westphal, O., O. Liideritz, and F. Bister. 1952. Z. Naturfrasch. Teil B
7, 148—155.

Whitfield, C., J.C. Richards, M.B. Perry, B.R. Clarke, and LL.
MacLean. 1991. J. Bacterial. 173, 1420-1431.

Wilkinson, S.G. 1983. Rev. Infect. Dis. 5, $941-$949.
Wilkinson, S.G., and L. Galbraith. 1975. Eur. J. Biochem. 52, 331-343.
Wright, B.G., and RA. Rebers. 1972. Anal. Biochem. 49, 307—319.

Yokota, S., S. Kaya, Y. Araki, E. Ito, T. Kawamura, and S. Sawada.
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1987. Eur. J. Biochem. 167, 203-209.

CHAPTER 5

Common Antigen Lipopolysaccharide from Pseudamanas aeruginasa

AK1401 as a Receptor for Bacteriophage A7 .

122

ABSTRACT

Lipopolysaccharide (LPS) from Pseudamanas aeruginasa AK1401 was
fractionated by gel filtration chromatography into two major size populations: the
A-band or common antigen LPS and B-band or short chain (SC)-LPS. Purified
A—band LPS was mixed with bacteriophage A7 and analyzed on sodium dodecyl
sulfate-polyacrylamide gels. The results indicated that phage A7 recognizes the
A—band polyrhamnose in the A-band isolate and within 2 h hydrolyzed the
molecule to core-lipid A containing only two to three rhamnose repeat units. The

Phage A7 also hydrolyzed the A-band component in unfractionated (U f) LPS. This
Phage A7 showed a high specificity to A-band LPS but did not alter the O-
serotype-specific LPS. Chemical composition of the purified phage A7-digested
A—band LPS showed low levels of heptose, 2-keto-3-deoxyoctulosonic acid, and
amino sugars, and no phosphate. Reaction of lipid A from Uf-, SC-, and phage-
digested A-band LPS with monoclonal antibodies (MAbs) specific for lipid A
indicated that all the samples had common epitopes. Lack of reactivity of the acid
hydrolyzed A-band isolate with anti-lipid A MAbs suggest that this sample is either
“onereactive or is resistant to acid hydrolysis. In addition, all AK1401 LPS

Isolates were reactive with M Ab specific to the inner core region of P. aeruginasa,

123

124

indicating that a common epitope in the inner core is shared by these LPS isolates.

In contrast, an outer core-specific MAb raised against P. aeruginasa, bound only
the Uf- and SC-LPS and not the A-band isolates indicating that the O-serotype LPS
outer core structure is different from that of the common antigen LPS. We

propose that the outer core, inner core and lipid A regions of A-band LPS are

different from those of the serotype-specific LPS.

INTRODUCTION

Lipopolysaccharide (LPS) is a major component of the outer leaflet of the
Gram negative outer membrane, comprising 20 to 40% of this structure by weight
(2.5). These molecules are heteropolysaccharides covalently linked to lipid A
(16,29). The heteropolysaccharide consists of three regions: a diglucosamine
backbone, the oligosaccharide core, and the O-antigenic polysaccharide chain.
Specific structures within the LPS molecule serve as receptors for a variety of
bacteriophage (23,38). Phage absorption to its receptor is highly specific (23).
Phage resistance that results from changes in LPS structure usually indicate that
the LPS is the surface receptor (38), and the structural change identifies the region
0f the LPS comprising the receptor.

Since the structure of the O—serotype specific antigen varies from strain to
Strain, the host range of a phage whose receptor is the O-polysaccharide is rather
narrow (16,23). One characteristic of O-specific phage is that during infection

they often hydrolyze the O-antigen destroying the initial receptor (38). This
enZYmatic activity generally is localized in the tail-like phage attachment complex
(23 ,3 8). Thus, adsorption of these phage involves the formation of an enzyme-

subStrate complex, the enzyme being an integral part of the phage tail and the

125

126

substrate being the O-antigen receptor.

In many strains of Pseudamanas aeruginasa a second LPS species is present
whose polysaccharide chain differs serologically and structurally from O-antigen
chain (17,21,24,30—32,4l,42). This LPS has been termed A-band or common
antigen LPS. The common antigen polysaccharide of P. aeruginasa is a regular
homopolymer of rhamnose. On the basis of NMR and chemical analyses the
structure has been shown to consist of the repeating unit [- > 3)-a-D-Rhap-(l- > 3)-
oz-D-Rhap-(l->2)-a:-D-Rhap-(1->]n (17,41,42; T.L. Arsenault, D.W. Hughes,
D-B. MacLean, W.A. Szarek, A.M.B. Kropinski, and J.S. Lam, Can. J. Chem.,
in press). Interestingly, the structure proposed for the repeating unit of the
rh amnan chain in the common antigen LPS of P. aeruginasa is identical with that

reported for the O-polysaccharide chain in the LPS of P. syringae pv.
morsprunarum C28 (34).
The O-polysaccharide of P. syringae pv. morsprunarum C28 LPS, which
is composed entirely of rhamnose (34), is specifically cleaved and released as
<>1i.g<)saccharides by the action of a rhamnanase borne on the typing phage A7 (33).
This phage uses the LPS as its initial binding site (28,33). Thus, it is expected
that the common antigen (A-band) LPS from P. aeruginasa will also serve as a
substrate for phage A7. In this paper we present evidence to show that phage A7
binds to and hydrolyzes the polysaccharide chain of the A-band LPS from P.

aeruginasa AK1401.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

P. aeruginasa AK1401 is an LPS defective rough mutant of the
restrictionless mutant isolate OT684 (leu-l lys-l res-4; 2). This strain produces
A-band LPS but is defective in the synthesis of O-antigen. P. syringae pv.
morsprunarum strain No. 2168 was obtained from the National Collection of Plant
Pathogenic Bacteria (Plant Pathology Laboratory, Hatching Green, Harpenden,
Hertfordshire, England).

P. aeruginasa AK1401 was cultured at 37°C in tryptone (1%, w/v)-yeast
CXtract (0.2% , w/v) broth (TYE). The cultures were harvested late in logarithmic
growth phase. The phytopathogenic strain of P. syringae pv. morsprunarum was
Cultured in nutrient broth (NB) (1% tryptone, 0.2% yeast extract, 0.4% NaCl) at
25 °C. An aliquot of an overnight culture was transferred to fresh NB media and
tl'le Cells were grown to mid-logarithmic phase. This culture was used as a host

for phage A7 propagation (see below).

127

128
Pr0pagation of phage A7 .

P. syringae pv. morsprunarum phage No. 2377 was obtained from the
National Collection of Plant Pathogenic Bacteria (Plant Pathology Laboratory,
Hatching Green, Harpenden, Hertfordshire, England). Phage were propagated on
host cells of P. syringae pv. morsprunarum in nutrient broth-agar (NBA) (NB/1%
agar) plates. Plates with 2 ml of the host (approx. 109 cells ml‘l) were overlayed
with 0.2 ml of the A7 phage so as to give confluent lysis over the whole plate.
After 18 to 24 h, 5 m1 of sterile NB was poured onto the surface of the plate and
left for 4 h to allow the phage to diffuse into the broth. The resulting phage
SUSpension was centrifuged at 13,300 x g for 10 min and the supernatant solution
Was stored at -80°C. Yields were determined by a phage plaque counting

technique (11). Titres of the order of 1010-1011 p.f.u. ml'1 were obtained by this

method. No significant loss of titre was observed during storage.

LPS isolation.
P. aeruginasa AK1401 or P. syringae pv. morsprunarum cells from 2 to
5 liters of medium were washed with distilled water and extracted with hot
a(ll-"i=ous phenol (36). The aqueous phase was extensively dialyzed and lyophilized
as described previously (24). In addition, the O-serotype-specific LPS from P.
aeruginasa PA01715 (peak 2 isolate) was isolated and purified by gel filtration on

a Sephadex G-200 column as described elsewhere (30).

129
Column chromatography.

LPS isolates were fractionated on a Sephadex G-200 (Pharmacia Fine
Chemicals, Piscataway, NJ.) column (69 cm by 25 mm) at room temperature by
using the column buffer system of Peterson and McGroarty (pH 8.5) (27), unless
otherwise noted. Between 10 and 30 mg of LPS were applied to the column, and
4-ml fractions were collected at a flow rate of 6.2 ml/h. To remove detergent and
buffer, pooled fractions were extensively dialyzed (12,000- to 14,000-molecular-
weight cutoff membranes), at room temperature, against a buffer composed of 50
mM Tris, 10 mM EDTA, 50 mM triethylamine, and 0.02% sodium azide, pH
8 -0. The pooled fractions were dialyzed further against 100 mM KCl and then
against distilled water. The dialyzed fractions were lyophilized and stored at 4°C

for further analysis.

Monoclonal Antibodies.
In order to produce core-specific or lipid A-specific monoclonal antibodies
(M Abs), immunogen was prepared according to the method of Bogard et al (3).
Briefly, cells of a rough P. aeruginasa strain AK1401 were suspended at a
corlcentration of 5 x 109 cell per ml in 1% acetic acid, heated for 1 h at 100°C,
Was bed in distilled water and were ly0philized. This treatment should kill the cells
and strip off cell surface polysaccharides. Core fractions of LPS from strain
AI(1401 were prepared by standard methods including hot aqueous phenol

extraction and gel filtration fractionation using Sephadex G-50 (Pharmacia,

130
Uppsala, Sweden). Five mg of this core-LPS was then suspended in 5 ml of 0.5 %

(w/v) triethylamide followed by addition of 5 mg of the acid-treated bacteria. The
mixture was stirred slowly for 30 min at room temperature and dried in vacuo
with a Speed Vac centrifuge (Savant Instrument Inc., Hicksville, N.Y.). These
core—LPS-coated cells were then used to immunize Balb/c mice intraperitoneally
at a dose of 50 pl of core-LPS-coated cell suspension per injection in a 1:1 mixture
with Freund’s Incomplete adjuvant. The animals were immunized initially on days
0, 4, 9, 14, and 28. The injections were kept up once every two weeks until day
5 6 - A test bleed was done to test for positive response against core bands of LPS
with Western immunoblotting techniques. Upon detection of positive reaction to
Core region bands, the animals were immunized once more and sacrificed three
days later to extract splenocytes for fusion with myeloma cell line N81. The
filsion protocol and isolation of hybridoma clones were precisely as described
Previously by Lam et al (20). Screening of the hybridoma cell lines was facilitated
by ELISA and Western immunoblots and the use of LPS purified from the
following rough LPS strains, including AK1401 (core-plus-one 0 side chain),
AK1012 (core deficient mutant; 14), and AK44 (O-antigen deficient but with
complete core; 18). A more detailed characterization of the MAbs 101 (outer-
coreesmcific), 7-4 (inner-core-specific), and 177 (lipid A-specific) is described
elsewhere (T .R. Chivers, L.A. MacDonald, and J .S. Lam, Ann. Meeting of the

Can- Soc. Microbiol. 1991, Abst. No. MS4p; Manuscript in preparation). The

131
other MAbs 4A10 and 8A1 are specific to lipid A of enterobacteria (3) and were

a kind gift of RT. Coughlin.

Gel electrophoresis, dot blots, and Western blots (immunoblots).

LPS samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) by using the buffer system of Laemmli (19). The
gels were silver-stained by the method of Dubray and Bezard (10). Western
immunoblots of SDS—polyacrylamide gels were prepared as described previously
(5,30,31,35). The gels were electrotransferred with a model TE Transphor
Electrophoresis apparatus (Hoefer Scientific Instruments, San Francisco, CA) at
a constant current of 290 mA for 28 h. For reaction with anti-lipid A MAbs, the
nitrocellulose blots were hydrolyzed in 10% acetic acid for 2.5 h at 100°C
immediately after electrotransfer. After hydrolysis the nitrocellulose blots were
washed 5 times (5 min) with T ris-saline (0.9% NaCl in 10 mM Tris-HCl, pH 7.4).
Lipid A was visualized on the blots, as described by Otten et al. (26), with either
MAb 4A10 and 8A1 , specific for lipid A of enterobacteria (3) or anti-lipid A MAb
177 raised against P. aeruginasa. In addition, Western immunoblots of the LPS
samples were incubated with outer core-specific MAb, 101, or inner core-specific
MAb, 7-4, both of which were raised against P. aeruginasa. Likewise, dot blots
were analyzed by applying known quantities of LPS isolates directly on

nitrocellulose with or without acid hydrolyzing the samples as above. The dot

132
blots were reacted with anti-lipid A MAbs 4A10 or 8A1 (3), outer core-specific

MAb 101, or inner core-specific MAb 7-4.

Hydrolysis of LPS by phage A7 .

Phage A7 (5 .0 x 1011 p.f.u.) was pelleted from a stock suspension in NB
by centrifugation at 148k x g for 1.5 h at 4°C and then mixed with 2 mg LPS in
2 m1 of distilled water. The mixture was incubated with mild agitation at 20°C
for 2, 3, 4, 5, and 24 h. An aliquot of 50 pl of the digested LPS was mixed with
an equal volume of electrophoresis sample buffer (containing 4% SDS) and applied
to an SDS-polyacrylamide gel. Electrophoresis was performed as previously
described, and the gels were silver-stained.

In addition, a sample of approximately 10 mg of A-band LPS purified from
isolated LPS of P. aeruginasa AK1401 by gel filtration, was resuspended in 2 ml
of distilled water. Phage A7 (5.0 x 1011 p.f.u.) was added, and the mixture was
incubated for 24 h as outline above. The digested LPS was treated for 24 h at
4°C with RNAse and DNAse ( both at 22 pg ml’l) followed by treatment with
proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, IN) (20 pg ml’l)
for 5 h at room temperature. The digested LPS sample was separated on
Sephadex G-200 as described previously. The pooled phage A7-digested A-band

fractions were extensively dialyzed, lyophilized, and suspended in distilled water

133

to a known concentration for further chemical analysis. A parallel experiment was

performed using unfractionated LPS from P. syringae pv. morsprunarum.

Chemical assays.

Assays for total carbohydrate (9), amino sugar (13), heptose (39), and
phosphate (1) were performed as described previously. The 2-keto-3-
deoxyoctulosonic acid (KDO) (8) levels were determined after hydrolyzing the LPS
sample in 2.0 N H2804 at 100°C for 1 h in aqueous 1% SDS solution (6).
Protein concentrations were estimated by the Pierce BCA protein assay (Pierce

Chemical Co., Rockford, IL) with bovine serum albumin as the standard.

RESULTS

The LPS from P. aeruginasa AK1401 was separated by gel filtration
chromatography, and column fractions were monitored for total carbohydrate,
KDO, and phosphate. The elution profile showed three carbohydrate peaks
(Figure 1, peaks 1-3) while KDO and phosphate was detected only in the major
peak. The SDS-polyacrylamide gel of the column fractions, when applied in order
of elution, revealed a diagonal ladder pattern representing molecules of different
sizes (Figure 1, A and B bands). The A-band LPS corresponds to the slower
migrating set of bands (peak 2), while the second population of molecules,
designated the B-band or SC-LPS (peak 3), corresponds to the faster migrating set
of bands. As indicated in earlier studies (30,31), the SC-LPS fraction contains the
majority of the molecules, comprising core and lipid A with none or only one 0-
repeat unit similar to that of the main serotype-determining antigen. The first peak
in the elution profile (Figure 1, peak 1) did not appear to be LPS and was not
analyzed further. Analysis of the pooled fractions for protein after gel filtration
indicated a contamination of less than 1% (w/w).

To investigate the role of LPS from P. aeruginasa AK1401 as a receptor

for phage A7, phage—binding and infectivity studies were carried out. A mid-log

134

Figure l.

135

Fractionation profile of LPS from P. aeruginasa AK1401 separated
on Sephadex G-200. Fractions were analyzed for total carbohydrate
(O), KDO (.), and phosphate (‘). Silver-stained SDS-
polyacrylamide gels of column fractions are aligned under their
appropriate fraction number. A represents the slow—moving set of

bands, and B represents the faster-moving set.

136

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137
phase culture of AK1401 was tested as a host for the phage. A 0.1-ml aliquot of

saline containing the test phage at 103 or 104 p.f.u. ml'1 was incubated with 0.1
ml of saline for 10 min at room temperature. At that time, 0.1 ml of bacterial
culture was added with 3 m1 of melted soft agar at 44°C and mixed; the mixture
was then poured over the surface of an NBA plate and incubated at room
temperature for 18 to 24 h. A parallel experiment was performed using P.
syringae pv. morsprunarum as the host. The results showed no phage plaques
with P. aeruginasa AK1401 whereas, with P. syringae pv. morsprunarum as host,
phage plaques were observed (results not shown). We also tested the ability of
purified A-band LPS from P. aeruginasa to inactivate phage A7 and prevent
infection of P. syringae pv. morsprunarum. A 0.1-ml aliquot of saline containing
the phage at 103 p.f.u. mrl was incubated with 0.1 ml of purified A-band LPS
(100 pg) for 10 min at room temperature and was then diluted and mixed with the
host cells as described above. Phage infectivity in the absence of added LPS was
used as a control. The results indicated that A-band LPS produced a slight
decrease in phage A7 plaque formation (less than 35 % inhibition; data not shown).
In addition, the SC-LPS isolate from P. aeruginasa was tested for its ability to
inactivate phage A7 of P. syringae pv. morsprunarum, and the results indicated
that there was no inhibition of phage infection with this sample (data not shown).

To show that phage A7 hydrolyses the polysaccharide chain of the A-band
LPS from P. aeruginasa AK1401, phage A7 (5.0 x 1011 p.f.u.) was mixed with

purified A-band LPS (~2 mg) at room temperature for 2, 3, 4, 5, and 24 h. The

138
SDS-polyacrylamide gels of the digested samples showed that the polysaccharide

chain of the A-band LPS was completely hydrolyzed by phage A7 within 2 h
(Figure 2A, lanes 2-7). Note that the higher molecular weight bands detected in
Figure 2A (lanes 2-7) results from phage A7 protein bands (*); these samples were
applied to the gel at high concentrations to detect minor LPS components. When
lower amounts of LPS were applied to the gel, only the fastest-migrating bands
were detected; the results show that, following hydrolysis with phage A7, the LPS
consists predominantly of molecules composed of core-lipid A with presumably
one to three rhamnose repeat units (Figure 2B, lanes 2-5). To demonstrate that
phage A7 was specific to common antigen LPS, phage hydrolysis was also done
using isolated O-serotype-specific LPS from the smooth strain of P. aeruginasa,
PA01715 (peak 2 isolate) (30). The results show that phage A7 does not degrade
the P. aeruginasa O-serotype-molecule (Figure 2A, lanes 8 and 9). To define the
minimum concentration of phage A7 required to completely hydrolyze 2 mg of A-
band LPS from P. aeruginasa AK1401, samples of 1 m1 of phage A7 containing
3.3 x 109 p.f.u. mr1 (1:10), 1.4 x 109 p.f.u mrl (1:25), or 7.1 x108 p.f.u. mrl
(1:50) were used and hydrolysis carried out as described above. Results indicated
that a concentration of at least 109 p.f.u. ml'1 is required for complete degradation
of 2 mg of the polyrhamnose after 24 h (results not shown).

The hydrolysis of A-band LPS by phage A7 was further characterized using
unfractionated LPS (Uf-LPS) from strain AK1401. A 0.5 mg sample of Uf-LPS

was mixed with 1 ml of either 3.3 x 109 or 1.4 x 109 p.f.u. ml'1 at room

Figure 2.

139

Silver-stained SDS—polyacrylamide gels of phage A7-digested A-band
LPS (lanes 2-7) from P. aeruginasa. (A) Purified AK1401 A-band
LPS was mixed with phage A7 for 2h (lanes 2 and 3), 3 h (lane 4),
4 h (lane 5), 5 h (lane 6), and 24 h (lane 7). Also, a sample of
PA01715 serotype-specific LPS was mixed with phage A7 for 2 h
(lane 9). Samples of AK1401 A-band and Uf-LPS (lanes 1 and 10,
respectively) as well as PA01715 serotype specific LPS (lane 8)
were applied to the gel. Samples of either 10 pg (lane 2), 4 pg
(lanes 1, 3-9), or 0.5 pg (lane 10), were applied to the 15%
acrylamide gel which had been polymerized overnight with a butanol
overlay. A represents the slow-moving set of bands, and B
represents the faster-moving set, and * indicates the phage A7
protein bands. (B) SDS-gel of AK1401 LPS after incubation with
phage A7 for 2 h (lane 2), 3 h (lane 3), 4 h (lane 4), and 5 h (lane
5). Samples of either purified AK1401 A-band LPS (lane 1) or
phage-digested A-band LPS (lanes 2-5) were applied to the gel at

low concentrations (0.33 pg and 0.07 pg, respectively).

11 A-band
l A-band
(lane 4),
ample of
7 for 2 h
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(lane 3)
2), 4 F!
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and B
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140

Figure 2

 

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141

 

Figure 2

142

temperature for 24 h. Figure 3A shows the silver-stained gel of these samples.
The results indicate that phage A7 hydrolyzes the A-band component in the Uf-
LPS to the same extent as it does the purified A-band LPS (Figure 3A, lanes 2,
3, and 4, respectively). As indicated above, phage A7 does not appear to modify
the SC-LPS (Figure 3A, lane 5). A parallel experiment was performed using P.
syringae pv. morsprunarum LPS. A 1 mg sample of LPS was mixed with 1 ml
of phage A7 (2.4 x 1010 p.f.u. ml'l) at room temperature for 24 h. The SDS-
polyacrylamide gel of the digested sample showed that the P. syringae pv.
morsprunarum LPS was not completely digested after 24 h (Figure 3B, lane 2)
suggesting that the phage was inactivated by this LPS isolate. Notice that the
banding pattern of P. syringae pv. morsprunarum phage-digested LPS is well
defined and can be used to count up to a maximum of approximately 30 rhamnose
repeat units in the AK1401 A-band LPS as well as the Uf-LPS (Figure 3B, lanes
3 and 4, respectively).

It has been reported that the core-lipid A structure of A—band LPS from P.
aeruginasa contains low levels of reactive KDO, lacks phosphate, but contains
sulfur (31; M. Rivera and E.J. McGroarty, J. Bacteriol., manuscript submitted).
Therefore, the lipid A component of this LPS may be antigenically different from
that of the phosphorylated lipid A of the SC~LPS fraction. Samples of Uf-LPS,
SC-LPS, A-band LPS, and phage A7-digested A-band LPS from P. aeruginasa
AK1401 was separated by SDS-PAGE and then electrotransferred to nitrocellulose.

A sample of an O-serotype—specific LPS from P. aeruginasa PA01715 was used

Figure 3.

143

Silver-stained SDS-polyacrylamide gel of LPS fractions from P.
aeruginasa AK1401 and P. syringae pv. morsprunarum after
incubation with phage A7. (A) A 2 mg sample of AK1401 Uf-LPS
from P. aeruginasa was mixed with either 3.3 x 109 (lane 2) or 1.4
x 109 p.f.u. (lane 3) for 24 h. Also, 2 mg of purified A-band LPS
(lane 4) and SC-LPS (lane 5) was incubated with phage A7 (1.4 x
109 p.f.u.) for 24 h. Samples of 5 pg of AK1401 Uf-LPS (lane 1)
and phage-treated LPS (lanes 2-5) were applied to the gel. A
represents the slow-moving set, and B represetns the faster-moving
set. (B) A 1 mg sample of LPS from P. syringae pv. morsprunarum
was mixed with 2.4 x 1010 p.f.u. for 24 h (lane 2). Samples of5 pg
of P. syringae pv. morsprunarum LPS (lane 1) and 2.5 pg of phage
A7-digested LPS (lane 2) were applied to the gel. Samples of 5 pg
from P. aeruginasa AK1401 Uf-LPS (lane 3), A-band LPS (lane 4),
and phage A7—digested A-band LPS (lane 5) were also applied to the

gel.

144

 

Figure 3

145

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Figure 3

146

as a control. After acid hydrolysis of the samples on the blot, the nitrocellulose
was incubated with MAb 8A1, specific for lipid A of Escherichia coli (3). A dot
blot of the same samples was also developed under the same conditions. Figure
4 shows the Western blot (Figure 4A) and the dot blot (Figure 4B) of these LPS
fractions. The Western blot indicated that MAb 8A1 reacted strongly with
AK1401 Uf- and SC-lipid A derivatives, whereas phage digested AK1401 A-band
lipid A derivative showed only moderate reactivity (Figure 4A, lanes 4, 3, and 1,
respectively). Interestingly, AK1401 A-band isolate did not show reactivity
(Figure 4A, lane 2), suggesting that this isolate may be resistant to the acid
hydrolysis. The lipid A derived from the O-polymer-containing isolate showed
weak reactivity (Figure 4A, lane 5). The results from the dot blot confirmed these
findings (Figure 4B). Note that the AK1401 A-band isolate gave a weak reaction
when 4 pg were applied; this reactivity may have resulted from the sample
containing a low molecular weight contaminant, as seen in the silver-stained gel,
which is immunoreactive with MAb 8A1 (Figure 2A, lane 1). In addition, this
antibody reactivity might be due to non-specific binding of the A-band isolate. We
also tested the binding of the MAb 4A10, specific to lipid A of Salmonella
minnesota, to the same samples using the procedures described above. The results
from the Western blot using MAb 4A10 showed that only AK1401 Uf- and SC-
lipid A derivatives, and lipid A derived from O polymer gave a positive reaction
(results not shown). We also tested these same samples using anti-lipid A MAb

177 specific to P. aeruginasa by using these same procedures. The results from

Al'

Figure 4.

147

Western blot (A) and dot blot (B) of lipid A’s derived from LPS
fractions of P. aeruginasa reacted with MAb 8A1. (A) Samples of
AK1401 phage A7-digested A-band LPS (lane 1, 40 pg), AK1401 A-
band LPS (lane 2, 80 pg), AK1401 SC—LPS (lane 3, 20 pg),
AK1401 Uf-LPS (lane 4, 25 pg), and PA01715 O-serotype—specific
LPS (lane 5, 40 fig) were separated by SDS-PAGE,
electrotransferred to nitrocellulose, and hydrolyzed as described in
Materials and Methods. The lipid A derivatives were incubated with
anti-lipid A MAb 8A1. (B) Samples of PA01715 O-serotype-
specific LPS (lane 1, 4.0 pg), AK1401 Uf—LPS (lane 2, 2.5 pg),
AK1401 SC-LPS (lane 3, 2.0 pg), AK1401 A-band LPS (lane 4, 4.0
pg), and phage A7-digested A-band LPS (lane 5, 2.5 pg) were
applied onto a nitrocellulose strip, and was hydrolyzed as described
in Materials and Methods. The lipid A derivatives were incubated

with MAb A1; * indicates very weak reactivity.

148

 

Figure 4

149
the Western blot showed that AK1401 Uf-, SC-, and phage-digested A-band lipid

A derivatives had moderate affinity toward the MAb (Figure 5, lanes 2, 3, and 5,
respectively). PA01715 long chain LPS-derived lipid A showed very weak
reactivity (Figure 5, lane 1). Again, the AK1401 A-band isolate did not show
reactivity (Figure 5, lane 4) similar to the results with MAb 8A1. In addition, we
performed a Western blot and dot blot immunoassay with Uf-LPS, SC-LPS, A-
band LPS and phage A7-digested A-band LPS from P. aeruginasa as well as the
O-serotype-specific LPS from P. aeruginasa PA01715 using core—specific MAbs.
The blots were incubated with either outer core-specific MAb 101 or inner core-
specific MAb 7-4 raised against P. aeruginasa. When the LPS samples were
incubated with the inner core-specific MAb 7-4, all the LPS samples reacted
suggesting that a common epitope in the inner core is shared by these LPS isolates
(Figure 6). Interestingly, when these same samples were incubated with the outer
core-specific MAb 101, only the AK1401 Uf- and SC—LPS showed reactivity
which indicates that the O-serotype-like LPS has an outer core structure different
from that of the common antigen or A—band LPS (Figure 7).

To purify phage-digested A-band LPS for chemical analysis, the sample was
treated with DNase and RNase, and then with proteinase K and separated on
Sephadex G-200 column at room temperature eluting with column buffer at pH
8.0. Fractions were characterized by SDS-PAGE. The silver-stained gel revealed
only one p0pulation of molecules (Dig-A-band), containing core-lipid A alone or

core—lipid A with one to three rhamnose repeat units (results not shown). The

l..."

Figure 5.

150

Western blot of lipid A’s derived from LPS fractions of P.
aeruginosa reacted with MAb 177. Samples of PA01715 O-
serotype-specific LPS (lane 1, 7.5 pg), AK1401 Uf-LPS (lane 2, 10
pg), AK1401 SC-LPS (lane 3, 5 pg), AK 1401 A-band LPS (lane 4,
15 pg), and AK1401 phage A7-digested A-band LPS (lane 5, 10 pg)
were separated by SDS—PAGE, electrotransferred to nitrocellulose,
and hydrolyzed as described in Materials and Methods. The lipid A
derivatives were incubated with anti-lipid A MAb 177 specific to P.

aeruginasa. Arrow indicates very weak reactivity.

....._ rav‘?

.-. firm-'fl “w. .

151

 

Figure 5

 

Figure 6.

152

Western blot (A) and dot blot (B) of LPS fractions of P. aeruginasa
reacted with inner core—specific MAb 7-4. (A) Samplesof PAO
1715 O-serotype-specific LPS (lane 1, 7.5 pg), AK1401 Uf-LPS
(lane 2, 10 pg), AK1401 SC-LPS (lane 3, 5 pg), AK1401 A-band
LPS (lane 4, 15 pg), and AK1401 phage A7-digested A-band LPS
(lane 5, 10 pg) were separated by SDS-PAGE, electrotransferred to
nitrocellulose and incubated with the MAb 7—4. Arrow indicates
very weak reactivity. (B) Samples of PA01715 LPS (lane 1, 4 pg),
AK1401 Uf-LPS (lane 2, 2.5 pg), AK1401 SC-LPS (lane 3, 2 pg),
AK1401 A-band LPS (lane 4, 4 pg), and phage A7-digested A—band
LPS (lane 5, 4.5 pg) were applied onto a nitrocellulose strip and

reacted with MAb 7-4.

153

'12345

 

 

 

 

 

 

Figure 6

 

154

Figure 7. Western blot (A) and dot blot (B) of LPS fractions of P. aeruginasa
reacted with outer core-specific MAb 101. The LPS isolates
analyZed and the amounts applied are identical to those described in

Figure 6.

12345

 

a...
E;

155

Figure 7

(”Poona

.1,

 

 

 

I!

156

fractions corresponding to the major peak were pooled, dialyzed, lyophilized, and
suspended in water to a known weight concentration for further analysis.
Chemical composition of the AK1401 LPS isolates were analyzed and the
results are shown in Table 1. In previous studies (30,31) we showed that all size
fractions of LPS from P. aeruginasa PAO strains contained heptose, a component
of the inner core of most LPS isolates. The heptose content of the Uf- and SC-
LPS were similar whereas that of A-band LPS was 10 times lower. The Dig-A-
band LPS showed a dramatic decrease in the heptose level compared to that of A-
band LPS isolate suggesting that rhamnose was interfering with the assay.
Reaction with pure rhamnose was shown to be significant in the heptose assay
(data not shown) raising the question as to whether heptose is present in the A-
band isolate. Yokota et al. (40) have reported that the LPS of Thiobacillus
versutus contains neutral sugars, glucosamine, KDO, and phosphorus, but is
devoid of heptose. The quantity of KDO appeared to be very low in the A-band
and Dig-A-band isolates confirming previous results (Table 1) (30,31). In
Acinetobacter calcoaceticus, an octulosonic acid isomer resembling KDO is
attached to lipid A and is also resistant to acid hydrolysis and does not give a
positive thiobarbituric test (15). We cannot discard the possibility that an
octulosonic acid isomer might be replacing the KDO residue in P. aeruginasa A-
band LPS. Interestingly, the level of amino sugar also appeared to be very low
in the A-band isolates. The molar amounts of amino sugars in Uf- and SC-LPS

were approximately twice the KDO content. Furthermore, as shown previously

157

Table 1. Chemical composition of LPS fractions from Pseudomonas

aeruginasa AK1401”.

 

 

LPS sample Heptoseb KDO Phosphate Amino sugar
Uf—LPS 440 280 1940 550
SC-LPS 300 280 1490 480

A-band LPS 44 20 NDc 23

Dig-A-band LPS 18 30 ND 26

 

aLevels given in nM/mg dry weight and are reported as the average of analyses

from two or more isolates.

bLevels of heptose were corrected for D-glycero-D—manno-heptose (39).

cND, not detected.

A"

158
no detectable levels of phosphate was observed in the A-band and Dig-A-band LPS

(30,31; Rivera and McGroarty, unpublished results).

DISCUSSION

The LPS of P. aeruginasa AK1401 was separated by gel filtration
chromatography into two populations, the common antigen or A-band LPS and an
O-serotype-like LPS which is composed of core-lipid A with none or one O-repeat
unit (SC or B-band LPS). The column elution profile confirmed earlier studies
which indicated that this strain is defective in the synthesis of O-antigen (2). The
advantage of using this bacterial strain to isolate pure A-band LPS for chemical
analysis is that the sample is not contaminated with small amounts of serotype-
specific LPS as seen in previous studies (30,31).

It has been reported that the polysaccharide chain of the common antigen
LPS from P. aeruginasa is a D-rhamnan trisaccharide unit, whose structure is
similar to that of the O-antigenic polysaccharide chain of P. syringae pv.
morsprunarum (17,33,34,41—43). The bacteriophage A7 is a typing phage for P.
syringae pv. morsprunarum, and its primary receptor is LPS (28). Furthermore,
phage A7 possesses a rhamnanase that specifically hydrolyses the O—antigenic
polysaccharide chain of P. syringae pv. morsprunarum releasing it as
oligosaccharide (33). Thus, we tested phage A7 for its ability to bind to cells of

P. aeruginasa AK1401 and to digest its LPS. The results indicated that phage A7

159

160

cannot infect strain AK1401 and, when high concentrations of purified A-band
LPS was added to the phage prior to mixing with host P. syringae pv.
morsprunarum cells, only a moderate inhibition of phage infection was observed.
Since phage A7 does not infect AK1401 cells, the moderate phage-inactivation
using A-band LPS suggest that the phage may recognize the A—band LPS, but does
not have the receptor needed for ejection of the nucleic acid (23,38). Smith et al
(33) reported that the final binding site for phage A7 is within the residual core-
lipid A region of P. syringae pv. morsprunarum.

Analyses of phage-digested A-band LPS from P. aeruginasa AK1401
(Figure 2) indicated that phage A7 recognizes the A-band polysaccharide chain as
an initial receptor, hydrolysing the molecule to core-lipid A containing only two
or three rhamnose repeat units. Furthermore, phage A7 reacted with Uf-LPS
from AK1401 (Figure 3), indicating that the LPS of AK1401 acts as a primary
surface receptor but does not have the final receptor for infection. Our results
indicate that phage A7 does not completely hydrolyze all of the rhamnose
polysaccharide side chain to core plus lipid A, as was suggested by Smith et al.
(33). In fact, their results showed that the LPS from P. syringae pv.
morsprunarum after phage treatment contained 2 to 4 times as much rhamnose as
did the core oligosaccharide (peak 11 after acid hydrolysis). Our results also
indicated that phage digestion of P. syringae pv. morsprunarum LPS was not
complete (Figure 3B, lane 2). In the initial studies of this phage-catalyzed

hydrolysis of the host LPS, the side chain digestion ended within the side chain

161

polysaccharide (33). Perhaps this region contains a secondary phage receptor site
which induces ejection of the nucleic acid, inactivating the phage. From the
digestion of the isolated host LPS we could determine that P. aeruginasa AK1401
A—band LPS has approximately 25 to 30 rhamnose repeat units (Figure 3B).
McGroarty and Rivera (24) reported that when P. aeruginasa PAO strains were
grown in high concentration of either MgClZ or NaCl, an increase in the amount
or length of the common antigen was induced. Furthermore, Lightfoot and Lam
(22) observed that the LPS from a derivative of P. aeruginasa AK1401 containing
the pFV3 plasmid, produced A-band molecules of higher molecular weight (30 or
more repeats) than that of the parent strain (max. of 20 repeats). They proposed
that the pFV3 plasmid contains an A-band polymerase gene which when present
in high copy number changes the A-band distribution and chain length.

Using the rhamnanase on phage A7 we can obtain an A-band LPS isolate
devoided of most of the rhamnose polysaccharide to analyze the core
oligosaccharide structure. In previous studies, we showed that, in contrast to the
serotype LPS, the core-lipid A region of A-band LPS from P. aeruginasa contains
low levels of reactive KDO and lacks phOSphate, but contains sulfur (30,31; M.
Rivera and E.J. McGroarty, unpublished results). The chemical composition of
AK1401 A-band and Dig-A—band LPS showed low levels of heptose, KDO, and
amino sugars, and no phosphate when compared to the serotype-specific or SC-
LPS (Table 2). Nevertheless, all AK1401 LPS isolates reacted with the MAb 7-4,

specific to the inner core region of P. aeruginasa (Figure 6). The differences

.Jpr

162

observed in the amounts of heptose and KDO between A-band, Dig-A-band, and
SC-LPS apparently does not affect the binding of the MAb 7-4 to the common
epitope shared by these LPS isolates. We propose that the inner core and lipid A
regions of A-band LPS show some structural similarities as well as some
differences compared to the serotype-specific LPS. Results from the Western blots
(Figures 4 and 5) using the MAbs 8A1 and 177 indicated that all the samples have
common epitopes in the lipid A. Lack of reactivity of the AK1401 A-band isolate
presumably is the result of either non-reactivity or lack of acid hydrolysis.
Interestingly, the phage-digested A—band lipid A derivative showed reactivity to
MAb 8A1. This is unexpected since phosphate is reported to be part of the
binding site for this MAb (3); the observed reactivity suggests that sulfate may be
substituted as part of the epitope. Reactivity of MAb 4A10 with AK1401 Uf- and
SC-lipid A derivatives, and with O-serotype-LPS derived lipid A but not lipid A
from A-band isolates suggests that this MAb recognizes determinants exclusive to
lipid A of the B-band type (3).

In addition to differences in the lipid A region, AK1401 A-band LPS and
O-serotype-specific LPS showed structural differences in the outer core region.
This was clearly shown with outer core- specific MAb 101 (Figure 7). Yokota et
al (42) reported that the core oligosaccharide to which the common polysaccharide
antigen is attached, while similar in composition to O-antigen-containing-core, also
contains xylose and an unidentified 3-O-methyl-6-deoxyhexose. They proposed

that there could be a separate oligosaccharide chain containing seven residues of

163

the O-methylated sugar and two residues of xylose. Multiple core oligosaccharide
structures have been reported for other gram-negative bacteria. For example,
adherent enteropathogenic E. coli 0119 strains are reported to have a larger LPS
core than non-adherent strains, although the O-polysaccharide chains are identical,
and the inner core of adherent strains reportedly has an atypical structure
containing equimolar amounts of L-glycero-D-manno-heptose and D—glycero-D-
manna-heptose (4).

LPS has been shown to be an important virulence factor and to have a role
in pathogenesis (7). While it is not clear at present whether the A—band type of
LPS found in P. aeruginasa serves as a virulence factor, it appeared to be a
prominent surface antigen on organisms isolated from clinical sources. Lam and
coworkers (21) reported that 11 of the 17 serotype strains from P. aeruginasa
possessed A-band LPS. They also observed that 68% of the clinical isolates from
patients with cystic fibrosis had A-band LPS. In cystic fibrosis, during the course
of infection with P. aeruginasa, isolates have been shown to become nontypeable,
and the O-antigen is replaced with A-band as the major LPS antigen (21). In a
separate study, Sawada et a1 (32) described a MAb, E87, that bound to 80% of P.
aeruginasa strains of various serotypes. They showed that the antigen recognized
by this monoclonal antibody consisted mainly of rhamnose. As mentioned
previously, the structure of this common antigen polysaccharide is a D-rhamnose
trisaccharide repeating unit (17,41 ,42; Arsenault et al, unpublished results). This

same structure is found in P. syringae pv. morsprunarum LPS O-polymer (34).

164

In P. syringae pv. morsprunarum there is a correlation between phage sensitivity
and host specificity (12). Zamze et al (43) reported that loss of virulence in P.
syringae pv. morsprunarum was associated with changes in the LPS structure;
either modification of the side-chain polysaccharide or complete loss of the
sidechains to yield a rough LPS.

In conclusion, our results show that A-band LPS from P. aeruginasa
AK1401 serves as the initial receptor for phage A7 and is hydrolyzed by the phage
to core-lipid A with only two or three rhamnose repeat units. The chemical
composition of the phage A7-digested A—band LPS showed low levels of heptose,
KDO, and amino sugars, and no phosphate confirming earlier work (30,31).
Furthermore, the results from the immunoblots with MAbs indicate that even
though the inner core region of all AK1401 LPS fractions appeared to share a
common epitope, the outer core region of the A-band LPS is different from that
of the B-band or SC-LPS. Further structural studies will be performed on this

phage A7—digested A-band LPS fraction.

10.

11.

12.

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Laemmli, U.K. 1970. Nature (London) 227, 680-685.

Lam, J.S., A. MacDonald, M.Y.C. Lam, L.G.M. Duschesne, and G. G.
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Lam, M.Y.C., E.J. McGroarty, A.M. Kropinski, L.A. MacDonald, S.S.
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Lightfoot, J ., and J .S. Lam. 1991. J. Bacteriol., in press.

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Smith, A.R.W., S.E. Zamze, and RC. Hignett. 1985. J. Gen. Microbiol.
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Smith, A.R.W., S.E. Zamze, S.M. Munro, K.J. Carter, and R. C. Hignett.
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Yokota, S., S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito.
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131, 1941-1950.

CHAPTER 6

Summary and Perspectives

168

SUMMARY

In the past six years studies from several laboratories have demonstrated
that many strains of Pseudomonas aeruginosa produce a second lipopolysaccharide
(LPS) whose polysaccharide chain differs serologically and structurally from 0-
antigen polysaccharide (17,19,23,29,30,33,40,41). The common antigen
polysaccharide is a regular homopolymer of rhamnose, and the structure has been
shown to consist of the repeating unit [- > 3)-oz-D-Rhap-( 1- > 3)-oz-D-Rhap-(1- > 2)-
tit-D-Rhap-(1->]n (17,40,41; T.L. Arsenault et al, Can. J. Chem., in press). In
my initial studies the size heterogeneity of LPS isolates from several P. aeruginosa
strains was defined by both gel filtration and SDS-PAGE (29, Appendix A).
When column fractions were applied to SDS-polyacrylamide gels in their order of
elution, molecules of decreasing size were resolved, and the ladder of molecules
with different-length O-antigens formed a diagonal across the gel (Appendix A).
The LPS from the PAOl derivatives contained two distinct sets of bands,
distinguished on the gels as two set of diagonals. The set of bands with the faster
mobility, the B bands, represents 85 to 90% of the total LPS sample by weight;
this is the O—antigen-containing LPS which determines the O-specificity of the

bacterium. The slower-moving set of bands, the A-bands, represents 10 to 15%

169

.l'

170

of the total LPS sample and contains shorter chains of predominantly neutral
polysaccharide. Reaction of isolated fractions with monoclonal antibody specific
for the PA01 O—antigenic side chain indicated that only the B bands from the
PAOl strains were bound. These observations led to the proposal that PA01
strains synthesize two types of molecules that are antigenically different.

Further chemical characterization was performed on the A band and B band
isolates (Chapter 2). The fatty acid profile showed that indeed the A band isolate
is an LPS-like molecule. In addition, chemical analysis of the A-band and B-band
LPS revealed that not only are these two LPS molecules antigenically distinct but
they also differ in composition (Appendix A and Chapter 2). This is reflected in
the amount of amino sugars, rhamnose, KDO, and phosphate. An important
finding from this study was that A-band LPS lacked phosphate but contained
sulfate groups (Chapter 2 and 4). Furthermore, the A-band LPS contains high
levels of rhamnose and reacted with the polyrhamnose-specific monoclonal
antibody E87. Reactivity with the E87 monoclonal antibody is found in a large
number of different serotype strains (33,40) suggesting that this antigen is common
among P. aeruginosa, perhaps as a common antigen LPS. Lam and co—workers
(19) have produced seven monoclonal antibodies to the A-band LPS. Using
Western immunoblot analysis, they have shown that the A band molecule is
present as a common antigen on strains of many serotypes.

The size heterogeneity of LPS from P. aeruginosa as well as other gram-

negative bacteria is reported to be altered by changes in the growth conditions,

elf"

171

such as temperature (2,22,25) and medium composition (10,38). Alterations in
outer membrane structure induced by specific growth conditions may change
cation-binding sites (10). Since a major site of cation binding in the outer
membrane is with LPS, a study was initiated to analyze the influence of various
growth conditions on the size heterogeneity of LPS in P. aeruginosa (Chapter 3).
The results indicated that cells grown at near-growth-limiting conditions including
high temperature, high concentrations of salt, sucrose, or glycerol, low phosphate
concentration, and low pH produced decreased amounts of the very long chain
population of O-antigen LPS molecules. The size and amount of common antigen
LPS was either unaffected or increased slightly when the cells were grown under
the above stress conditions. Also, cells grown under stress conditions, in which
the long O-polymer LPS was absent, were agglutinated by both serotype-specific
and common antigen-specific monoclonal antibodies. The results indicate that the
long O-polymers cover and mask the shorter common antigen. However, specific
growth conditions limit the production of the long O-polymer, allowing the
exposure of the common antigen on the cell surface. Under certain conditions,
such as prolonged antibiotic therapy, clinical isolates from P. aeruginosa are found
to be nontypeable and appear to loose the O-polymer-containing B bands
(3,11,14). Lam and coworkers (19), using immunoblot analysis, have shown that
A bands were present in a high percentage of clinical isolates and appeared to be
a main antigen on nontypeable strains deficient in high molecular-weight serotype-

specific LPS. For such clinical isolates the A bands may become exposed and

172

serve as an important antigenic determinant.

Antigenic as well as chemical differences have been observed between the
A and B bands, but the novelty of A—band LPS is the presence of sulfate. To
corroborate that sulfur is incorporated into A—band LPS in stoichiometric amounts,
P. aeruginosa were grown in modified basal medium containing 35S-labelled
sulfate (Chapter 4). We observed that the A—band LPS isolated from smooth
strains of P. aeruginosa were always contaminated with small amounts of an
intermediate chain-length serotype-specific LPS (Appendix A and Chapter 2).
Therefore, the bacterial strain used for detailed structural analysis was P.
aeruginosa AK1401 which is a rough mutant that produces A—band LPS but is
defective in the synthesis of O-antigen (2). The 358-1abelled LPS was separated
by gel filtration chromatography into two major populations of LPS; A-band or
common antigen LPS and a serotype-specific short chain LPS (SC or B-band). The
elution profile as well as the autoradiogram showed that the A—band and B-band
LPS contained labelled-sulfur. Chemical analysis of the AK1401 A-band LPS
indicated low levels of KDO and no phosphate. In addition, A-band LPS showed
a 1:1 molar ratio of sulfur to heptose suggesting that two to three sulfur atoms are
present in the A-band molecule. The molar amounts of sulfur detected in
unfractionated and SC-LPS isolates were one half the heptose content. Thus, A-
band LPS contains covalently bound sulfur perhaps as sulfate. This functional
group can be identified by FT-IR, 1H NMR, and 13C NMR spectroscopy. It is

reasonable to propose that the biosynthetic pathways of the A-band and B-band

173

LPS molecules diverge at some point. It has been shown that the synthesis or
expression of P. aeruginosa A- and B-band LPS appear to be partially independent
(J. Lightfoot et al, Abstr. Annu. Meet. Am. Soc. Microbiol. 1991, D-69, p.90).
Genes for A-band synthesis have been shown to map at a location different from
the B-band genes.

Arsenault et al (Can. J. Chem. , in press) characterized the structure for the
rhamnose polysaccharide portion of P. aeruginasa AK1401 A-band LPS, and it
was shown to be identical with that reported for the common antigen LPS
(17,40,41). Interestingly, the O-polysaccharide chain in the LPS of Pseudomonas
syringae pv. morsprunarum is reported to have the same structure as the
polysaccharide of A-band LPS (36). The bacteriophage A7, a typing phage for P.
syringae pv. morsprunarum, has LPS as its primary receptor (35). This phage A7
possesses a rhamnanase that specifically hydrolyzes the rhamnose polysaccharide
chain to oligosaccharides (35). In our studies (Chapter 5) we demonstrated that
A-band LPS from P. aeruginasa AK1401 serves as the initial receptor and is
hydrolyzed by phage A7 to core-lipid A with only two or three rhamnose repeat
units. The chemical composition of the core components of this phage A7-digested
A—band LPS was similar to that reported previously for undigested A-band LPS
(29,30; Chapter 4). This phage-digested A-band isolate was also incubated with
monoclonal antibodies against either inner core or outer core epitopes of P .
aeruginasa LPS, and the results were compared to that of the serotype SC-LPS.

The results from the immunoblots indicated that, even though the inner core region

174

of all AK1401 LPS fractions share a common structure, the outer core region of
the A—band LPS is different from that of the B—band or SC-LPS.

The identification of two chemically distinct forms of LPS in P. aeruginasa
raises the question regarding the roles of these two fractions in virulence,
antigenicity, and antibiotic sensitivity. It has been proposed that LPS is an
important virulence factor and has a role in pathogenesis (6). Perhaps the A-band
type of LPS found in P. aeruginasa common antigen serves as a virulence factor
during infection in cystic fibrosis patients. Thus, it is important to characterize the
structure of this A-band LPS to understand the mechanism of virulence and

antibiotic resistance.

PERSPECTIVES

To analyze the core-lipid A region of A-band LPS, P. aeruginosa AK1401
strain will be grown at 37 ° C in either TYE or 3SS-labelled MBM (Chapter 4).
The cells will be harvested late in logarithmic growth phase and washed with
distilled water. LPS will be extracted from the cells, separated by gel exclusion
chromatography, and fractions purified as indicated in Chapter 5. Purified A—band
LPS (5-15 mg) will be suspended in water and phage A7 will be added (1010-1011
p.f.u.) and incubated for 24 h at 25 ° C. The phage A7-digested A-band (Dig-A-
band) LPS will be further purified as described previously (Chapter 5). Even
though the A-band LPS is resistant to acid hydrolysis, the Dig-A-band LPS can be
acid hydrolyzed to lipid A under conditions described earlier (Chapter 5). To
prevent loss of sulfate, the Dig-A-band as well as B-band LPS will be acid
hydrolyzed, under more gentle conditions, treating the samples either with 1% or
5% acetic acid at 100 ° C for l or 2.5 h (15), or with 20 mM sodium acetate (pH
4.5) at 100°C for 1h (1). Lipid A release will be monitored by thin layer
chromatography (TLC; 32). The hydrolyzed products will be separated on a
Sephadex G-50 column using deionized water as the eluant. The fractions will be

monitored for total carbohydrate (9) and sulfate (7, Chapter 4). The fatty acid

175

176

composition of the lipid A isolate from A-band and B-band will be monitored by
gas chromatography (24,39) for losses in fatty acid during the hydrolysis
procedure. In addition, TLC will be used to determine the heterogeneity of the
lipid A isolates; using preparative TLC we can separate and purify the lipid A
mixture (16).

The structure of the lipid A as well as the 35S-labelled lipid A fractions will
be analyzed by soft ionization methods, namely, fast atom bombardment-mass
spectrometry (FAB-MS) (16,20), plasma desorption MS (5,26), or laser desorption
MS (18,34). In addition, 1H and 13C NMR will be used, in conjunction with
Fourier transformed infra red (FTIR) spectroscopy, to analyze the structure of the
lipid A isolates (12,18,21,26,27).

The complete core structure from Dig-A band LPS will be determined by
using methylation analysis and degradation methods (4,28,31). The neutral sugars
will be determine as their alditol acetate derivative using GC and GC-MS (28).
Quantitation of amino sugars will be performed on an automatic amino acid
analyzer as described elsewhere (28,31). Methylation analysis will be performed
according to Hakamori (13) and Stellner et al (37). Methylation of the core
oligosaccharide will be preceded by a reduction of the uronic acid residues using
NaBD4, and then will be subjected to methylation and the methylated sugars will
be quantitated and identify by GC and GC-MS as the alditol acetate derivatives
(28). Smith degradation of the isolated core oligosaccharide will be carried out

as described elsewhere (28). Sulphation as the mode of incorporation of the 35S-

177

label into both core oligosaccharide and lipid A isolates can be confirmed by mild
acid hydrolysis and precipitation of the released counts as barium sulfate
(Hollingsworth, R.l., Analyt. Biochem., submitted). Glycosyl linkages will be
determined by a previously described procedure (4).

In addition, the core oligosaccharide of the Dig-A-band as well as the B-
band fraction will be examined by the physical methods described above. Mass
spectrometry of 35S-labelled oligosaccharide can be used to define the substituents
(20). Proton and 13C NMR in conjunction with FTIR will reveal the anomeric
configuration, the conformation of the monosacharide units, and will give relevant
structural information concerning further substitution and group functionalities
(4,12,20,21,28).

It has been shown previously that the cell-free preparation from
Acanthamoeba castellanii has the ability to digest LPS from Salmonella Rd
mutants (8). A cell-free preparation has been shown to have esterase, amidase,
and phosphatase activity but no cleavage of glycoside linkages could be detected.
The crude enzyme preparation acted on LPS in a specific way in vitro,
quantitatively releasing the O—acyl residues; approximately 50% of the N-acyl
residues and 70% of the original amount of phosphate was removed. Therefore,
the activity of this mixed enzyme preparation can be tested on the Dig-A-band LPS
isolate from P. aeruginosa AK1401 which will avoid unwanted chemical

degradation of the LPS molecule. After purification of the enzymatic products by

178

extraction procedures and gel filtration, the carbohydrate fraction will be examined

by both chemical and physical methods as described above.

10.

11.

12.

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Caroff, M., R. Chaby, D. Karibian, J. Perry, C. Deprun, and L. Szabo.
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1987. Eur. J. Biochem. 167, 203-209.

APPENDIX A

Heterogeneity of Lipopolysaccharides from Pseudomanas

aeruginasa: Analysis of Lipopolysaccharide Chain Length

182

ABSTRACT

Lipopolysaccharide (LPS) from smooth strains of Pseudomanas aeruginasa
503, PAZl, PA01715, PAOl7l6, and Z61 was fractionated by gel filtration
chromatography. Lipopolysaccharide samples from the first four strains, all PAOl
derivatives, separated into three major size populations, while strain Z61, a Pae
K799/WT mutant strain, separated into two size populations. When column
fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order
of elution, molecules of decreasing size were resolved, and the ladder of molecules
with different length O-antigens formed a diagonal across the gel. The LPS from
the PAOl derivatives contained two distinct sets of bands distinguished on the gels
as two sets of diagonals. The set of bands with the faster mobility, the B bands,
was found in column fractions comprising the three major amino sugar containing
peaks. In the sample from 503, a fourth minor peak which contained B bands was
resolved. The slower moving set of bands, the A bands, were recovered in a
minor peak. LPS from strain Z61 contained only one set of bands, with the higher
molecular weight molecules eluting from the column in a volume similar to that
of the B bands of the PA01 strains. Analysis of the fractions of LPS from all

strains indicated that less than 8% of the LPS molecules had a long attached 0-

183

184

antigen. Analysis of the peak that contained mainly A bands indicated a lack of
reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxy-
octulosonic acid were detected. Reaction of isolated fractions with monoclonal
antibody which is specific for the PAOl O-antigen side chain, indicated that only
the B bands from the PAOl strains were antigenically reactive. The bands from
strain Z61 showed no reactivity. The data suggest that the A and B bands from
PA01 strains are antigenically distinct. We propose that PAOl strains synthesize

two types of molecules that are antigenically different.

INTRODUCTION

Lipopolysaccharide (LPS), a major component of the outer membrane of
gram-negative bacteria, is important in the structure (34,38) and function (34,37)
of this membrane. Structural microheterogeneity has been demonstrated in several
regions of LPS molecules from the Enterabacteriaceae (3,15,23,38,39,48,52) and
Pseudomonas aeruginasa (34,58). Of the several methods used to separate the
subclasses of LPS from individual strains, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) (21,25,46,48) and gel filtration
(10,27,31,33,35,48) are the best. Either of these two methods by themselves,
however, may be insufficient to completely characterize the high and low
molecular weight fractions of LPS. Peterson and McGroarty (48) demonstrated
that the SDS-PAGE of the column fractions of samples from Salmonella
typhimurium, Salmonella minnesata, and Escherichia coli was instrumental in
characterizing the various sized fractions. Analysis of the isolated fractions
allowed for the estimation of the average number of O—antigen repeat units per
LPS from each of the size fractions.

Compositional analysis of LPS from P. aeruginasa has indicated that the

LPS molecules are structurally similar to enterobacterial LPS molecules, but

185

186

possess several distinctive features (34,58). The most outstanding differences
include the unusually high phosphate content (34,59), the presence of L-alanine in
the core (34,58), and the high levels of amino sugars and uronic acids in the 0-
side chain (9,34). The characterization of P. aeruginasa O-specific
polysaccharides has been complicated in some cases by chemical heterogeneity of
the polysaccharide chains (9,10,31,59). In some instances, the polymeric material
has been resolved into amino sugar-rich and neutral sugar-rich fractions (31,59).
The biological significance of such fractions is unclear, but a possible explanation
is that P. aeruginasa strains produce multiple types of molecules with chemically
distinct polysaccharide chains.

In this study, we have analyzed the size heterogeneity of LPS isolates from
several P. aeruginasa strains by both gel filtration and SDS-PAGE. These studies
have revealed that LPS isolates from PAOl strains contain two distinct sets of
bands, suggesting that PAOl strains are capable of synthesizing more than one
type of chemically and antigenically distinct molecule. We also present evidence

that the percentage of core oligosaccharides carrying the O-specific polymer is less

than 8%.

MATERIALS AND METHODS

Bacterial strains.

P. aeruginasa stain 261 was a mutant derived from strain Pae K799/wt
selected for antibiotic supersusceptibility; and PAZl met-28, ttp-6, lysA—12, his-4,
ile-226, and absA was a PA0222 derivative into which absA mutant gene from
Z61 encoding antibiotic sensitivity has been conjugated (2). Strains PAOl7l6 ade-
136, leu-8, rif-l (revertant) and PA01715 ode-136, lea-8, rif-l, talA-12 (an
aminoglycoside supersensitive mutant) were described previously (41). Strain
PA0503 met-9011 is a methionine auxotroph of P. aeruginasa PA01. Strains
PAZl, 1715, 1716, and 503 were 0-5 serotype. Escherichia coli strains D21 and
D21f2 are derived from strain K-12 and were characterized as Ra and Re

Chemotype, respectively (4).

Growth media.

Strain PAOl7l6, PA01715, Z61 and PAZl were grown at 37°C to mid-
logarithmic phase in a 100 l fermentor containing 80 l of protease peptone no. 2
medium from 1 l overnight culture grown in the same medium. Strain 503 was

grown as previously described (5).

187

188
E. coli strains D21 and D21f2, grown as described by Coughlin et al. (11),

were harvested in late log phase.

Isolation of LPS.

LPS from P. aeruginasa strain Z61 and PAOl derivatives 1715, 1716,
PAZl, and 503 were isolated by the method of Darveau and Hancock (13),
followed by two extractions in chloroformzmethanol (1 :1 v/v) resulting in recovery
of approximately 80% of the total LPS. The LPS from E. coli strains D21 and
D21f2 was isolated using the hot aqueous phenol (57) and the chloroform-

petroleum ether (17) extraction procedures, respectively.

Gel electrophoresis.

Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels were prepared
and run using the buffer system of Laemmli (36). Unless otherwise noted,
separating gels were formed with 15% acrylamide, 0.1% SDS, with a 7.5%
acrylamide stacking gel. Samples were mixed 1:1 with sample buffer (containing
4% SDS) and applied to the gel. Electrophoresis was performed with a constant
current of 15 mA per gel until the tracking dye entered the separating gel and then
at 30 mA per gel until the tracking dye reached the bottom of the gel. LPS bands

were detected by the silver staining method of Dubray and Bezard (l6).

189

Column chromatography.

Samples were fractionated with a Sephadex G-200 (Pharmacia Fine
Chemicals) column (64 cm by 25 mm) at room temperature suing the column
buffer system of Peterson and McGroarty (48). Approximately 30 mg of LPS was
applied to the column, and 5 ml fractions were collected at a flow rate of 8 ml per
h. To remove detergent and buffer, pooled fractions were extensively dialyzed
(12,000 to 14,000 molecular weight cutoff membranes) against column buffer
without deoxycholate at 37°C and then against distilled water at 4°C. The
dialyzed fractions were lyophilized and resuspended to a concentration of 10

mg/ml in water. All fractionations were done at least twice.

Western blots.

Western blots of SDS-polyacrylamide gels were prepared as previously
described (6,55). The gels were electrotransferred with a model TE Transphor
Electrophoresis apparatus (Hoefer Scientific Instruments) at a constant current of
150 mA for 18 h using the electrode buffer described by Otten and co-workers
(45) unless otherwise noted. The nitrocellulose blots were visualized as described
previously (45) with monoclonal anti-503 antibody (20, titer ~ l:100,000) diluted
l:10,000 in blocking solution. In addition, dot blots were performed by applying
known quantities of LPS isolates directly on nitrocellulose. The blots were washed

and visualized using horseradish peroxidase conjugated goat anti-mouse IgG

190

antibody (Sigma Chemical Co.) as described above or using silver stain protocol

identical to that described by Dubray and Bezard (16).

Assays.

Assays for amino sugars (18), heptose (60), and 2-keto-3-deoxyoctulosonic
acid (KDO) (29) were performed as described previously except where noted.
Phosphate analysis were performed using either a colorimetric assay (1) or by
inductively plasma emission spectroscopy (11). Protein concentrations were
estimated with the Pierce BCA protein assay (Pierce Chemical Co., Rockford, IL)

using bovine serum albumin as a standard.

RESULTS

Silver staining of LPS from strains of P. aeruginasa separated by SDS-
PAGE revealed a progressive ladder-like pattern of bands up the gel (Figure 1A).
For the Enterabacteriaceae and P. aeruginasa, these bands have been reported to
represent LPS molecules containing increasing lengths of O-antigen (5,21,46).
The intensity of staining indicated three to four regions of bands representing as
many as four populations of molecules (band sets 1, 2, 2a and 3, Figure 1)
differing in O-antigen length. The electrophoretic pattern of LPS from strain 503
showed a set of bands (set 2a) which were closely spaced and slower migrating
bands immediately above the 2a set which had greater spacing (Figure 1A, lane
2). In the banding pattern of LPS from PA01 derivatives 1715, 1716, and PAZl
(Figure 1A, lanes 1, 3, and 5, respectively), we observed irregularities in the
spacing and intensities of bands up the gel. In contrast, LPS from strain Z61
appeared to have a regular spacing and intensity in the banding pattern (Figure 1A,
lane 4). The average length of the highest molecular weight LPS of strains Z61
and PAZl seemed shorter than that of the other PAOl derivatives, a phenomenon
observed previously and ascribed to the absA (antibiotic supersusceptibility) mutant

locus (2). When low amounts of LPS were applied to the gel, only the fastest

191

Figure l.

192

(A) Silver-stained SDS-polyacrylamide gel of LPS from P.
aeruginasa strains 1715 (lanes 1 and 8), 503 (lanes 2 and 9), 1716
(lanes 3 and 10), Z61 (lanes 4 and 11), PAZl (lanes 5 and 12), and
from E. coli strain D21 (Ra, lanes 6 and 13) and D21f2 (Re, lanes
7 and 14). Samples of either 5 pg (lanes 1 to 7) or 0.1 pg (lanes 8
to 14) were applied to a 15 % acrylamide gel which had been
polymerized overnight with a butanol overlay. Arrows indicate the
four intensively stained regions of the P. aeruginasa samples: band
sets 1, 2, 2a and 3. (B) Western blots of LPS from P. aeruginasa
strains 1715 (lane 15), 1716 (lane 16), 503 (lane 17), and PAZl
(lane 18) reacted with monoclonal anti-503 antibody. Samples of 2.5
pg were applied to at 12% acrylamide gel which had been
polymerized overnight with a butanol overlay. The gel was blotted

as described in Materials and Methods.

193

 

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194

migrating bands were stained, and there was no difference in migration pattern of
LPS of strains 1715, 503, 1716, and PAZl (Figure 1A, lanes 8, 9, 10, and 12
respectively). On the other hand, the low molecular weight bands from strain Z61
migrated faster than that of the other P. aeruginasa strains (Figure 1A, lane 11)
as previously observed (35), due to an apparent truncation in the rough core of the
short chain LPS molecules in this strain. LPS from E. coli D21 (Ra chemotype)
and D21f2 (Re chemotype) was used to compare and characterize the
electrophoretic mobilities of the short chain populations.

Using antibodies specific to the PAOl O-antigen, Western blots of the LPS
separated with SDS-PAGE were analyzed to help clarify the irregularities in the
banding pattern. The blots of LPS isolated from P. aeruginasa strains 1715,
1716, 503 and PAZl revealed a ladder pattern of molecules that consisted of
doublet bands (Figure 1B). Furthermore, the level of one of the bands in the
doublet was in lower amounts in the isolates from strains 1715, 1716, and PAZl
than from strain 503. Presumably, this reflects a difference in substoichiometric
modification within the core-lipid A region of the molecules. Interestingly,
spacing and intensity of the ladder pattern seen in the Western blot was much more
regular than that of the silver stained gel. This suggested that the stained sample
may contain bands superimposed on the ladder pattern of the main antigen.

If the irregular silver-stained banding pattern was a result of heterogeneity
in the LPS samples, this heterogeneity could have been due to contamination of

the culture or to true heterogeneity of the sample. The possibility that the cultures

 

195

were contaminated is very low since after growth, all cultures were streaked out
onto protease peptone no. 2 agar plates to observe characteristic colonial
morphologies and pigmentation and, in the cases of Z61, PAZl and PA01715,
tested for characteristic antibiotic supersusceptibilities. Furthermore, the irregular
banding patterns were seen in samples from PAOl derivatives (Figure 1A) isolated
in two different laboratories and from several independently isolated LPS samples.

To further characterize the heterogeneity of the LPS isolates from the PAOl
strains, the samples were separated on a Sephadex G200 column. The elution
profile showed three major amino sugar-containing peaks for strains 503 (Figure
2), 1715 (Figure 3), 1716 and PAZl (results not shown). In contrast, the elution
profile of the LPS sample from the Z61 strain showed only two major peaks
(Figure 4). Both gel permeation chromatography and SDS-PAGE separate
molecules on the basis of size; therefore, a diagonal banding pattern should be
expected across SDS-PAGE gels of column fractions when applied in the order of
elution. SDS-PAGE of the column fractions of samples from each of the PAC]
derivatives studied revealed two distinct ladder patterns of apparently different
sizes, the A bands (later eluting ladder) and the B bands (earlier eluting ladder)
(see Figures 2 and 3 for elution profiles of strain 503 and 1715, respectively). In
contrast, the LPS isolate from strain Z61 showed only one ladder set (Figure 4).
Peterson and McGroarty (48) reported that the SDS-PAGE profiles of fractionated
LPS from Salmonella species separated in this type of column as molecules of

decreasing size. The presence of two distinct ladder patterns suggests that either

Figure 2.

196

Fractionation of LPS from P. aeruginasa strain 503 on Sephadex G-
200. Fractions were analyzed for KDO (0) and amino sugar (A).
Silver-stained SDS-polyacrylamide gels of column fractions are
aligned under their appropriate fraction number. A represents the

slow-moving set of bands and B the faster-moving set.

197

 

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198

Fractionation of LPS from P. aeruginasa strain 1715 on Sephadex
G-200. Fractions were analyzed for KDO (0) and amino sugars (A).
Silver-stained SDS-polyacrylamide gels of column fractions are
aligned under their appropriate fraction number. A represents the

slow-moving set of bands and B the faster-moving set.

199

 

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60

Fraction Number

wt

2’/

 

 

H 029v

200

Figure 4. Fractionation of LPS from P. aeruginasa strain Z61 on Sephadex G-
200. Fractions were analyzed for KDO (0) and amino sugar (A).
Silver-stained SDS-polyacrylamide gels of column fractions are

aligned under their appropriate fraction number.

A548. '

201

 

0.5 -

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0.3 --

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Figure 4

2
20 4O

Fraction Number

 

0.3

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1 UOEGV

202

stable aggregates were present (48) or there were two types of molecules with
different charges; electrophoresis separates molecules on the basis of both size and
charge. To eliminate the possibility that A bands were aggregates of the B bands
stabilized by hydrogen bonding between O—polymers, SDS-PAGE of the column
fractions of samples from strain 503 was run in the presence of 4 M urea (final
concentration). The same two sets of bands were observed in 4M urea-SDS gels
(results now shown). In addition, two-dimensional electrophoresis of column
fractions of LPS from strains 503 and 1716, which contained approximately equal
amounts of A and B bands, showed that both the A and B bands ran with the same
mobilities in the second dimension. Since the gels in both dimensions consisted of
standard SDS gels and the gel strip from the first dimension was heated before
running the second dimension, the results indicate that the bands do not
interconvent (results not shown). This again indicates that one set of bands is not
an aggregate or different conformational state of the other. Amino sugar analysis
of our column fractions indicated that the fractions containing mainly A bands
(peak X, Figure 3) lacked reactive amino sugars, while the other fractions (peaks
1, 2, 2a, and 3; Figures 2 and 3) showed reactivity.

The individual fractions from the elution profile of each of the LPS samples
separated in a Sephadex G-200 column were also analyzed for KDO and phosphate
content, and the relative molar ratios of amino sugar to KDO and amino sugar to
phosphate were determined for the 3 or 4 major peaks. Also, the fractions

corresponding to each of the major peaks in the elution profile of strains 1715 and

 

203

TABLE 1. Chemical Analysis of P. aeruginasa B-Band LPS Fractions

 

Peak No. % P Amino Amino P/KDO
Recovereda Sugar/KDOb Sugar/Pc (mole/mole)

 

Strain Z61Cl
2 2.8 5.5 9.3 N.D.e
3 97.0 1.0 1.0 5.5
SuanPAzfi
1 2.3 24.8 30.8 4.8
2 3.2 19.5 18.8 6.1
3 94.3 1.0 1.0 5.9
Strain 1715f
1 3.4 29.3 31.4 5.4
2 1.4 17.0 18.2 5.4
3 95.1 1.0 1.0 6.0

 

a Percentage of total amount in each of the peaks.

b Relative molar ratio; KDO and amino sugar levels were normalized to a value
of 1.0 for peak 3 samples.

c Relative molar ratios; phosphate levels were determined for the individual
fractions of the Z61 sample by using the colorimetric assay and for the pooled
fractions from samples of strains 1715 and PAZl by inductively coupled plasma
emission spectroscopy, as described in Materials and Methods. Levels were
normalized to a value of 1.0 for peak 3 samples.

d Individual fractions from the elution profile of the LPS samples separated on a
Sephadex G-200 column, as described in Material and Methods, were analyzed
and the amount in all the fractions in each peak were added together.

3 ND, not determined.

f Pooled fractions corresponding to each of the peaks in the elution profile of LPS
samples separated on a Sephadex G-200 column were dialyzed, lyophilized, and
resuspended in distilled water for analysis.

204

PAZl were pooled, dialyzed, lyophilized and resuspended in water to a final con-
centration of 10 mg/ml and were analyzed as above. The data shown in Table 1
for the pooled fractions and data from the individual fractions (not shown)
indicated that, using phosphate as an indicator of molar amounts of the LPS, the
short chain peak 3 sample represented 92 to 97% of the total LPS molecules; peak
2a, 0.4 to 2%; peak 2, 1 to 3%; and the very long chain fraction, peak 1, 3 to
4%. The fifth population of molecules, the A bands, were resolved as a separate
population on the column (peak X) in the samples from 1715 (Figure 3), 1716, and
PAZl, whereas A bands overlapped with the 2a peak of the main ladder set in the
503 fractionation (Figure 2). As stated above, strain Z61 showed only two
different LPS size populations, the short chain fraction, peak 3 (97% of total), and
the long chain fraction, peak 2 (2.8% of total) (Figure 4).

The O—antigen of P. aeruginasa is reportedly rich in amino sugars
(34,35,40,58), although the reported sequence of the 05 serogroup (equivalent to
Lanyi type 3a,d) indicates that most of the amino groups are acetylated (30). The
phosphate and KDO assays measure residues in the core region of LPS (35),
whereas the assay for amino sugars detects residues both in the core-lipid A region
and O-antigen repeat region. The data reported in Table 1 show that the molar
ratios of PzKDO for the three major B band-containing peaks of the pooled
fractions are similar for strains PAZl and 1715. Also, this ratio for peak 3,
calculated from the individual fractions for all strains, appeared to be relatively

constant from strain-to-strain and was determined to be between 5.5 and 6.6

205

(mole/mole, data not shown). Since the level of phosphate or KDO can be used
as a measure of the relative molar amount of LPS in each peak, comparison of the
ratios of amino sugar to KDO and/or amino sugar to phosphate in the three major
peaks of the pooled fractions should reflect the O-antigen length. For the
individual fractions a discrepancy existed in these ratios where the values are lower
than for the pooled fractions. This may be due to the elution buffer present in the
fractions before dialysis. Since the pooled fractions were dialyzed to remove the
detergent and salts and resuspended in H20 to a known concentration, the pooled-
fraction data were more reliable. Furthermore, the low amount of sample in the
individual fractions decreased the sensitivity of the assays.

The chemical composition of the dialyzed A band samples from strains 1715
and PAZl was analyzed, and the results were compared with those of the three
major amino sugar-containing peaks (Table 2). The results indicated that peak X
(A bands) comprised 10-15% of the total LPS sample by weight. Furthermore,
under the normal conditions of the assay, it appeared to have low levels of reactive
KDO, amino sugars and phosphate. When the pooled samples were hydrolyzed
for a longer period of time (20 min) and at a higher acid concentration (0.5 N
H2804), the KDO levels detected in the B band fractions were similar to previous
assays, but the KDO levels in the A band fraction increased 10-fold. Thus, the
A band material appeared to contain KDO residues which are much less reactive
than those in the B band isolates. Analysis of the isolates from strain 1715

indicated that the A band material had levels of heptose in between that found in

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207
peaks 2 and 3 (Table 2). Analysis for protein in the pooled fractions indicated less

than 1.5 % (w/w) in all fractions. From the SDS-PAGE of the separated fractions
(e.g. Figure 3) and from the chemical characterization of the pooled samples, it
was observed that the A bands of strains 1715 and PAZl were contaminated with
only minor amounts of the B band material. The A band material is probably a
glycolipid since the isolate, suspended in the absence of detergent at 10 mg/ml,
was cloudy and not completely water soluble, and since it contained heptose and
KDO.

To better compare the migration patterns of equivalent LPS populations
from different strains, comparable column fractions of LPS containing both A and
B bands from the different strains were run together on SDS-PAGE (Figure 5).
The A bands from all four PAOl strains had very similar spacings (lanes 2—5),
and the spacings of the B bands of strain PAZl, 1716, and 1715 (Figure 5, lanes
2, 4 and 5, respectively) appeared very similar. In contrast, the B bands of strain
503 (Figure 5, lane 3) appeared much more closely spaced compared to the same
bands of the other strains due to an increased amount of the second band in the B
band set of doublets (see also Figure 18, lane 17). Fractions from peak 3 of LPS
from strains Z61, 503, PAZl, 1715, and 1716 were run on an 18% SDS-
acrylamide gel. No differences were seen in band mobilities of the short chain
isolates except for that from strain Z61 which migrated faster (results not shown),
corroborating the results with unfractionated LPS (Figure 1A, lanes 8-12).

To determine the antigenic reactivity of the A and B bands, the LPS

Figure 5.

208

Silver-stained SDS-PAGE (11% acrylamide) (lanes 1 to 5) and
Western blots (lanes 6 to 10) of LPS fractions from P. aeruginasa
reacted with monoclonal anti-503 LPS antibody. Lanes: 1 and 6,
LPS from strain Z61 (fraction 36); 2 and 7, strain PAZl (fraction
44); 3 and 8, strain 503 (fraction 40); 4 and 9, strain 1716 (fraction
42); 5 and 10, strain 1715 (fraction 48). Samples of 100 pl were
applied to the SDS-polyacrylamide gel, which had been polymerized
overnight with a butanol overlay. The gel was blotted as described
in Materials and Methods. Arrows indicate the A and B bands of

the respective fractions.

209

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and
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210

fractions containing approximately equal amounts of A and B bands were subjected
to Western immunoblotting. Figure 5 (lanes 6-10) shows a Western blot of these
LPS fractions from strains PAZl, 503, 1716, and 1715 and a fraction of Z61
whose LPS had an electrophoretic mobility similar to that of the A bands of the
other four samples. Reaction with monoclonal anti-503 LPS antibody (specific for
the O-antigen side chain) indicated that only the B bands were antigenically
reactive and not the A bands. There was a weak antigenic reactivity of closely
spaced bands above the region corresponding to A bands which presumably was
due to reaction with aggregated B band-type LPS (Figure 5, lanes 7-10). Peterson
and McGroarty (48) have shown that the hi gh-molecular weight fractions of LPS
from E. coli can migrate as multimers. As expected, the bands from strain Z61
also showed no reactivity with the antibody since it is known that the LPS from
strain Z61 is antigenically distinct from that of PAOl derivatives (2).

To show that the lack of immunoreactivity of the A bands was not due to
lack of transfer or recovery of samples on the nitrocellulose, Western blots were
performed under different conditions. We used 150 and 400 mAmp and also
transferred for 24 h. The gels were stained for LPS with and without transblotting
to determine the level of electrotransfer of the different bands. The results
indicated that, at the lower voltage, A bands did not transfer as well as B bands,
consistent with their mobility on the gel. However, at the higher transblotting
voltage where A band-type of molecules were removed from the gel no

immunoreactivity was detected on the nitrocellulose (data not shown).

211

We also performed a dot blot immunoassay using the pooled column
fractions to characterize differences in the antigenic reactivity of the two types of
molecules. The four pooled fractions of LPS from strain 1716 (peaks 1, 2, 3 and
X) were dialyzed, lyophilized and resuspended in water. A 10 pg portion of each
of the fractions was spotted along the top of a nitrocellulose strip. Subsequent
rows were spotted with the same volume of sample serially diluted 10-fold each
row. The spots were dried and visualized with the anti-503 monoclonal antibody
as described for the Western blots. The results (Figure 6A) indicated that the very
long chain and long chain populations of B bands had similar reactivity, and as
little as 1 ng could be detected. In contrast, the short chain population, containing
no or only one O-repeat unit per molecule, showed no reactivity. Interestingly,
the A band sample showed weak reactivity when 10 pg and 1 pg were applied, but
no reactivity with lower amounts. The reactivity seen with 10 pg of the A bands
was similar to that seen with 10 ng of long chain and very long chain fractions,
suggesting that the A band fraction was contaminated with < 1% (w/w) of B
band—type molecules. That the A band fraction remained bound to the
nitrocellulose was shown by staining the dot blot with the silver stain used for the
polyacrylamide gels. We found the intensity of staining indicated the approximate
molar amount of material applied (Figure 6B). The levels detected were not
changed if the blots were washed extensively with buffer or the blocking solutions

used in the Western blots prior to staining (data not shown).

Figure 6.

212

Dot blots of pooled column fractions from P. aeruginasa 1715 LPS.
Peaks 1, 2, 3 and X were separated on Sephadex G-200, dialyzed,
and lyophilized as described in Materials and Methods and suspended
in distilled water to a final concentration of 10 mg/ml. In the top
row, 10 pg of each samples was applied on the nitrocellulose strip,
and subsequent rows were spotted with an equal volume of 10-fold
serially diluted samples. The sample spots were dried, and the
nitrocellulose was either reacted with monoclonal anti-503 antibody
and developed as described in Materials and Methods (A) or washed
with 10 mM Tris, 150 mM NaCl (pH 7.0) and reacted with the
silver stain as indicated in the Materials and Methods for

polyacrylamide gels (B).

'0 9a“

Figure 6

N

Oh 11:92:

 

213

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<—|ng

DISCUSSION

The LPS isolates of P. aeruginasa strains 503, PAZl, 1715, 1716, and Z61
were separated by SDS-PAGE into as many as four major size populations. The
ladder-like banding patterns represent molecules with increasing numbers of 0-
antigen repeat units (21,25,46). Other investigators have reported similar
heterogeneity in the LPS from strains 503 and Z51 , as well as other P. aeruginasa
smooth strains (5,25,34,35). An O-antigen ladder pattern has recently been
described for several smooth strains of P. aeruginasa using silver staining and
immunoblotting techniques (54). Our Western blot of unfractionated LPS (Figure
1B) revealed a ladder-like banding pattern with regular spacing. The bands were
resolved as doublets (Figure 1B, lanes 15-18) suggesting substoichiometric
modification in the core-lipid A similar to that seen with Salmonella LPS (43,56).
The irregular spacing that we observed in the SDS-PAGE silver-stained ladder
pattern of LPS from strains 503, PAZl , 1715, and 1716 (Figure 1A) suggested the
possibility that PA01 derivatives may be producing LPS with more than one type
of O-polymers.

Peterson and McGroarty (48), using strains of Enterabacteriaceae, have

shown that LPS molecules of different sizes can be partially separated with

214

215
Sephadex G-200 in the presence of deoxycholate and EDTA. Two or three major

populations of LPS could be resolved as detected by sugar analysis. These
populations represent sets of molecules with O-antigens of different lengths which
are made in high amounts. Other investigators have also demonstrated, using gel
permeation chromatography in combination with other methods, that the LPS from
the Enterabacteriaceae and other gram-negative bacteria could be resolved into at
least two main populations of LPS differing in the length of their O-polysaccharide
chain (10,27,31,33,35). In the results presented here, we found that the LPS from
strains 503 and 1715 (Figures 2 and 3, respectively), as well as strains 1716 and
PAZl, separated into three major populations (peaks 1, 2, and 3) and two minor
populations (peaks 2a and X). Interestingly, the short chain population comprised
more than 90% of the total sample on a molar basis (Table 1). In addition, there
were greater amounts of the very long chain population from the PAOl strains
compared to the long and intermediate chain populations. These results agree with
those of Wilkinson (58) and Hancock et al. (24), where they estimated the mole
percent of S-form LPS to be between 0.2-14%. On the other hand, the
Enterabacteriaceae show a distribution of 44-60% of the LPS molecules in the low
molecular weight population of 30-50% in the high molecular weight fractions
(37,48). It has been demonstrated that S. typhimurium synthesizes LPS molecules
with over 80 O—antigen repeating units and this population constitutes about 6%
of the total LPS sample (48). Since the hydrophilic O-polysaccharides extend from

bacterial surface into the aqueous environment, the observed heterogeneity of O-

216

chain lengths suggests that the surface topography of the gram-negative bacteria
is irregular and that accessibility of the lipid A head group of the LPS could vary
in different regions on the surface and on different bacterial species. It has been
shown that the presence of O-antigen-containing LPS influences various cell
surface phenomena, including antibiotic binding to LPS (49), antibiotic
susceptibility (2,5,19), LPS aggregate structure (49), bacteriophage recognition
(26,28), immunochemical characterization (9, 10,50), virulence (12,51), protection
against the bactericidal action of serum (22,42), polyclonal B cell activation and
macrophage cytotoxicity (44). The low level of LPS on P. aeruginasa that
contains a long O-polymer, however, may be sufficient to form a uniform cover
over the cell since the surface is inaccessible to rough core specific monoclonal
antibodies (53).

A striking feature of PAOl strains from P. aeruginasa is the presence of
the A bands which constitute a significant amount of the isolated LPS (Table 2).
This set of bands was observed as a slow moving diagonal banding pattern across
the SDS-PAGE of the column fractions (peak X, Figures 2 and 3). LPS from
strain Z61 contained only a single diagonal banding pattern corresponding to the
bands in peaks 2 and 3 (Figure 4). Although SDS-PAGE separates LPS molecules
according to size (27,46,48), we propose that the anomalous migration in SDS-
PAGE of the A bands is due to a difference in the charge in the core-lipid A
region of the molecules. The lack of phosphate substituents in the A band sample

would make the molecules much less negatively charged than the B band fractions '

217

which are high in phosphate groups. To further explore this possibility, we
compared the mobility of the A and B bands of the PAOl strains on an 11% SDS-
PAGE (Figure 5). We observed a difference in the spacing between the A and B
bands, and that the B bands consisted of sets of doublet bands in which one of the
two differ in their staining intensity (Figures 1B and 5), while the A bands seemed
to lack this doublet pattern. This doublet probably represents substoichiometric
modification in the core or lipid A of the B band components. It has been
reported for different gram-negative bacteria that there is microheterogeneity in the
structure of lipid A (phosphate levels and types and numbers of fatty acids)
(3,52,58), in the substituents in the core (15,23,37-39,58), and in modifications
of the O-antigen side chain (37,38,58). The heterogeneity of LPS molecules
presumably depends, in part, on the strain and on growth conditions (14,37).
We have presented several pieces of evidence which indicate that the
molecules represented by the A and B bands are chemically distinct. Only the B
bands from the PA01 strains reacted with anti-O-antigen antibodies in Western
blots (Figure 5). Also, the A bands lacked reactive amino sugars detected in the
B band fractions. Since the pooled fractions corresponding to A bands (peak X)
contained very low levels of reactive KDO unless hydrolyzed with high
concentrations of acid, and no phosphate or amino sugar was detected (Table 2),
there is the possibility that these A bands represent another type of molecule
different from LPS, that is, an O-repeat attached to a molecule which is not lipid

A. In the past 10 years the chemical structure of the lipid A’s from gram-negative

218

bacteria other than the Enterabacteriaceae have been studied, and the existence of
"unusual" lipid A’s has been noted (40). For instance, the lipid A from
Pseudomonas paucimabilis contains a number of sugars, in addition to
glucosamine, in the "bound lipid" fraction, and phosphate as well as KDO appears
to be lacking (40). Also, species of Ihermus have been reported to make LPS that
lacks detectable heptose, KDO, glucosamine, and phosphorus (40). However,
negative reactivity in the thiobarbiturate assay may not reflect the lack of KDO
residues. Recently, Parr and Bryan (47) demonstrated that more rigorous
hydrolysis conditions were required to release KDO from Haemaphilus influenza
LPS compared to other LSP species. Subsequently, Caroff et al. (8) demonstrated
that after treatment with aqueous hydrofluoric acid, the presence of KDO could
readily be demonstrated in LPS of Bortedellas, Bacteroides, Aeromonas, and
Vibrios which had been reported to be KDO-deficient. Thus, we hydrolyzed our
A band sample with a higher acid concentration for longer times and observed a
10-fold increase in the KDO level. This suggests that the A bands are resistant to
hydrolysis due to substitutions of the KDO units in position 4 and 5, or 5 and 7
(7). Other evidence that suggests that the A bands represent LPS molecules is the
ability to silver stain the A band molecules; Kropinski et al. (32) and Lam
(personal communication) reported than the silver staining reaction for P.
aeruginasa LPS occurs in the lipid A rather than the core sugars.

Analysis of the Western and dot blots (Figures 5 and 6) yielded several

additional interesting observations. The pooled fractions containing the higher

219

molecular weight LPS (peaks 1 and 2) from the PAOl derivatives reacted with the
antibody (Figure 6), indicating that the B bands comprise the serotype-specific
LPS. However, P. aeruginasa synthesizes a significant amount of A band type
molecules (Table 2) with presumably a different antigenicity. It has been shown
that S. paratyphi B and S. typhimurium can synthesize a T1 polysaccharide and an
O-polysaccharide attached to the same core (38,42), and that the synthesis of the
two molecules is independent (38). Other P. aeruginasa strains may also have the
ability to synthesize more than one type of LPS with different O—antigenic side
chains (9,31,58,59). The high molecular weight polysaccharide released from LPS
of these strains has been resolved into amino sugar-rich and neutral sugar-rich
fractions when separated by gel permeation chromatography (31,58,59). And
finally, Caroff et al. (7) have shown that Bordetella pertussis produces two
lipopolysaccharides, one of which does not give a positive reaction for KDO under
normal thiobarbituric assay conditions. This is very similar to our findings for the
A and B band-type LPS of the PAC] derivatives.

The effect of the presence of a unique, A band-type of LPS on the physical
interactions within the outer membrane, as well as the immunological reaction with
the cell surface, may be important. It has been proposed that bacteria synthesizing
O-side chains of an unusual structure might escape the immune system of the host,
which might have difficulties in producing effective antibody molecules against
these O-side chains (42,51).

In summary, our data suggest that PAOl strains from P. aeruginasa are

220

capable of synthesizing more than one type of LPS—like molecule differing in their
antigenic reactivities. Although the LPS isolated from the different strains used
in this study was shown to be heterogeneous on SDS-PAGE, this method by itself
does not have the power to predict the presence of more than one type of LPS-like
molecule with a different O-polymer. However, by combining SDS-PAGE with
gel permeation chromatography, Western blots, and sugar analysis, we have been
able to distinguish chemically distinct subclasses of molecules from individual
strains. We have also demonstrated that the percentage of core oligosaccharides

carrying the O-specific polymer is less than 8%.

10.

ll.

12.

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