L

[T

 

P
cm;

LAm

10F

 

W

   

STU

 

 

 

 
 
 
 

 
 

 

 

LA . . , .
. i _ . A. ,, ,
, . .. V 4
. . . .. , u,. .
. A J ,
:3 A . . .1 . _ .

 

 

    

 

 

 

 

gum

 

N6

Li

 

9T3

 

iU

 

 

1

E:
L
VAN

 

A

   

V P13" _
"Duamsn

G

M8

1370’: .
H 3

mo

AN ~

 

1 A .

a . fl —
”flag;

 

 

 

 

 

    

      

 

.z...

 

.J.
. : .... w;
c . 4

“mi; _

                 

   

 

     

      

. .
4 L551. .3 .
3:4...65... 40

     

     

a.........w.x...r._ .r. .r

    

...,.,....

A011,.

                  
     

   
   

  

53%
I'm” 1"

 

   

1.4.;
l . )1 a... .NJJ.
.= J .a .rltazb
.. .2 1.1.1.»; 5.. .

, I

                       

. .4
I
(1)::

1.: .

.p.”m“.3figsm‘ .n.
35:)“. 2......
£11!

     

quqxvz

        

. .I.
V 4r ’ .1?!» 3/ (I
) far-5...; . In... . .

J .h‘ra, linolirlflfo I

 

   

    

r...

    

, . l . f:
. , I . III! 3...!
Primrl.7

        

 
   

 

.21..
.r... , (Ire-L3.
. .3. r. 1!. . .
51 r r r

if; 1(J: .-
n.lry\.l.. I)
. n ‘ A. o...’

   

 

        

Riv:

    
 
 

WWf'f} ‘

LIBRAR Y ‘
Michigan State J

University

  

This is to certify that the

thesis entitled

Comparative Study of Protein Fractions and
Enzymes in the Culture Filtrates of
Hist0plasma capsulatum and Histo-

plasma duboisii
presented by

 

 

Parimondh Khanjanasthiti

has been accepted towards fulfillment

of the requirements for
Medical
EhD degree in Wong)

 

A.» viz/(19%“z,

Major professor
Everett 8. Beneke

Date June 16, 1971

 

0.7639

ABSTRACT
COMPARATIVE STUDY OF PROTEIN FRACTIONS AND ENZYMES

IN THE CULTURE FILTRATES OF HISTOPLASMA CAPSULATUM
DARLING AND HISTOPLASMA DUBOISII VANBREUSEGHEM

BY

Parimondh Khanjanasthiti

The purpose of this research was to study the

taxonomic relationship between Histoplasma cgpsulatum

 

Darling, 1906, and E. duboisii Vanbreuseghem 1952,

based on an investigation of the protein fractions and
certain enzymes in the culture filtrates. This informa-
tion may be useful in a more definitive classification
of the etiological agent of the large-form of histo-
plasmosis, E. duboisii which has been considered by

some as a stable variety of E. capsulatum.

 

Disc electrophoresis was performed on soluble
proteins extracted from culture filtrates after 10, 20,

and 30 days at 25 C and 37 C from E. capsulatum type

 

"A" and "B" and E. duboisii. More fractions were
produced in the older culture filtrates. After 30 days

at 25 C, E. capsulatum type A had 3 fractions; type B,

 

7; and E. duboisii had 5 fractions. The fractions of

type A were common with 3 in B, while 3 of the fractions

 

IIIIIIIIIIIIIIIIII::I____________________________——f7Eggmwm 7
Parimondh Khanjanasthiti

of E. duboisii were common with 3 in B, but different
from A. At 37 C, type A had only 5 fractions, type B
had only 2, both of which were homologous with type A,
while E. duboisii produced 3 fractions, 1 of which was
common for type A and B.

Structural membrane proteins in the cell extract
of the two species grown in CYPG medium at 37 C produced
patterns very similar for 9 strains of E. capsulatum

and 5 strains of E. duboisii. Histoplasma capsulatum

 

type A and type B had almost identical patterns with a
range of 8 to 10 homologous bands.

Seven paranitrophenol substrates were used to
assay the culture filtrates and agar media for extra-
cellular enzyme activities from E. capsulatum and
E. duboisii. The mycelial phase of both species produced
high enzyme activities in PNP-alpha- and PNP—beta-D—
glucoside substrates. Histoplasma capsulatum type A
had 2 to 8 times greater activity in BHI agar than
type B for alpha-and beta—PNPGase. In the yeast phase
only, E. duboisii had high activity for these enzymes.
The kinetics, inhibitors, optimal temperature and pH
were studied for alpha— and beta-PNP—glucosidases

produced by E. duboisii.

IIIIIIIIIIIIII::I_______________________________’——TTTTTTTTT

Parimondh Khanjanasthiti

Histoplasma duboisii produced high PNPralkaline

 

phosphatase activity in CPYG agar at 37C while it was
low for E. capsulatum.

The study of fine structures of young hyphae
from E. duboisii showed similar organelles to those in
E. capsulatum. Woronin bodies were found near the septum
in the hyphae of E. capsulatum but were not found in the
sections made of hyphae of E. duboisii.

Phenol red in a modified CPYG medium differen-
tiated most strains of E. capsulatum from E. duboisii
by a more rapid change in the pH, resulting in a color
change in the medium from yellow to red.

The similarities in enzyme studies and electro—
phoretic protein patterns of a number of strains of the
two species appears to support the proposal that the
large yeast cell form of histoplasmosis should be
considered E. capsulatum Darling, 1906 var. duboisii

(Vanbreuseghem, 1952) Ciferri 1960.

'..'.. ’ «T ’5,‘

 

COMPARATIVE STUDY OF PROTEIN FRACTIONS AND ENZYMES
IN THE CULTURE FILTRATES OF HISTOPLASMA CAPSULATUM

DARLING AND HISTOPLASMA DUBOISII VANBREUSEGHEM

BY

Parimondh Khanjanasthiti

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1971

PLEASE NOTE:

Some Pages have indistinct
print. Filmed as received.

UNIVERSITY MICROFILMS

To my wife Monta
and my son Ton

ii

ACKNOWLEDGMENT

The author wishes to express his sincere apprecia-
tion to Dr. E. S. Beneke and Dr. A. L. Rogers who have
used their efforts to encourage and assist the author
during the research and writing of this thesis and
throughout his graduate program.

Sincere thanks are extended to Dr. R. W. Wilson
who gave advice and materials for the enzyme experiments,
Drs. L. W. Mericle, W. J. Hooker, and G. C. Spink who
kindly instructed and provided for electron microscopy
facilities.

I am indebted to Drs. R. W. Wilson, G. C. Spink,
and W. B. Drew for serving on the Graduate Committee
and for their suggestions.

The author is very grateful to Drs. L. C. Georg,
M. D. Berliner and M. L. Furcolow, M.D. who provided

strains of Histoplasma capsulatum, and E. duboisii.

 

This research was supported in part by the China
Medical Board of New York.

I want to express sincere thanks and appreciation
to the China Medical Board and the Agency for International

Development for financial support.

iii

TABLE OF CONTENTS

DEDICATION . . . . . . . . . . . . . .

ACKNOWLEDGMNT O O O O O O O O O O O O 0

LIST OF TABLES. . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . .
REVIEW OF LITERATURE. . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . .

Disc Electrophoresis of Soluble Protein from
Cultures of Histoplasma capsulatum and
Histoplasma duboisii. . . . . . . . .

 

 

 

Enzyme Activities . . . . . . . .
Comparison of Ultrastructure of Histoplasma
capsulatum and H. duboisii. . . .

 

Attempts to Develop a Differential Medium for
Histoplasma capsulatum and E. duboisii . . .

 

RESULTS . . . . . . . . . . . . . . .

Disc Electrophoresis of Soluble Protein
Extracted from the Culture Filtrates of E.
ggpsulatum Strain G—184 Type "A" and "B" and
E. duboisii MSU Strain . . . . . . . .

 

Enzyme Activ1ties . . . .
Fine Structure in Young Hyphae of E. duboisii
and H. capsulatum. . . . . .

 

Attempts to Develop a Differential Medium
for E. capsulatum and E. duboisii . . . .

 

DISCUSSION . . . . . . . . . . . . . .

Disc Electrophoresis of Soluble Protein in
Culture Filtrate . . . . . . . .

Survey of Enzyme Activities in Liquid and
Solid Culture Media of E. g_psulatum and

H. duboisii. . . . .
A Pbs§TBI§“foferential Medium for

Histoplasma capsulatum and E. duboisii. . .

 

 

iv

Page
ii
iii
vi

viii

16

16
21

3O
31

34

34
46

82
82

89

89

95
102

Page
SUMMARY . . . . . . . . . . . . . . . . 108

BIBLIOGRAPHY . . . . . . . . . . . . . . lll

LIST OF TABLES

Table Page

1. Enzymes in the filtrate of CPYG medium
after 10, 20 and 30 days at 25 C . . . . . 48

2. Enzymes in the filtrate of CPYG medium after
10, 20 and 30 days at 37 C . . . . . . . 49

3. Enzymes in the filtrate of BHI medium with
0.1% cysteine after 10, 20, and 30 days
at 25 C O O O O O O O O O 0 O C O 50

4. Enzymes in the filtrate of BHI medium with
0.1% cysteine after 10, 20 and 30 days
at 37 C . . . . . . . . . . . . . 52

5. Enzymes in the filtrate of Sabouraud medium
after 10, 20 and 30 days at 25 C . . . . . 53

6. Alpha and beta-PNPGases production in the
filtrate during growth of E. duboisii
in CPYG medium at 37 C . . . . . . . . 55

7. Enzyme activities of alpha and beta-PNPGases
produced by H. capsulatum and H. duboisii
in hte CPYG agar plates over a—30 day
period at 37 C . . . . . . . . . . . 57

 

8. Effect of temperature on activity of alpha
and beta-PNPGases produced by E. duboisii
MSU strain . . . . . . . . . . . . 59

9. Effect on pH on activity of alpha and beta-
PNPGases from the filtrate from E. duboisii
MSU strain . . . . . . . . . . . . 61

10. Kinetics of alpha and beta-PNPGases for E.
duboisii MSU strain at 37 C. . . . . . . 63

ll. Inhibition effects of glucose and mannose
on alpha and beta—PNPGases from the culture
filtrate of H. duboisii MSU strain in CPYG
medium at 37_C . . . . . . . . . . . 66

vi

 

 

Table Page

12. Effects of casein and amounts of glucose
on enzyme production of E. duboisii in
CPYG liquid medium in 15 days at 37 C . . . 68

13. Effects of the amounts of glucose and
casein in solid CPYG medium on enzyme
production by H. duboisii in 10 days at
37c..."...........70

14. Effects of different sugars and carbohydrates
in liquid CPYG medium on enzyme productions
by E. duboisii in 15 days at 37 C. . . . . 71

15. The production of alpha and beta-PNPGases in
Sabouraud dextrose agar on additional
strains of H. capsulatum and H. duboisii
for 30 days-incubation at 25 E. ".'—. . . 75

 

16. The distribution of alkaline-PNPPase in
polacrylamide gels from disc electrophoresis
of lyophilize culture filtrates of 3 strains
of E. capsulatum and E. duboisii in CPYG
media for 30 days at 25 C . . . . . . . 77

 

17. Comparison of alpha ane beta-PNPGases and
alkaline PNPPase activities produced by 3
strains of E. capsulatum type "A" and "B"
on BHI agar plates with 0.1% cysteine for
21 days at 25 C . . . . . . . . . . 80

 

18. Activities of alpha and beta-PNPGases and
alkaline-PNPPase of strains of H. capsulatum
and H. duboisii on CPYG agar plEEes for
9 days at 37 C . . . . . . . . . . . 81

 

vii

 

LIS T OF FIGURES
Figure Page

1. Electrophoresis patterns of protein extract
from culture filtrate of H. capsulatum
strain G-184 type "A" and—“B“Iand a strain
of E. duboisii in CPYG medium for 10 days
at 25 C . . . . . . . . . . . . . 35

 

2. Electrophoretic patterns of protein extract
from culture filtrate of H. capsulatum
type "A", "B" and H. duboISii In CPYG medium
for 20 days at 25 E‘ . . J . . . . . . 36

3. Electrophoretic patterns of protein extract
from culture filtrate of H. capsulatum type
"A", "B" and H. duboisii In CPYG med1um for
30 days at 25_C. . . . . . . . . . . 38

4. Electrophoretic patterns of protein extract
from culture filtrates of E. capsulatum
type "A", type "B" and H. duboisii in CPYG
medium for 30 days at 37 C . . . . . . . 39

 

 

5. Electrophoretic patterns of protein extract
from yeast cells of 3 isolates of E.
capsulatum and l isolate of H. duboisii
WHen grown on CPYG medium at—37 C. '. . . . 41

 

6. Photographs of electrophoretic patterns of cell
extracts from the yeast phase of additional
strains of E. cgpsulatum and E. duboisii . . 42

 

7a. Diagrams of electrophoretic patterns in
acrylamide gels of protein extracts from
the culture filtrates of additional strains
of E. capsulatum and E. duboisii for 30
days at 25 C. . . . . . . . . . . . 43

 

7b. Photographs of electrophoretic patterns in
acrylamide gels of protein extracts from
the culture filtrates of additional strains
of H. capsulatum and H. duboisii in CPYG
medium for 30 days at_25 C . . . . . . . 44

 

viii

 

 

Figure Page

70. Photographs of electrophoretic patterns in
acrylamide gels of protein extracts from
the culture filtrates of additional strains
of H. capsulatum and H. duboisii in CPYG
medium for 30 days at—25 C . . . . . . . 45

 

 

8. Growth curve and enzyme productions of
Histoplasma duboisii in CPYG medium at
37 C O O O O O O O O O O O O O O 56

 

9. Effects of temperature of alpha and beta-
PNPGases from the culture filtrate of
E. duboisii at 37 C . . . . . . . . . 60

10. Effects of pH on alpha and beta-PNPGases
from the culture filtrate of E. duboisii
at 37 C O O O I O O O O O O O O O 62

 

11. Kinetics of alpha and beta-PNPGase activities
in the culture filtrate of E. duboisii
in CPYG medium at 37 C . . . . . . . . 64

12. Inhibition effects of glucose and mannose
on alpha and beta-PNPGase from the culture
filtrate of H. duboisii in CPYG medium
at 37 c . T . . . . . . . . . . . 67

13a. Diagram of electrophoretic patterns in
acrylamide gels of the lyophilized filtrate
from E. duboisii in CPYG for 30 days at
37 C compared with the band produced by'
alpha and beta-PNPGase activities. . . . . 73

13b. Photograph of electrophoretic patterns in
acrylamide gels of the lyophilized filtrate
from H. duboisii in CPYG medium for 30 days
at 37-C, compared with the band produced

 

by alpha and beta-PNPGases . . . . . . . 74
14. Graphic diagram showing the distribution of
alkaline-PNPPase in polyacrylamide gels. . . 78
15. A segment of a hyphal cell of Histoplasma
duboisii showing three nuclei . . . . . . 83
ix

 

Figure

16.

17.

18.

19.

Page

segment of a hyphal cell of Histoplasma
duboisii showing intracytoplasmic membrane
system near the plasma membrane . . . . . 84

septum in a segment of a hypha of E.
duboisii, Woronin bodies were not found. . . 84

segment of a hyphal cell of Histoplasma
capsulatum showing a septum witfiitwo
Woronin body-like structures . . . . . . 85

 

 

photograph of a purple colony of H. duboisii

MSU strain and a colony of H. cap§u1atum

strain G-184A on a modifiedjmedium for 18

days at 37 C. . . . . . . . . . . . 87

 

 

INTRODUCTION

The relationship of the two species, Histoplasma

 

capsulatum Darling 1906 and E. duboisii Vanbreuseghem

 

1952, have been studied since the latter was described
by Vanbreuseghem (1953). Histoplasma capsulatum, a
parasite of man and animals occurs in many countries

while E. duboisii is apparently restricted to the African

 

continent. In an extensive literature review of the
comparative morphology and immunology of these two
species Ajello (1968) concluded that E. duboisii should

be referred to as E. capsulatum Darling, 1906 var.

 

duboisii (Vanbreuseghem, 1952) Ciferri, 1960.

Many attempts have been made over the last 15
years to differentiate E. duboisii from E. capsulatum,
but all of these have shown few distinct differences.
The only certain way, up to the present time to separate
these two species, is by animal inoculation to demonstrate
the larger yeast form typical of E. duboisii (Blumer and
Kaufman, 1968). Surpisingly, within the same strain

(isolate) of E. capsulatum, great variations were found

 

in the microscopic and macroscopic morphology of the
mycelial phase in a primary subculture from a patient.

Berliner (1968) gave the name type "A" to the organisms

 

that formed white cottony colonies having hruad hyphnv
and few macroconidia and type "B" to tho brownish colonies
having narrow hyphae and numerous tuberculate chlamydospores.

Thus, the species of E. capsulatum itself seems to be

 

composed of a heterogeneous group of variants.

The objectives of this research are to further
study the differences and similarities in E. ggpsulatum
type "A", type "B", and E. duboisii by means of disc
electrophoresis, enzyme assays through the use of electron
microscopy, and by development of a differential medium.

A comparison of the protein pattern and certain extra-
cellular enzymes that are produced by these organisms in
the mycelial and yeast phase may aid in a definitive
separation of E. duboisii from E. capsulatum or support
the proposed combination of E. duboisii into E. capsulatum

Darling 1906 var. duboisii (Vanbreuseghem, 1952) Ciferri,

 

1960.
Any added knowledge concerning the differentiation
of these two closely related organisms would be especially

helpful in a diagnostic laboratory.

REVIEW OF L ITERATURE

Histoplasma capsulatum was first discovered by

 

Darling (1906). This organism, studied in detail by

De Monbreun (1934), Howell (1939), Conant (1941),

Dowding (1948), Pine (1960) and G005 (1964), is a dimorphic
fungus capable of transforming into yeast-like cells,
usually 2—4U in diameter, when grown in animal tissue

or on blood agar medium at 37 C and mycelial form on
Sabouraud glucose medium at 25 C. This fungus is a

human pathogen, the causative agent of histoplasmosis.

The parasitic yeast-like form is found in tissue, while

the mycelial form growing in Sabouraud glucose agar
produces a white cottony colony which later becomes brown
or tan in color. Microscopically, it has septate branch-
ing hyphae, 1.5-2.5u thick. On these hyphae characteristic
tuberculate, thick wall chlamydospores of variable sizes
are produced. Based on microscopic and macroscopic
morphology of the mycelial phase of primary isolates and
their subcultures, Berliner (1968) found that there were
two types of colonies. Type "A" was designated for albino
type colonies, characterized by producing white, cottony
broad aerial hyphae. Macroconidia are smooth and not

numerous while microconidia are smooth or spiny.

 

Diffusible pigment may be seen after the colony is at
least one month old. This type fails to produce spores
on modified Sabouraud agar. In type "B" or brown form,
the colony is flat becoming light tan to dark brown
within a few days. Hyphae are narrow and pigmented with
enormous numbers of tuberculated macroconidia which are
characteristic 0f the species. Rapid development of
diffusible brown pigment may be seen in the agar medium.
The primary colony, from which type "A" and "B" are
derived, is named type "P" for parent which consists of
a mixture of type "A" and "B", and eventually type "A"
overgrows type "B". Several varieties of spore shapes
and sizes were found in type "P". The macroconidia of
both types are usually borne singly on simple or branched
conidiophores but may be in chains or so attached as to
suggest budding. When these three types are converted

to yeast-like phase, they are microscopically and macro—
scopically indistinguishable. The virulence of type "A",
"B" and "P" are different (Daniels, Berliner and Campbell,
1968). When the same amount of yeast-like cells of

type "A", "B" and "P" are injected intravenously into
rabbits, animals receiving type "P" and "B" yeast cells
died within 10 to 14 weeks. Those with type "A" survive

considerably longer or recover.

 

Conant (1941) studied the life cycle offE. capsulatum.

 

He cultured the mycelial form of the fungus in sealed
blood agar tubes at 37 C. After 10-14 days, the yeast-
like form of the fungus was recovered. He also demon—

strated the development of the mycelial form of E. capsulatum

 

from a yeast cell by using the slide culture technique and
incubating the culture at 24 C.

Histoplasma duboisii Vanbreuseghem 1952, a species

 

separated from E. capsulatum by Van breuseghem (1953) is

 

the causative agent of a form of histoplasmosis distinguish-
able from the classical histoplasmosis of Darling by the
relatively large intracellular parasite. This form of the
disease occurs only in parts of tropical Africa, notably
Nigeria, Ghana, the Belgian Congo and the French colonies
of Senegal and Soudan (Duncan, 1958). This species was
studied in detail by Vanbreuseghem (1953) and Duncan (1958).
From their reports, the agent of African histoplasmosis

is a dimorphic fungus, similar to E. capsulatum except

the parasitic yeast form of E. duboisii is larger, measur-
ing 8-12u and occasionally up to lSu in the longer axis.
In the lymph nodes of patients, the cells were ovoid,
measuring lO-l3.5u. They had thick double contoured walls
and contained very large amounts of fat in the form of
single round or smaller globules. Vanbreuseghem (1953)
pointed out that the chlamydospores of E. duboisii were

perfectly round while those of E. capsulatum were sometimes

 

pyriform. This comment was argued by Drouhet (1962) as

he noted that cultures of E. duboisii formed oval and
pyriform as well as spherical tuberculated chlamydospores
on Sabouraud agar. Duncan (1958) found that large
parasitic yeast cells of E. duboisii could only be obtained
five months after inoculation into adult English mice,
while Vanbreuseghem (1953) succeeded in obtaining these
cells in two months after inoculation of guinea pig
testes.

In culture media, Pine 35 3;. (1964) found that
strains of E. duboisii formed yeast cells of two sizes

within a strain. One was similar to E. capsulatum, having

 

a thin wall and containing no lipoidal globule, the
other was larger and tended to develop large, thick-wall
cells with lipid granule(s) or "duboisii form"
(Vanbreuseghem, 1953). They also found that when the
cultures were maintained at a young age by frequent sub-
culturing, the cell population could be classified as

being the same as E. capsulatum.

 

Because of the small size of the yeast-like cells
of the organisms in the parasitic form as well as in
cultures at 37 C, it is not possible with either the
ordinary light or phase contrast microscope to obtain
a precise picture of the cellular components. Edwards E; ii-
(1959) studied the fine structures of the yeast phase of

E. capsulatum and E. duboisii by electron microscopy using

 

90% butyl metacrylate and 10% methyl metacrylate as
embedding material with 1.5% of lucidol as the initiator
at 48°C for 36-40 hours. They reported that the capsule
or slime layer on the outside of the cell wall of both
species does not exist. The cell wall of E. duboisii

is slightly thicker than E. capsulatum, but the cell

 

structures of both species are similar, consisting of
endomembrane systems, the same numbers and forms of
mitochondria and the same nuclear fine structure.
Apparently the first comparison of fine structure of

the mycelial phase has been done by Garrison EE.E£°
(1970). They investigated the ultrastructural changes
during the conversion of the yeast-like cells to mycelial

phase of E. capsulatum and described some fine structure

 

of the developing mycelium or primary hyphae from a

yeast-like cell. They noted among the hyphal fine

structure, intracytoplasmic membrane systems were in
association with glycogen-like bodies. Woronin bodies (Reichel,
1965) were found near the septa. The ultrastructure of micro-

conidia or chlamydospores of E. capsulatum was investi-

 

gated by electron microscopy by Edwards, Hazen and
Edwards (1960). They discovered that the tuberculate
spore is the result of the thickening of the spore wall
while the tubercle is a part of the cell wall formed by
expansion of the outermost layer of the cell wall.

Developing spores, according to Edward EE.§$° (1960)

 

have various organels, endoplasmic reticulum, mitochondria,
nucleus, microdroplets, irregular clumps of lipid and a
cytoplasmic membrane. The mature spores have increasing
amount of lipid, while the mitochondria and reticulum are
invisible and the residual cytoplasmic matrix becomes
homogenously granular.

As previously mentioned, the only way according to
Blumer and Kaufman (1968) to separate E. duboisii from
E. capsulatum is by animal inoculation. After two months
the large yeast forms may be found in animal tissue for
E. duboisii. Contradictary works have been also reported.
Crumrine and Kessel (1931) noticed the large forms of
E. capsulatum in the spleen of a patient. Drouhet and
Schwarz (1956) have shown that large forms measuring 6
to 27p, similar to the organism observed in African human
and animal histoplasmosis were found when 12 American and
3 African strains were inoculated into hamsters. Large yeast
forms could be induced by culturing the organism in
blood agar base at 37C for 1 week as they grew from pieces
of mice which had been inoculated intravenously 3-4
weeks previously with the mycelial phase of E. capsulatum
(Schwarz, 1953). Binford (1955) reported from the
study of 22 fatal cases of disseminated histoplasmosis
and from lesions obtained surgically in non-disseminated
cases that large yeast-like cells of E. capsulatum measur—

ing up to 15p were also found in necrotic tissue.

 

 

Many mycologists have tried to differentiate
E. duboisii from E. capsulatum by biochemcial means but
these organisms vary in their enzymatic reactions and
their nutritional requirements so that no definite
patterns in biochemical reactions can be used to differ—
entiate these two organisms at present. Montemartini
and Cifferi (1953) first reported that E. capsulatum
utilized histidine and ammonium sulphate as nitrogen
sources but E. duboisii did not. They used only a
single strain from the two species, so their results
need to be confirmed. Coremans (1963) reported that
E. capsulatum produced urease in 24-48 hours while
E. duboisii failed to do so after 48 hours, however,
this could not be confirmed by Rosenthal and Sokolsky
(1965) but they found that E. capsulatum hydrolyzed
tyrosine, not gelatin while E. duboisii hydrolyzed gelatin
but not tyrosine. These findings were more significant
when Berliner (1967) confirmed that the mycelium of
E. duboisii liquified 15% plain gelatin in 24-96 hours
whereas E. capsulatum did not in 6 weeks. She used 70
strains of E. capsulatum from various sources and 7
strains of E. duboisii. Blumer and Kaufman (1968) could
not confirm the work of Berliner nor all previous reports
when they used 10 strains of E. capsulatum

and E. duboisii. Nine of ten strains of E. duboisii

 

hydrolyzed 0.4% gelatin within 3 weeks, 6 strains hydrolyzed
15% gelatin in 96 hours, however, 5 strains of E. capsulatum
were able to hydrolyze 0.4% gelatin and all 10 strains
hydrolyzed 15% gelatin. Only 7 strains of E. capsulatum
hydrolyzed tyrosine but 3 strains of E. duboisii did too.
Three strains of E. duboisii were also found to produce
urease within 48 hours on the solid media.

Although the two organisms are almost indistin-
guishable £2 KEEEQ' striking difference in their clinical
manifestations were pointed out by Olurin,

Lucas and Oyediran (1969). They found E. capsulatum
infections may cause (1) acute pulmonary and disseminated
disease which is frequently fatal, or (2) a benign sub—
clinical form leading to pulmonary calcification and a

positive histoplasmin skin test. Histoplasma duboisii

 

infections cause either (1) a localized form with solitary
skin nodule, (2) bone lesion, or (3) a disseminated form
involving skin, subcutaneous tissues, lymph nodes, bones,
joints and abdominal viscera, but rarely lungs.

Most of the previous biochemical investigations
included studies on production of enzymes by E. capsulatum
and E. duboisii were detected by indicators such as
phenol red for the detection of pH change in media,
(urease test; sugar fermentation media), and hydrolysis

of the media (gelatin liquefaction,casein hydrolysis and

 

 

11

etc.). Enzyme production in fungi has been found to

be affected by nutritional and environmental factors
(Wilson and Niederpruem, 1967). Since strains of

E. capsulatum differ in Vitamin and amino acid require-
ments (Rowley and Pine, 1955), enzyme production should

be studied in a medium rich in vitamins and amino acids.
Beneke, Wilson and Rogers (1969) used both liquid and solid
casein-peptone-yeast extract—glucose (2.0 g, 2.0 g, 2.0 g
and 5.0 g respectively/500 ml. water) media for study of

enzymatic activities of Blastomyces dermatitidis. After

 

5, 10, and 15 days of incubation, the liquid filtrate and

agar medium of the yeast and mycelial phases were assayed for

enzymatic activities. High activities of paranitrophenyl

acid and alkaline phosphatases were found in both media

for the yeast phase in comparison to the mycelial phase.
Regarding the antigenic structures of E. capsulatum

and E. duboisii, Drouhet (1967) studied the antigenic

structures of these two organisms. He used antigens

prepared from lyophilized histoplasmin and obtained

the sera from rabbits hyperimmunized with

 

 

 

12

ground mycelium. By the use of agar double diffusion

technique, he found at least four common fractions in the

two types of histoplasma and two fractions specific for

E. capsulatum. With immunoelectrophoresis, the serum

from ground mycelium hyperimmunized rabbit showed at

least eight antigenic fractions in the histOplasmin of

E. capsulatum and five or six fractions in the histoplasmin

of E. duboisii. Two specific fractions of E. capsulatum

were also demonstrated by exhausting the immune serum

with heterologous antigens. Andrieu et al.(l969) also

applied agar gel diffusion and immunoelectrophoresis in

their comparative study of E. capsulatum and E. duboisii,

but the results were not identical with that of

Drouhet. They concluded that antigenic structures of

these two organisms were the same, at least qualitatively.
Kaufman and Blumer (1961) studied extensively

the antigenic structures of 56 strains of E. capsulatum

and 11 strains of E. duboisii by fluorescent antibody

specific for the yeast phase of E. capsulatum by means

of adsorbed labeled antisera with cells of known antigenic

structures and staining titers. All 56 strains of

E. capsulatum tested were subdivided into 5 serotypes

represented by the combination of numbers 1, 2; 1, 4;

1, 2, 3; 1, 2, 4; 1, 2, 3, 4. One serotype 1,4 was

closely related antigenically to E. duboisii and

Blastomyges dermatitidis. These discoveries later

 

 

 

tn“ CI;
13

resulted in the development by Kaufman and Blumer (1968)
of a polyvalent conjugate to differentiate Histoplasma
from other organisms but this conjugate failed to
separate E. duboisii from E. capsulatum.

When Pine (1970) analysed the yeast phase cell
wall amino acids of E. capsulatum serotype 1,4 and
E. duboisii, he found that the relative molar concentra-
tions of the individual amino acids were similar with
a higher threonine content in E. duboisii. He also
discovered that the amino acids in the mycelial cell
walls of the two types of Histoplasma were not signifi-
cantly different.

Leone (1964) has reported that protein electro—
phoresis can be used as a valuable tool for the study
of phylogenetic relationships, as protein structure is
primarily an expression of the genotype of an organism.
Since species may be determined by their genotypes,
the study of proteins by electrophoresis is important
in identifying of the species. Characteristic of
electrophoretic mobilities which can be demonstrated
by protein staining is unique and reproducible for a
particular species. Disc electrophoresis (Ornstein
and Davis, 1962) is the sensitive technique used to
separate protein components by means of a charge and
molecular sieving. It is a highly reproducible method
for the study of proteins (Whipple, 1966). This

technique has been applied to taxonomic studies of

 

 

14

several microorganisms. Chang 3; EL. (1962) demonstrated
the difference among species of Neurospora by electro-
phoretic fractions of soluble protein extracted from
mycelium by disc electrophoresis on polyacrylamide gels.
They also showed that a mutant strain of E. crassa
possessed a number of slow-moving bands not present in
the pattern of the wild type strain used. A similar
report by Clare (1963) has demonstrated that Pythium
species have the same protein pattern even when isolated
from different locations. Durbin (1966) in his investi-
gation of electrophoretic patterns of many isolates of
Septoria, found that the majority of the patterns were
similar among strains in the same species although
variation in patterns within each species also existed.
The complexity of dermatophytes has been clarified by
Shechter g3 il' (1966) by the application of disc
electrophoresis of the proteins extracted from mycelial
mats or culture filtrate; each species has specific
electrophoretic pattern and has common fractions in

the same genus. Furthermore, Shechter, Landau and
Newcomer (1967) found that culture filtrates of

Coccidioides immitis from different media or the same

 

media at different incubation periods yielded different
protein patterns by disc electrophoresis. Thus, if
E. capsulatum and E. duboisii are grown in the same

media with the same amount of inocula, and under the

 

 

15

same environmental conditions, the culture filtrates of
both organisms should yield characteristic protein
patterns by disc electrophoresis.

Recently, Rottem and Razin (1967) proposed that
electrophoretic patterns of membrane proteins be used

as finger prints of Mycoplasma and later, they

 

recommended the same method he used to identify other
microorganisms (Razin and Rottem, 1967). Since the
phenol-acetic acid-water was found to be very active in
solubilizing the structural membrane proteins of the
cells (Takayama g3 §$°r 1966), it might be used to

dissolve membrane proteins of Histoplasma and the

 

protein solution could be subjected to disc electro-
phoresis. Since it is believed that the synthesis of
membrane proteins is directed in part by heredity (Korn, 1966),
the electrophoretic patterns of membrane proteins of

the two types of Histoplasma may reflect phylogenetic

 

relationships.

 

 

MATERIALS AND METHODS

Disc Electrophoresis of Soluble Protein
from Cultures of Histoplasma capsulatum
and Histoplasma duboisii

 

Extraction of Soluble Protein

 

Histoplasma capsulatum strain 184 G type "A"
and type "B" obtained from Berliner, Harvard University,
and Histoplasma duboisii from the M.S.U. stock culture
collection were grown in 250 m1 Erlenmeyer flasks with
100 mls of liquid medium consisting of 0.4 gm. each of
casein, neopeptone, yeast extract and 1.0 gm. of glucose
(CPYG medium). The inoculum consisted of approximately
1800x106 yeast cells per flask from a 6 day culture
which was growing on solid medium of the same composition.
Each organism was cultured in triplicate and incubated
at 25 C and at 37 C on rotary shakers at 160 rpm. At the
end of 10, 20, and 30 days of incubation, one culture from
each group of flasks was checked microscopically for
contamination and then filtered through seitz sterilizing
filters. Two volumes of cold acetone were added gradually
into one volume of culture filtrate and uninoculated broth,
and stirred gently. The acetone broth filtrate mixtures were
left for several hours in the cold room. Protein precipitate

was then removed by centrifugation at 18,000 g for 15

16

 

17

minutes and redissolved in distilled water. Protein
determinations of the solutions were performed by
Lowry's method (Lowry, g5 EE. 1951). The protein extracts

were kept frozen at -20 C for periods up to 4 weeks.

Disc Electrophoresis Procedure

 

The method for disc electrophoreses as described
by Davis (1964) was adopted. Polyacrylamide gel columns
were prepared in 5 x 100 mm. cylindrical glass tubes.

The gels were composed of two layers. The upper layer
having the total length of 1 cm, was the large pore gel
consisting of 2.8% acrylamide, N,N,N',N', tetramethylethyle—
nediamine, N,N' methylenebisacrylamide, riboflavin, Tris-
HCl and water. The lower layer with a total length

of 6.5 cm. was the small pore gel in which electrophoretic
separation took place. It consisted of 7.5% acrylamide,
N,N,N',N', tetramethylethylenediamine, N,N' methylene
bisacrylamide, ammoniumpersulfate and buffering systems.

For convenience, 6 stock solutions had been previously

prepared as follows:

Stock A Stock B
l N PKH.48 m1. 1'N HCl approximately 48 ml.
TRIS 36.6 g. TRIS 5.98 g.
TEMED 0.23 ml TEMED 0.46 ml
Water to 100 ml Water to 100 ml

pH 8.9 pH 6.7

Stock C Stock D
Acrylamide 28.0 g Acrylamide 10.0 g
BIS 0.735 g BIS 2.5 g

Water to 100 m1 Water to 100 ml

 

 

18

Stock E Stock F
Riboflavin 4 mg Sucrose 40 g
Water to 100 ml Water to 100 ml

For the preparation of the lower gel, a solution
of 1 part A,2 parts C,l part water, 4 parts small pore
solution (0.15 g ammonium persulfate in 100 ml water)
was made and then poured into an upright cylindrical
glass tube by a capillary pipette up to 6.5 cm high.
Before polymerization, the upper top surface was flattened
by a thin layer of water. The solution of the upper gel
was a mixture of 1 part B, 2 parts D, 1 part E and 3
parts F. It was layered 1 cm high over the lower gel
after the water had been drained away.

For operation of electrophoresis, 300-400u g of
soluble protein extract was dissolved in 0.2 ml of 40%
sucrose solution and carefully layered on top of the large
pore gel. The acrylamide columns connected the upper
and lower jars which were filled with tris-glycine buffer
at pH 8.6 Direct current of 200—350 volt and 5 milliamperes
per gel were connected with the cathode on the upper jar
and the anode at the lower jar. Both electrodes were
immersed in the tris-glycine buffer. In the upper buffer,
a few crystals of bromphenyl blue were dissolved as
tracking dye. The protein samples were concentrated in
tine upper gels; moved and separated in the lower gels.
Thee front boundary, as seen by tracking dye, was allowed

to lnove down for 5 cm from the upper gel. The gels were

 

 

l9

removed from the glass tubes and cut away at the tracking
dye. They were stained by Buffalo Black (0.5% in 7%
acetic acid) for one hour and destained by submerging in

7% acetic acid overnight.

Disc Electrophoresis of Protein
Extract from the Culture Filtrates
of the Mycelial Phase of Additional
Strains of H. capsulatum and

H. duboisii

 

After 3 to 4 weeks of growth on Sabouraud dextrose
agar at 25 C, mycelial mass and spores of each strain
were scraped from an agar slant into 4 ml of sterile
physiological saline. Four ml were inoculated to 100 ml
of CPYG broth in 250 m1 flask and incubated on a rotary
shaker for 30 days at 25 C. After checking for contamina-
tion, microscopically and culturally, the liquid medium
was filtered through seitz filters. Protein extraction,
protein determination and disc electrophoresis was performed
as previously described.
Disc Electrophoresis of

Phenol Acetic Acid
Water Cell Extracts

 

 

 

Ten day-old cultures of Histoplasma capsulatum

 

 

G—184 type "A" and "B", and E. duboisii grown on CPYG
agar slants at 37 C were scraped into physiological
saline, centrifuged and washed with saline solution again.
One milliliter of phenol—acetic acid-water (2:1:0.5 w/v/v)
was added to 150 mg of wet weight of the organism. After
the mixtures were left overnight at 4 C then, the

insoluble materials were removed by centrifugation at

 

 

20

30,000 g for 15 minutes. One tenth of the clear super-
natant was mixed with 0.05 ml of a 40% (w/v) sucrose
solution in 35% (v/v) acetic acid. The sample sucrose
mixtures were layered on top of gels for electrophoresis.
The same procedure was used for 10 day-old yeast
phase broth cultures of different strains of E. duboisii

and E. capsulatum. A modification of Razin and Rottem

 

(1967) method for the acrylamide gel was made. In
preparation of the gels, stock solution of A and B

were made as follows:

Solution A Solution B
Acrylamide gel 3.5 g Urea 6 g
Urea 6 g Ammonium persulfate 0.15 g
N,N'methylbisacrylamide 0.08 g Water to 10 ml

47% acetic acid V/V to 30 m1
Three volumes of solution A were mixed with one volume
of solution B, then 0.02 volume of N,N,N'N' tetramethyl-
ethylenediamine was added to the solution. The final
mixture was put into 100 x 5 mm cylindrical glass tubes
and overlayered with 75% (v/v) acetic acid. Polymeriza-
tion of acrylamide gel was carried out at 37 C for
45 minutes.

After the sample sucrose mixtures had been put
on top of the gels, 0.5 m1 of a 75% acetic acid was
carefully layered on tOp. Both upper and lower jars of

the electrophoretic apparatus were filled with 10% (v/V)

 

 

 

21

acetic acid. The lower electrode was cathode with
direct current of 5 milliamperes per tube. The front
boundary was allowed to move down for 5.5 cm with
approximately 70 minutes of running time. The gels were
taken out of the glass tubes and stained with 0.5%

(w/v) Buffalo Black in 7% acetic acid. After 1 hour of
staining, the gels were destained by submerging in 7%

acid (V/v) overnight.

Enzyme Activities

 

Survey for Enzymatic Activities in
Three Kinds of Liquid Media at
37°C and 25°C

 

 

 

Yeast phases of Histoplasma duboisii and

 

E. capsulatum strain G-184 type "A" and "B" in CPYG

 

agar slants were transferred to 100 ml of each of the
following: Sabouraud dextrose broth; brain heart
infusion with 0.1% cysteine; and CPYG broth. Each
6

inoculum consisted of 2 m1 of approximately 900 x 10

cells per ml. One set of cultures in 250 m1 flasks was

 

22

incubated on a rotary shaker at 37 C, the other, at 25 C.
Cultures in Sabouraud dextrose broth were incubated at

25 C, only. At the end of 10, 20, and 30 days of incuba-
tion a 10 ml portion from each culture was filtered through
a seitz sterilizing filter. The culture filtrates were
used as the enzyme sources to assay for enzymatic
activities. The following chemicals were used as substrates:
paranitrophenol—alpha D-glucopyranoside (1.0 mg/ml in
sodium acetate buffer, pH 5.4), PNP-beta-D-glucopyranoside
(1.0 mg/ml in sodium acetate buffer, pH 5.4), PNP-alpha-D—
galactoside (1.0 mg/ml in sodium citrate-phosphate buffer,
pH 7.0), PNP-beta-D-galactoside (1.0 mg/ml in citrate-
phosphate buffer, pH 7.0, PNP-N-acetyl-beta-glucosaminide
(1.0 mg/ml in sodium acetate buffer, pH 5.4), PNP—beta-D-
glucuronide (1.0 mg/ml in sodium acetate buffer, pH 4.5),
PNP—phosphate (1.0 mg/ml in Tris-HC1 buffer pH 8.6 for
alkaline phosphatase, and in sodium acetate buffer, pH 5.2
for acid phosphatase). The concentration of each buffer
was 0.1 M. Nine-tenth ml of each substrate solution was trans-
ferred into test tubes, 0.2 ml of culture filtrate was
added into each tube of substrates, mixed well and
incubated at 37 C for two hours. Enzymatic reactions

were stopped by adding 2.0 ml of 1.0 M Tris pH 9.8.

The activity of the enzymes was proportional to the
){ellow color of paranitrophenol released from the sub-

EStrates which was measured with a spectrophotometer at

 

23

410 mu (Turner, model 330). Enzymatic activities were
expressed as optical density per milliliter of broth
filtrate per one hour and multiplied by 1000.

PNP-alpha and beta glucosidase were found to be
greatest in the CPYG culture filtrate of E. duboisii
at 37 C. On this basis, filtrates of cultures between
10 to 30 days of incubation were used to investigate
some characteristics of these enzymes.

Some Characteristics of
PNP-Alpha and Beta Glucosidase

 

 

Enzyme Production in Relation to the Age of

Culture in Liquid Media at 37 C.--A ten day-old culture

 

of E. duboisii in liquid CPYG at 37 C containing approxi-
mately 829 x 106 cells per ml was used as inoculum.
Two mls of this inoculatum were inoculated into 100 ml
of the same medium in 250 ml flasks and incubated on
a rotary shaker at 37 C. Each day over a ten day
period, total cell counts and enzymatic activities for
PNP-alpha and beta glucosidase were determined and
recorded. This was continued periodically over the
entire 30 day period.

Enzyme Production in Relation to the Age of

Culture on Solid Medium.--Six day-old cultures of the

 

E. duboisii growing on CPYG agar plates were used as

the inocula. The organism was heavily streaked in

a 9 square cm area on CPYG agar and incubated at 37 C.

 

24

Small plugs of agar medium near the edges of the growing
colonies were removed for testing enzyme activity. A
cork borer was used to insure that constant amounts of
medium were taken for each sample and 0.9 m1 of substrate
was added per plug of agar. The enzymatic reaction was
stopped by additional 2 ml of 1.0 M Tris after 1 hour of
incubation at 37 C. Enzymatic activities of the organisms
were recorded at various times during the 30 day incuba-
tion period.

Optimal Temperature and Optimal pH.--Nine tenth

 

ml of PNP-alpha and beta glucopyranoside in sodium acetate
buffer pH 5.4 and 0.1 ml of broth filtrate were mixed

and incubated at 4 C, 25 C, 59 C, 64 C, 69 C and

75 C for 1 hour. Activities were recorded. Enzyme
activities were also checked after 3 minutes in the
steamer.

Substrate (PNP-alpha and beta glucopyranoside)
were dissolved in Tris-citrate-phosphate buffer (1 mg/ml)
in ranges of pH from 3.0 to 9.0. From a preliminary
test, the optimal pH of both enzymes from E. duboisii for
the highest activities were between pH 4.0 to 5.0.

For a more critical determination of pH, the buffers
were adjusted to the following pH values: 4.2; 4.4; 4.6;
4.8; 5.0 and 5.3. Substrates were dissolved in buffer
(1 mg/ml) at each pH, and 0.1 m1 of broth filtrate for

each 0.9 ml substrate for the various pH values was

25

used for assay. The enzyme activities at the different
pH readings were recorded.

Effect of Storage at -20 C.--Enzyme activities

 

were first investigated on the filtrate before storing
in the freezer. After a period of 90 days of storage
in the freezer at -20 C, the enzyme activities were
rechecked. The assay method was the same as previously
described.

Investigation of the Kinetics.--A similar method

 

of enzyme assay for PNP-alpha and beta glucosidase was
used with varied times of incubation at 0, 5, 10, 15, 25,
40, 60, 90, 212 minutes. The rise of enzyme activities
in comparison to the time factor were plotted graphically.

Inhibition Test.--The following sugars:

 

glucose, mannose, galactose and d-levulose were tested

as possible inhibitors of PNP—a1pha-and-beta-glucosidase

in the concentration of 0.46 M, 0.09 M, and 0.04 M.

The substrate concentration was 1 mg/ml in sodium acetate
buffer pH 5.4. One tenth m1 of broth filtrate from

E. duboisii was used as the enzyme source. The reaction

temperature was 37 C for 1 hour. The inhibiting effects

were calculated as percentage activity over control

(water was used as a control to replace inhibitors).

26

Effects on Enzyme Production due to the Modifica-

tions in Liquid CPYG Medium.--

 

a. Casein and the amount of glucose.

 

The glucose in the CPYG medium was varied in
amounts from 0, 0.5%, 1.0%, 1.5% to 2.0%. Another
test medium was CPYG, but vitamin free casein

was replaced by casamino acids in the same pro-
portion. Two ml of a 10 day-old culture of E.
duboisii in CPYG broth (casein was replaced by
casamino acids) having approximately 828.7 x

106 cells/ml was inoculated into 250 m1 flasks
containing 100 ml of test medium and incubated

on a rotary shaker at 37 C. After 15 days, all
cultures in the test medium were filtered through
seitz sterilizing filter. The broth filtrate

of each culture was assayed for activities of

PNP-alpha and beta glucosidases by the same

method as previously described.

b. Sugars and Carbohydrates.

 

The glucose in CYPG medium was replaced by

either d-lactose, d-cellobiose, soluble starch,

or dextrin, in the same amount by weight. Two

ml of a 4 day-old culture of E. duboisii in CPY
broth containing approximately 60.4 x 106 cells/m1
was inoculated into 100 m1 of the test media in

250 ml flasks and incubated on a rotary shaker at

27

37 C for 15 days. Activities of PNP-alpha and beta

glucosidasefi were assayed by the method given above.
Identification of PNP-alpha and Beta-glucosidases

Fraction of Lyophilized Culture Filtrates of H. duboisii

in CPYG Broth at 37 C by Disc Electrophoresis.--Histop1asma

 

duboisii was cultured for 30 days in 100 ml of CPYG broth
inoculated with 1 ml of a cell suspension containing
approximately 900 x 106 cells ml from a six day old
culture on CPYG agar. The broth filtrate was lyophilized
and used as the protein source for disc electrophoresis.
Six milligrams of lyophilized material was dissolved in
0.2 ml of 40% (W/V) sucrose solution and put on top of
the large pore gels. Electrophoresis of this material
was performed in triplicate. One of the gels was stained
with Buffalo Black for 1 hour and destained in 7% acetic
acid electrophoretically. While the first gel was
staining, the other two were put in test tubes, one
containing PNP-a1pha-D-glucopyranoside the other con-
taining PNP-beta-D-glucopyranoside, for 1 hour. The
substrates were replaced by 1.0 M Tris. The yellow

bands of paranitrophenol were visible immediately,
indicating the location of PNP—alpha and beta glucosidase.
The position of the yellow bands were compared with

the homologous bands that stained with Buffalo Black.

 

28

Enzymatic Activities of PNP-alpha and Beta

 

Glucosidases in the Mycelial Phase of Other Isolates of

Histoplasma Capsulatum and H. duboisii.-—Two more isolates

 

of E. duboisii strain E 937 and B 939 and three isolates

of E. capsulatum strain B 621 from soil, B 293 from human

 

lung, B 815 from dog, were obtained from Dr. B. Georg,
Communication Disease Center, Atlanta Ga. Four isolates

of_E. capsulatum strain 186—G type "A" and "B", strain

 

219 type "A" and "B" were obtained from Berliner,

Harvard University. Three isolates of E. capsulatum

 

strain D—213, 68-46 and 488 were obtained from Dr. Furcolow,
University of Kentucky. All isolates were cultured on
Sabouraud dextrose agar in plates at room temperature
(25 C). After 30 days, a portion of the agar medium
near the edge of the colonies was assayed for PNP-alpha
and beta glucosidase using the agar disc method as
previously described.

The Distribution of PNP-alkaline

Phosphatases in Polyacrylamide Gels

from Disc Electrophoresis of

Eyophilized Culture Filtrate

from Strains of H. capsulatum

and H. duboisii in CPYG Broth
at 25 C

 

 

 

 

 

 

Six milligrams per gel of lyophilized culture

filtrate of E. capsulatum strain 184 G type B, 186 G

 

type B, 219 type B and E. duboisii MSU strain, B 937,
B 939 were fractionated by disc electrophoresis as

previously described. The gels were then, cut into

 

29

sections 0.5 cm 10ng and assayed for alk—PNPPase activity
in each section. One ml of 1 mg/ml of PNP-phosphate in
Tris-HCl buffer at pH 8.6 was added to each section of
the gel and incubated at 37 C for two hours. Reactions
were stopped by adding 2.5 ml of a 1.0 M Tris. The
activities were measured by a spectrophotometer (Turner,

Model 330) at 410 mu.

A Study of Alpha and Beta-PNPGases
andFAlkaline-PNPPase‘1n HistopIasma

Capsulatum Type "A" and "B"

 

 

 

 

Three strains of E. capsulatum type "A" and

 

"B" were grown on BHI agar with 0.1% cysteine and
incubated at room temperature (25 C) for 20 days. A
portion of agar medium near the edge of the colony of
each culture was taken with a cork borer of 0.5 cm in
diameter to use as the enzyme sources. Nine tenth ml
from each of the substrates (PNP-alpha and beta-D
glucopyranosides 1 mg/ml in 0.05 M acetate buffer pH 5.4
and PNP-alkaline phosphate 1 mg/ml in 0.1 M Tris-HCL
buffer pH 8.6) was added to a plug of agar medium to
detect enzyme activity in solid medium cultures. The
procedure continued the same as previous enzyme

experiments.

 

30

Study of Alpha and Beta-PNPGases

and Alkaline-PNPPase ActiviEIes in Five
Strains of H. capsulatum and

H. duboisiI'In CPYG Agar PIates

for 9 days at 37 C

 

 

 

 

In order to confirm the differences in the three
enzyme productions on CPYG agar plates at 37 C of these
two organisms. Five strains of the yeast phase of E.

capsulatum strain B 913, B 329, 105, MSU, 222A and

 

E. duboisii strain B 650, B 651, B 652, B 939, MSU were
grown in CPYG agar plates to study the enzymes in the
agar medium. The method was the same as previously

described.

Comparison of Ultrastructure
of Histoplasma Cgpsulatum and H. duboisii

 

 

Histoplasma capsulatum and E. duboisii were

 

 

grown in 100 ml Sabouraud dextrose broth in 250 m1 flasks
on a rotary shaker at room temperature (25 C) for ten
days. The small pellets of mycelium were filtered out
and washed with phosphate buffer pH 7.4 and fixed with

3% glutaraldehyde in phosphate buffer pH 7.4 for 2 hours.
Then, the glutaraldehyde was drained away, the pellets
were washed 3 times with phosphate buffer. The

pellets were later postfixed with 1% osmium tetroxide

in phosphate buffer for 1 hour. The specimens were
dehydrated with 25%, 50%, 75%, 95% ethyl alcohol for

10 minutes each and 100% ethyl alcohol for 20 minutes

twice. After dehydration, propylene oxide was added to

31

the specimens twice for 30 minutes each. Then, propylene
oxide was replaced by propylene oxide plus EPON mixture
(7 parts A and 3 parts B) with the proportion of lv/lv

and left overnight.

EPON Mixture A EPON Mixture B
EPON 812 62 m1 EPON 812 100 ml
Dodecenyl succinic Methyl nadic
anhydride 100 m1 anhydride 89 m1

Then the specimens were ready to embed in EPON
complete mixture. Bean capsules were filled with EPON
mixture first, then the specimens that had been submerged
in EPON mixture plus propylene oxide overnight were
picked up by a capillary pipette, released and pushed into
the bottom of the capsules. The capsules with specimens
at the bottoms were left overnight in a dissicator for
complete penetration, then, put in 60 C oven for 2—3 days
for hardening. Sections were cut on a Reichert ultra-
microtome, mounted on 400 mesh copper screens, 3.0 mm in dia-
meter, and observed in the Zeiss EM9A transmission electron

microscope.

Attempts to Develop a
Differential Medium for Histoplasma
Capsulatum and H. duboisii

As a result of the investigation of the effect of
nutritional factors on the activity of alpha and beta-
PNP-glucosidase a modification of CPYG medium was made
in an attempt to demonstrate the difference in color

reaction in the colonies of E. capsulatum and E. duboisii.

 

Three sets of CPYG agar media were prepared. The first

32

set of media contained two different concentrations of

ferrous sulfate (0.2 g/l and 0.4 g/l). The second set
contained two concentrations of the combination of ferrous
sulfate and sodium thiosulfate (0.2 g/l and 0.4 g/l;

0.4 g/l and 0.5 g/l respectively). The third set contained
one concentration of the combination of ferrous sulfate
and cysteine (0.4 g/l and 0.5 g/l respectively). In

the same way, three sets of modified BHI agar were pre—

pared. All media were sterilized by autoclaving for

 

15 minutes at 121 C under pressure of 15 pounds/square
inch. Ten day old cultures of 7 strains of E. capsulatum,
G-184 A, G-l84 B, G-186 A, G-186 B, 222 A, B 621, B 875
and 1 strain of E. duboisii in CPYG agar were inoculated
heavily on these media and incubated at 37 C. The
colonies of both species on each plate were observed
periodically for the difference in color. Later, 4
additional strains of E. duboisii were used.

An indicator medium was prepared to observe the
changes in the pH in the medium during the growth of

E. cgpsulatum and E. duboisii strains for possible differ—

 

entiatflxiof the species. The medium had the following
composition: casaminoacids; neopeptone, 2 g/l; yeast
extract, 2 g/l; cellobiose, 10 g/l; phenol red, 0.05 g/l.
The pH was adjusted to 5.1 with HCl. Histoplasma capsulatum
strains 488, B—621, B-875, B-293, and E. duboisii strain
B-937, B-938, B—939, and MSU were used. All cultures

were inoculated with mycelial phase, and grown on slants

33

at 25 C. The color changes from yellow to red were noted

periodically over a 9 day period.

 

RESULTS

Disc Electrophoresis of Soluble Protein
Extracted ffom the Culture Filtrates
of H. capsulatum Strain G-184 TypeIwA"

and "B" and H. duboisii MSU Strain

 

 

 

 

Comparison of electrophoresis patterns of soluble
protein from the broth filtrates of a 10 day culture at

25 C is shown in Figure 1. Only one clear band at rf 43.63

 

was demonstrable in E. capsulatum type "A" and "B". Histo-

plasma duboisii MSU strain had 2 fractions, one with the same

 

rf as in type "A" and "B", the other had one at rf 21.81.
All three organisms produced good mycelial growth in the

culture medium. After 20 days in culture, E. capsulatum

 

type "A" still produced only 1 fraction as in the 10
day culture but type "B" produced 8 fractions, 4 of which
were very faint, located at higher rfs (60.0, 65.45,

74.54, 80.0). HistOplasma duboisii produced 4 fractions

 

(23.63, 41.81, 45.45, 65.45). All of these are common

to E. capsulatum type "B". Comparative electrophoretic

 

patterns for 20 day culture is illustrated in Figure 2.
In 30 day cultures, the protein fractions were most
clearly separated. Two more light bands (rf 40.00,

54.54) were visible in E. capsulatum type "A". Seven

 

fractions were produced in E. capsulatum type "B".

 

34

 

35

 

3‘ , B “Wow“
—_—W ___1
451.) __— use: —— 4305 ——

 

 

 

 

 

 

 

 

 

Duboisii

FIGURE l.—-Electrophoresis patterns of protein extract
from culture filtrate of E. capsulatum strain
G-184 type "A" and "B" and a strain of H. duboisii
in CPYG medium for 10 days at 25 C. —

“1.11
1’“,

 

 

 

 

“I“
4555

513‘

0635

"45‘
800

D u\o\s\\

fl

 

 

 

 

Duboisii

FIGURE 2.——E1ectrophoretic patterns of protein extract
from culture filtrate of H. ca Sulatum type "A"
type "B" and E. duboisii In CPYG med1um for

20 days at 25 C.

37

Four of the protein bands (rf 21.81, 40.0, 43.63, 50.9)
were intensely stained while those at rf 54.54, 61.81,
72.72 were very light. The fractions at rf 40.0, 43-45.45,
54.54 were common between type "A" and type "B" of

this strain. Histoplasma duboisii had 5 fractions, 3

 

of which were clearly visible (rf 21.81, 43.63, 47.27).
Three bands (21.81, 43.63 and 61.81) were common with
type "B". Figure 3 summarized these results. The
yeast phase cultures were incubated for 30 days at

37 C. The protein extracted from the culture filtrate

of E. capsulatum strain G-184, type "A" produced 5

 

fractions (rf 18.18, 27.27, 52.72, 65.45, 81.81). For
the same strain, type "B" produced only 2 fractions
(rf 18.18, 52.72) which were homologous for the two

types. Histoplasma duboisii showed one fraction (rf 41.81)

 

intensely'stained, the other two bands (rf 47.27, 52.72)
were very faint. The fraction at 52.72 appeared to be
common for all three isolates. The diagram of protein
patterns is shown in Figure 4.

Disc Electrophoresis of

Penol-Acetic Acid-Water
Cell Extracts

 

 

 

Cells, from 10 day old CPYG agar cultures at 37 C were
extracted by the Phenol—acetic acid-water method for
disc electrOphoresis. Three isolates of E. capsulatum
strain G-l84 type "A" and "B" and a strain from our

MSU stock culture, had almost identical patterns while

 

 

 

1‘ b “has“
'L\M 1.).“ —
—— “6.0

3%. pl: TflbI-ll
503 “11 —

S‘LN —— 515‘
“\8\ “m
TLJ‘I.

 

 

 

 

 

A B Duboisii

FIGURE 3.——E1ectrophoretic patterns of protein extract
from culture filtrate of H. ca sulatum type "A",
type "B" and H. duboisii In CPYG medium for
30 days at 25—C.

39

E) Ihhnw“

[Mr
[I
E

 

 

 

 

 

 

- \3.\%
firm
L“.B\ —
5m. —— sur- —- 2E1} ___—
(.53; —
9\.8\ —

 

 

 

 

A B Duboisii

FIGURE 4.-—E1ectrophoretic patterns of protein extract
from culture filtrates of H. ca sulatum type
"A", type "B" and H. duboigii 1n CPYG medium
for 30 days at 37 C.

40

E. duboisii had similar positions of protein bands
but some of them were more intensely stained. Both
species produced such compact patterns of 8 to 10
protein bands that their rf values were difficult to
calculate accurately. The photograph of the gels is
illustrated in Figure 5.

Disc Electrophoresis of Cell

Extracts from the Yeast Phase

of Additional Strains of

H. capsulatum and H. duboisii
Grown on CPYG Agar at 37 C

 

 

Protein extracts from the yeast cells of E.

cgpsulatum, strain G-186 type "AN, strain B-621,

 

B 913, B 329, 105,strain 222 type "A".and E. duboisii,
strain B-939, B—650, B-651 and B 6 were subjected to
disc electrophoresis by the method of Razin and Rottem
(1967).

The electrophoretic patterns of all strains of
both species appeared to be similar although some faint
bands at the lower rf values were not clear as illustrated
in Figure 6.

Disc Electrophoresis of Protein
Extract from the Culture Filtrates
of the Mycelial Phase of Additional

Strains of H. cgpsulatum and
H. duboisii

 

 

 

 

 

All strains grew very well in CPYG liquid medium
at 25 C. The electrophoretic patterns of both E.

capsulatum and E. duboisii appeared different, even

 

41

 

 

 

HC G184A HC G184B HC-MSU HD-MSU

FIGURE 5.——Electrophoretic patterns of protein extract
from yeast cells of 3 isolates of E. capsulatum
and 1 isolate of E. duboisii when grown on
CPYG medium at 37 C.

—————‘—

    

B650 B651 B652 B329 B913 HC105

 

 

 

 

HC B621 HC 222A ,HC 6186A HD B939

FIGURE 6 .—- Photograph(s) of electrophoretic patterns
of cell extracts from the yeast phase of
additional strains of E. capsulatum and E.
duboisii.

   

[>113

(.00, .
we

 

213:]

 

 

 

43

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'Z.\‘7 A. '2.\‘) b (AWN Gibb b 933. k 111'!)
232 — 2.5.2 — 25H ’LM —— m _‘
lflfl
AM A we __—- nu "" an
‘1 u “I‘ — J: l I:
5L5
5w
“53 ~— :9: u
65 ‘3 —— .55 fl .. '
a; ’
L__ \—' -;_. |__. —_ L—l
W -
‘ 9““ Wu 5195 A“ LEEAAM 693'! 593‘)
u s a...) I'D") 1U, — 2“" 1235 —
. “3.5 “.3! “‘ - —
"" ..._, w— :3: “:2?
51.25— 5R5 — 5 F ' —
4.9.6 MA — 59.5 57.5 ——
ow. —-— 0.; “a" ‘ “"
6°] 39 l
.. L L. L_
\\ Lavvhth H. D “ms“

FIGURE 7a.--Diagrams of electrophoretic patterns in
acrylamide gels of protein extracts from the
culture filtrates of additional strains of
E. capsulatum and H. duboisii in CPYG medium
for 30 days at 25 E. ——

 

 

219A 219B G—18 6A G-18‘6B

 

B621 B815 222A 222B

FIGURE 7b.--Photographs of electrophoretic patterns in
acrylamide gels of protein extracts from the
culture filtrates of additional strains of
H. ca sulatum and H. duboisii in CPYG medium
for 30 days at 25 5. _—___-——

45

; .n ‘

.-

 

 

 

 

D213 B293 68-46 B939 B-937

FIGURE 7c.-—Photographs of electrophoretic patterns in
acrylamide gels of protein extracts from the
culture filtrates of additional strains of
g. capsulatum and'H.'duboisii in CPYG medium
for 30 days at 25 E.

46

strains in the same species were different. Moreover
in the same strain with different types (Berliner, 1968)

there was a variation. Only 3. capsulatum, strain 219

 

type "A" and "B" showed the same pattern. However,
some strains of E. duboisii had electrophoretic patterns
which were more closely related than those in strains

of E. capsulatum. For the two strains of E- duboisii,

 

6 fractions (rf 24.5, 43.8, 55.5, 59.6, 68.4, and 80.7)

were in common. Forll strains of E: capsulatum, 10 had

 

one fraction (rf 21.0 - 23.2) in common. The fraction
at rf 41.8 - 43.8 and 65.4 - 68.4 were believed to be

homologous among seven strains of E. capsulatum and

 

two strains of 5' duboisii. The diagrams and photographs
of these gels with protein bands are illustrated in

Figure 7.

Enzyme Activities

 

Survey for Enzymatic Activities
in Three Kinds of Liquid Media
at 37 C and 25 C

 

 

 

CPYG Liquid Medium at 25 C (Mycelial phase).--

 

The filtrates were taken from liquid CPYG medium at the

end of 10, 20, and 30 days at 25 C for enzymatic activity

determinations. All isolates of g. capsulatum and

 

E. duboisii tested had relatively high activities for
PNP-beta-D-glucosidase (beta-PNPGase), PNP-alpha-D—
glucosidase (alpha-PNPGase), PNP-alkaline phosphatase
(alk PNPPase) and PNP—acid phosphatase (acid PNPPase)
for all three periods or at least for the period at

30 days. Two strains, fl. capsulatum G-184 type B, and

 

47

E. duboisii were especially high for PNP alkaline phosphatase
(alk PNPPase). All isolates of both species had moderately
high activity of PNP-N-acetyl-beta-glucosaminase (PNPPNAGase)
at 30 days. The activity for alpha and beta-PNPGase of

three isolates of E, capsulatum was about the same as

 

for E. duboisii except for "B" at the end of 10 days.
There was little or no enzyme activity for PNP-alpha-D-

galactoside, in straing "A" of E. capsulatum while strain

 

"B" and E. duboisii showed activity for the former and
for PNP-glucuronidase activity. The results are shown
in Table l.

CPYG Liquid Medium at 37 C (Yeast phase).--The

 

only marked anzyme activities for the strains tested

in filtrates taken from CPYG medium after 10, 20, and

30 days were alpha and beta-PNPGase in E. duboisii.

This same strain also had higher alkaline and acid
PNPPase. Table 2 shows the comparative enzyme
activities. All activities were higher in the filtrate
from the 25 C cultures except alpha and beta PNPGase from
E. duboisii.

Brain Heart Infusion Broth with 0.1% Cysteine

 

at 25 C.--Higher activity of PNPNAGase was found for

3 isolates of E. capsulatum in the filtrate for BHI

 

broth with 0.1% cysteine at 25 C, but there was variably
high activities for alpha and beta-PNPGases, and
for alkaline and acid-PNPPases while alpha and beta-

PNPGases of E. duboisii were consistently high. Table 3

48

 

 

 

comm come mmv omm cmv code ooov com om
com comm c c o cmhm omom o om Ham
com comm o c om omom omhm coH ca :Hooso
mmm cmm mom c mm cmwv cmnm coma om
mm mma c mnH mmm cmmv cmvv omH cm om:
c com o om cm ccHH cmmH c ca
moma come mew com coo ommo come cmHH cm
mod cmov mom com cm ocmv ccav mom cm m
mom com cmm com mo mom com o ca
mmca mnmm cow mma mm ommv cove coo om
mmm mmm o com o ccHN mood mo cm 4
com ocv c mm c come cmmv mmm ca Sound
tommmo
mpflcflfimm
moflcou mpwmou moflmou mpfim moan oosam
owom Mam Isosam OMHMDIQ omammlo oosamlo OODHmIQ Hmumom
-mmzm -mmzm umzm muwnumzm memamamzm mumnnmzm mamamumzm zumzm
mammam
mononumbdm m50eum> mo Acccaxusoz\HE\.o.oc mmflpfl>fiuom mEmNom woo oumflm

 

.0 mm pm mama om can om .oa umpum assume osmo mo mumuuaflm me» an msmuamuu.a momma

49

 

 

 

com cmm hca mm mm ommw ommv cmm cm
com cmcH cmm mo cmH omom comm c om Ham
omma omkm com 0 ma comm omam om OH -Honso
com com o mo NHH mma o mmm cm
c mm mm om om cm cm cmm cm om:
com o mm o mm cm c om ca
mmn mm mm mm mom mmv cmv mmm cm
mom o mNH c cmm com com o om m
cow c c c mmm mom cm c ca
com cmm mm com cma mmm mmm mmm om
mom mom mmm ocH c moa com o cm é
cmm moH cm om mod mom cm o ca Esuma
Izmmmu
mpflsfismm
GUHQOH mpflmOp moflmou mpflm mpflm oosam m
pflom xam Isosam omammlo omammuo OOSHmIo oosHolo Hmpwom
nmmzm ummzm :mzm mumnumzm mamamsmzm mumnumZm mnoamumzm .z:mzm
mammHm
mmpmumeSm mSOHHm> mo Aoccaxuson\as\.o.oc mepH>Hpom mfimwcm mmo oumflm

 

pm pm mwmp cm can cm .cH Hmpwm Edflpmfi wwmo mo mumupaflm mop CH mofi»~:mul.m mqmdB

 

50

 

 

 

coma mhma mom cmm cmm cmmm cmmo mmo cm
occm com cmm com mom comm cmmm mom cm Hem
cmmm omsm omm cow was comm cmmm cmm on -Honso
com omca mom ccm mom moma occm mom cm
comm cmmm cmm cmH com cmm omHH mmm cm sz
mom occm mmm cmm ocH mum och cmm oH
coca occm mmm cmm cmm ccmv comm coma cm
comm ccav mom mom ccv cmmm cmmm cmm cm m
comm comm cmm cmv cmm com com cmv ca
mom mmaa com mom com cmcv comm cmmm cm
coca cmmm com mom mmm com mmcH mmm cm a
mum cmmm cmm mo coo cow mmm com ca Enuma
Izmmmo
mofloflfiwm
mpflcou mpflmop mpflmou moflm comm Cosmo
Uflom xam Isosam omammlo omammlo oosHmIQ Conamlo Hmumom
ummzm nmmzm nmzm mumnnmzm mamamumzm mumnumzm mamamnmzm anzm
mammam
mmpmuquSm wSOflHm> mo Acccaxusoc\aa\.o.oc mmflpfl>fluom mammcm moo oumwm

 

.0 mm um mmmp cm
pom cm .cH Houmm mcflmummo wH.c nufl3 Esflpmfi Hmm mo mumupaflw or“ :H mwsmmcmnl.m mqmfie

 

51

illustrates these results. Most reactions were higher
in the CYPG agar except for two strains of "A" and "B".

Brain Heart Infusion Broth with 0.1% Cysteine

 

at 37 C.--Histoplasma duboisii had higher activities in

 

alpha and beta-PNPGase and alkaline-PNPPase than the

isolates of E. capsulatum when grown in BHI with 0.1%

 

cysteine at 37 C. The results were similar to those in
CPYG medium at 37 C but lower in the activities for
alpha and beta—PNPGase and higher for alkaline-PNPPase.

All isolates of E. capsulatum had low activity for

 

every enzyme tested. Table 4 summarizes the results.

Sabouraud Dextrose Broth at 25 C.--All isolates

 

had more or relatively low activities for all enxymes
tested until the end of the 30 days incubation in
Sabouraud dextrose broth at 25 C. Apparently the activity
for alpha and beta—PNPGase of all isolates increased

at the end of the 30 day incubation period being much

higher for E. duboisii than for strains of E.

 

capsulatum. Alkaline and acid-PNPPase activities of

 

all strains except E. capsulatum G-l84 type "A" rose

 

at 30 days. Table 5 shows the results.

Some Characterisitcs of PNP-Alpha
and Beta Glucosidase

 

 

Enzyme Production in Relation to Age of Culture

in Liquid Medium.--In 100 ml CPYG medium at 37 C with

 

2 ml of inoculum containing approximately 829 x 106 cells

per ml, the total cell counts of E. duboisii increased

 

 

52

 

cmm 0 II mom ccH occm cmmm omH cm

 

 

mmm occm II mmm cmH mmm com ocH cm Ham
mmm mkmm o cmm omH cmm cmm com OH -Honsw
mmm mmm c cm om cmH c cmm om
c cmm c mmm com coH cmm cmm cm sz
om mm o mmm cmm o c c ca
mmm cmm com cm ooa mmm cmm o om
cca cmm mm cm mmm o mmm mma cm m
com com I: II II c o ccm ca
mmm com I: c II mm cmv mmm om
mmm mmm l: me an com mmm c cm m
.cmm mom I: c cmm c c c ca EdumH
nommmu
moHsHEMm
mpflcou mpflmo¢ mpflmou mpflm mpflm oooam
pflom xam Isosam omammlo omammlo oosamno oosamlo kumom
ummzm :mmzm nmzm mumnumzm mgmamumzm mumnumzm msmamimzm z:mzm
mammam
mmumuquSm msOHHm> mo Acccaxnson\as\.o.ov mmflpfl>fluom wEmNcm mmm oumflm

 

.o um um mmmo om
cam om .oH umumm mqflmummo wH.o sues ssfloms Hmm mo mumuuaflm map :a mmsmwcmau.e mqm<e

 

 

53

 

 

 

mom cmm cmH ccH mom cmmm mmoa com om
cm com mmH cm moH c mmv com cm mmm
nachsp
mm c mm c mmm c c ccH ca .
com mmm mm c c cmm com o cm
c cmm c mmm moa c cm mmH cm cm:
o c c c mm o c c ca
mmv mmm moH mmm mmm mmm com com cm
c c c cod mmm c c cca cm m
com o c ccH mmH ccH c c ca
mmm com mm mmm c ocm mmm mmm cm
mmm coH mm mmH c c cmH cmm cm <
c c c c c c c cca ca Enuma
nommmu
oUHcHEMm
moflcou mommOp mpflmou mpwm mpflm oosam
Uflom me Isosam omHmmlo omammlo oosHmlo Conamlo Hmumom
ummzm nmmzm umzm mumnnmzm madamsmzm mumnum2m madam:mzm Zumzm
mammam
mmpmupmhom msoflum> mo Acccaxusoc\afi\.o.oc mmfiufl>fluow wfimwcm moo oumflm

 

.o mm um mmmo om cam om .oa Hmumm ssfloms osmusonmm mo mumuuaflu men an msmucmnu.m mamas

 

 

54

rapidly during the third to the fifth day of incubation.
The peak of growth occurred on the seVenth day. The
production of alpha and beta-PNPGase started to rise
rapidly on the ninth day or 2 days after the growth

peak and then, continued to increase until the end of

the 30 days incubation. The total cell count for the
organism decreased during the last 10 days of incubation.
Table 6 and Figure 8 show the results. In comparison,

E. capsulatum strain G-184 "A" and "B" had low alpha

 

and beta-PNPGase activities, never higher than 150 units
(0.D./m1.hour x 1000) at any time in contrast to over
11,000 units for_E. duboisii at the end of the 30 day
period.

Enzyme Production in Relation to the Age of

Culture on Solid Medium.--The production of alpha and

 

beta-PNPGases were detected in the culture medium

(CPYG agar plate) by the plug method after 4 day incuba-
tion at 37 C. The activities of these enzymes kept
rising in E. duboisii throughout the 30 day period.
Relatively low activities of these two enzymes were
found in the culture medium with E. capsulatum, strain
G-184, type "A" and "B" and MSU strain. The data are

shown in Table 7.

55

TABLE 6.--A1pha and beta-PNPGase production in the filtrate
during growth of E. duboisii in CPYG medium at 37 C.

 

 

 

 

 

No. of day _Ehzyme activities 7T3531=E§116
Incubation (O.D./m1/hourx1000) countx4x10 /m1
alpha—PNPGase beta-PNPGase
1 O O 3.36
2 0 0 7.43
3 0 O 19.90
4 O O 56.81
5 0 0 137.34
6 0 0 186.45
7 100 35 224.84
8 75 75 197.81
9 2550 2200 217.25
11 7500 7850 200.62
22 10950 11100 190.62
25 9750 9650 178.12
30 11600 11000 171.35

 

 

56

ENZYME ACTIVITY

10.000

5.000

«a

 

.0 um um Edfloma ommo ca HHmHOQSU mammamoumflm
mo mcofluosooum mEmmsm can m>nso huzouwnl.m mmDon

m><o
ON 0— 0.. Q— N— O— a o
. ..s..\:o _
a
..V
. , ..\
«39.2.. 2...: 9| ...
canes-.... 20m 0.... xx
7... son ...—:00 :00 _Bo0h 0' a».
m. 0
Q.
\...\.o.
\.....
\...A...

00! as
7 anOD 1133 1V101

Oil

00!

OlX
9

57

.mpflmoosamlolmumhlmzm m
an

umpflmoosaololmnmamlmzm H

 

 

 

 

cmm cmm cmm cmm cmm com cme ccv cmm com cam com cmH cma cm cm om cw Hflmfl0hdp .m
cm c cm c cm c c ca ca o c c c c cm ca cm om cm:
cm cm cm cm cm cm cH cm on cm cm c c c cH cH cm cH m «calm
cm cH cm ca cm cH cm cm c c c c c c o c o c m emalw l
EoumHSmmmo .m
N H_. N, H N .H N H N H N H N H N .H‘ ..N ..H
mmmp cm mmmp mm whom cm mmmo ea mmmp am what m mmmo m mmmp v mmmp m
Accoa x Hoo:\msam HomM\.o.Oc mpfl>flpo< Emflcmmuo

 

 

.0 hm hm poflnmm mmm cm m Hm>o mopmHm Hmmm ammo mnp cH

mmmmmmmm .m can Eupmasmmmo .3 mp UGUSUOHQ mmmmwmzmlmumh pom manm mo mmflufl>fluom mEmNGmlu.m mamma

 

58

Optimal Temperature and pH.--At 4 C alpha and

 

beta-PNPGases were low in activity in the liquid filtrate

of CPYG medium for E. duboisii MSU strain grown at

 

37 C. An increase in the incubation temperature increased
the activity of both enzymes. At 69 C, both enzymes
showed the highest activity for the temperatures
selected. Table 8 . and Figure 9 show the results.

The alpha and beta PNPGases in the filtrate lost
their activities after 3 minutes in a steamer, or after
one hour at 70 C. The optimal pH for alpha-PNPGase in
E. duboisii was found to be pH 4.6 and pH 4.8 for beta-
PNPGase. A rapid drop occurs in activity of these
enzymes above or below these points. Table 9 and
Figure 10 illustrate the effects of pH changes.

Effect of Storage at -20 C.--The alpha and beta-

 

PNPGases in the filtrate of E, duboisii MSU strain
in CPYG medium after storage in the freezer for 90
days showed no deterioration in enzyme activity.
Kinetic Plots of Alpha and Beta-PNPGases.--
Both enzymes produced a linear increase in activity up
to 90 minutes then leveled off. Figure 11 shows the
kinetics of both enzymes, Table 10 lists the data of
this experiment.

Inhibition Test.--Glucose, mannose, galactose

 

and d-levulose were tested for inhibition effects on
alpha and beta-PNPGases. Greater inhibition of
alpha and beta-PNPGase occurred at 0.46 M than 0.04 M

for glucose and mannose. No inhibition was evident

59

TABLE 8.--Effect of temperature on activity of alpha and
beta—PNPGases produced by E, duboisii MSU strain.

 

 

 

 

Temperature Activity (O.D./ml/hour x 1000)

1 hour alpha—PNPGase beta-PNPGase
4 C 200 750

25 C 2700 2100

37 C 11700 11200

59 C 55200 54000

69 C 120000 117300

75 C 12700 11800

 

 

 

ENZYME ACTIVITY

60

130.000

...... Alpha PNPGase

Beta P N PGase

 

, 90,000 110,000

70.000

50,000

 

I

30.000

 

10,000

 

20 . 40 00 so

TEMPERATURE (degrees centigrade)

FIGURE 9.--Effects of temperature on alpha and beta—
PNPGases from the culture filtrate of E.
duboisii at 37 C.

61

TABLE 9.--Effect of pH on activity of alpha and beta-
PNPGases from the filtrate from E. duboisii MSU strain.

 

 

 

pH activity (O.D./m1/hour x 1000)
alpha-PNPGase beta-PNPGase

3.0 9400 8600
4.0 12000 11000
4.2 11000 11000
4.4 12000 11100
4.6 12500 11100
4.8 12000 11800
5.0 12000 11100
5.3 11800 11100
5.0 12000 11000
6.0 11000 8600
7.0 7500 7200
8.0 4900 3800
9.0 2500 2400

 

62

.--- Alpha PNPGase

_ 8". PNPGO‘O

O'y

IVI

12.000

Enzyme Act
0000

4p00

 

IO 20 30 40 50 60 70 80 90

pH

FIGURE 10.--Effects of pH on alpha and beta-PNPGases

from the culture filtrate of E. duboisii at
37 C.

 

 

 

 

63

TABLE 10.--Kinetics of alpha and beta-PNPGasesfor E. duboisii
MSU strain at 37 C. ‘

 

 

 

 

Time(minute) ' Activity (O.DEZmlzhour‘x 1000)
alpha-PNPGase beta-PNPGase

0 0 O

5 0 600

10 600 1300

15 1500 1950
25 3200 3500

40 5400 5400
60 8400 7400
90 10000 9000

212

11000

9500

 

 

 

64

...... Alpha PNPGase

...—.... Beta PNPGase

12.000

 

ENZYME ACTIVITY

 

20 60 100 140 180 220

MINUTES

FIGURE 11.--Kinetics of alpha and beta-PNPGase
activities in the culture filtrate of E.
duboisii in CPYG medium at 37 C.

65

when d-levulose and galactose were used at a concentra-
tion of 0.46 M. Figure 12 and Tablejll summarizes
the results .

Effects on Enzyme Production due to the Modifica-

 

tions in Liquid CPYG Medium.--

 

a. Casein and the amounts of glucose

 

The amount of glucose in the medium had an
important effect on the production of alpha

and beta-PNPGases by E. duboisii MSU strain

 

at 37 C. It was found that 1.5% glucose in
CPYG liquid medium was the best for the high-
est activities of both enzymes. Vitamin free
casein (Difco) was one of the essential factors
for enzyme production in this medium and could
not be replaced by casamino acids, an acid
hydrolysis product of casein. Some essential
factor(s) in the casein for the two enzyme
productions is suspected to be lost during acid
hydrolysis of casein. Attempts to find this
factor(s) by adding several salts and amino
acids in casamino acids to check whether they
could induce enzyme production were not success-

ful. The results are shown in Table 12.

 

 

 

66

 

 

 

o commH c oomoH zoo.o 0moHs>0Huo
o oommH o oomoH 2m4.o 0m000MH00
cN.m coocH Ho.mH ocmcH zoo.o 0moccmz
oH.mH oonH mo.NN ommH zoo.o 0000002
mm.co occm mm.Nm comm soc.c mmoccmz
mm.Nc occm cc.mo coco soc.o mmoooHo
cm.om comm mm.mm oooN soc.c mmoooHu
cm.mc cmcH oH.oo cooH 2mm.o 0moous

c ocmoH o cccoH Ammumzo
coHanHmcH oz

aOHHHnHmch 0mmumzmnmumn cOHHHnHmaHm 0mmomzmummmHm .0606 ummsm
HoocH x Hsoo\HE\.c.oo m0H>Huom HoanHmcH

 

 

.6 cm 00 ssHoms ammo 0H chuum cos HHmHonso .m 00 wumanHm 0050H00 0:0 5000

mmmwwmzmlmumh pom mnmam so mmoccmE can mmoosam mo muommmm coHumhmst11.HH mqm<e

67

I .U um

um Edmpmfi ammo cm mmmmOhdc .3 mo mumuummw

mHopaso 0:0 Eoum oncomzmlmumh com mamam so
mmossmfi cam omoosam mo whoommm documhmccH11.mH mmcme

2502:1050. ..o 2925200200

no '0 no «0 —O

0000 0 . . u ‘
0005.65 5 0300.251 Ucflnal ®.1‘.1‘.‘“‘

0005.05 5 cacao-2.— ass—Eel.

Uncu3w cm Gunmen-‘5 0.060131,

0.03.0 s 03002.. 2.020--

 

.00l

Moulamm 6

O

 

68

 

com
com
ooa
ooa
on

o

o
comma
oooma
oomo
comv

coma

com
com
cmm
oma
com

o

o
oomha
oooam
oovm
ooam

ooma

mcaumm aE\mEa
:mQQOpmmuu aE\mEa
mumnmmocm EdapOm aE\mEa
mumnuao oaHHmm aE\mEa

mumcmmocm oanumm aE\mEa

nqu
oqu
mpHs
oqu

ang

mcaom
mpaom
mcaom
mpaom

mcaom

.mmo

.mmo

.mmu

.mmu

.mmu

cauuxmc aE\mEa cams mcaow ocafimwmu

sammmo omomamou mcaom OCaEMmmu

mo.m

mm.a

mmoosaw

mmoosaw

mmOOSaw

omoosao

mmoosaw

 

0mmomzmumumn

mmmwmzmumcmHm

 

HoccH x uson\Hs\.a.oo m0H>H00<

ESaUGE mo mCOaumoamacoz

 

I .6 mm 00 mmmo mH 0H ssHoms oHsmHH ammo :H HHmHonso
.m we soauoscoum mammsm so mmoodam mo mucsosm pom sammmo mo muommmm11.ma mamda

69

The same CPYG liquid medium in which 1.5%
agar (Difco) was added and the agar medium near
the edge of the colony was removed as agar
’blocks and assayed for alpha and beta-PNPGases.
Lower but somewhat similar results occurred for
effects on the amounts of glucose in the medium,
with 1.5% best for enzyme production. Casamino
acids could be substituted for casein as alpha
and beta-PNPGase activities were still found in

the agar medium as shown in Table 13.

b. Sugars

Comparatively, the production of alpha and beta-
PNPGase activities were higher in cellobiose than
in lactose, soluble starch, and dextrin. Cellobiose
was nearly as good for alpha and beta-PNPgase
activity but glucose was still better as shown

in Table 14.

Identification of alpha and beta—PNPGase Fractions

 

by Disc Electrophoresis of Lyophilized Culture Filtrates

 

from H. duboisii in CPYG Liquid Medium at 37 C.--Enzyme

 

fractions in the lyophilized culture filtrate gels
reacted with the substrates (PNP-alpha-D-glucoside and
PNP-beta-D-glucoside) which diffused into the gels
resulting in the liberation of free paranitrophenol at

the sites of alpha and beta-PNPGases in the gels. When

70

TABLE 13.-—Effects of the amounts of glucose and casein in
solid CPYG medium on enzyme production by E. duboisii in
10 days at 37 C.

 

 

Modifications of medium ACthlty

 

(O.D./agar plug/hour x 1000)
alpha-PNPGase beta-PNPGase

 

Glucose 0% 360 310
Glucose 0.5% 390 330
Glucose 1.0% 450 600
Glucose 1.5% 930 750
Glucose 2.0% 570 690

Casamino acids replaced casein
(glucose 1.5%) 310 250

 

71

.muzuaso xooum Uao mac 0 mo

 

 

 

 

aE\maamo mca x v.cm mo soamsmmmsm aamo 0 mo maE 030 was Edasooca 0:9

0.»
cm.ama cocoa cccaa mc.a mmmoosao
cm.cma ccvm comm mc.a «meaQOaamo
ce.mm cmmm comm mc.a sauuxmo
cc.mv comm comm mo.a commum 0ahsaom
cm.mv cmmm come mc.a mmouomano
cm.cma comma ccoma mc.a mmOaQOaamOIQ
mm.v com com mc.c mmoosao mosafi mmm

00000 000002010000 00000201000H0 000000 000000

aamo amuos Accca x unoc\aE\«o.oc mpa>auoal Edaomfi mo mcoaumoamapoz

 

 

.0 mm 00 mmmc ma ca aamHOQmm .m an mcoaposconm mEm~s0

so Edapmfi mmmo caswaa ca mmpMH©>£0hnmo cam mucosm psmummmap mo muoommm11.va mamfle

72

the gels were put into 1.0 M Tris, paranitrophenol
developed a yellow color band in the gels at the
site(s) of the enzymes.

The gel which was put into PNP-alpha-D-glucoside
developed one yellow band at the rf 40.0. When the
gel was put into PNP-beta-D-glucoside, one yellow
band developed at rf 40.0. The yellow band of free
paranitrophenol in each gel corresponded with the
stained protein band of the third gel of the triplicate.
Figure 13a and 13b show the diagram and the photograph
of the bands.

Alpha and beta-PNPGases in other isolates of

 

H. capsulatum and H. duboisii on Sabouraud Dextrose

 

Egar for 30 days at 25 C.--All 12 isolates of E. capsulatum

 

 

and E. duboisii were cultured on Sabourand dextrose

 

agar for 30 days at 25 C. Agar discs were used for

assay. Two isolates of E; duboisii gave higher

 

activities for both alpha and beta-PNPGases than the

10 isolates of E. capsulatum. One (B-937) of the

 

two isolates of E. duboisiiEnot previously tested for

 

alpha and beta—PNPGases gave a slightly higher activity
while the other (B—939) was significantly higher. The

results are shown in Table 15.

 

 

73

 

 

ti ‘ No.0
‘0». \00 . x = §\‘5k 5V mum \MA swam» 900.01
it‘s: :1 xi: ‘_"—‘ ‘_ _‘ ‘e\\0v ‘00“ ‘3 “\SK

\‘N‘\%§€3§.& \\ \\.R\\\~‘?\~\9 (716%.

(.2- msk. \vxmuoeo w. 0031.- “w one.

 

 

 

 

 

 

 

 

 

FIGURE 13a.--Diagram of electrophoretic patterns in
acrylamide gels of the lyophilized filtrate
from E. duboisii in CPYG for 30 days at

37 c compared with the band produced by alpha
and beta-PNPGase activities.

 

 

74

 

 

 

FIGURE 13b.~-A photograph of electrophoretic patterns in
acrylamide gels of the lyophilized filtrate from
E. duboisii in CPYG medium for 30 days at 37 C,
'compared with the band produced by alpha and
beta-PNPGases.

75

TABLE 15.--The production of alpha and beta-PNPGases in
Sabouraud dextrose agar of additional strains of E. capsulatum
and E. duboisii for 30 days incubation at 25 C.

 

 

 

 

 

organism (O.D./agar 31335051. x 1000)
alpha-PNPGase beta-PNPGase-
H. capsulatum B-875 130 130
H. capsulatum B-293 10 4O
H.capsu1atum B-621 30 100
H.capsu1atum G—186 type A 10 40
H. capsulatum G-186 type B 40 30
H. capsulatum 219 type A 160 100
H. capsulatum 219 type B 30 70
H. capsulatum 488 60 60
H. capsulatum D-213 180 170
H. capsulatum 68—46 70 70
H. duboisii B—937 200 200

H. duboisii B-939 370 280

 

76

The Distribution of PNP-alkaline
Phosphatase in Polyacrylamide Gels

from Disc Electrophoresis of Lyophilized
Culture Filtrates of Strains of H.
capsulatum and H. duboisii in CPYG

Broth for 30 days at 25 C

 

 

 

 

 

 

Three strains of E. capsulatum 184 G type B,

 

186 G type B, and 219 type B, and three strains of E.
duboisii MSU strain, B 937, and B 939 were fractionsted
by disc electrophoresis, from lyophilized culture
filtrates after growing in CPYG broth at 25 C. The
activity of PNP—alkaline phosphatase produced by 3
strains of H. capsulatum and 3 strains of E. duboisii

was variable. Histoplasma capsulatum strain G-186

 

type "B" and E. duboisii strain B-937 had high activi—

ties while E. capsulatum strain 222 type "B" produced

 

low acitivity for alkaline-PNPPase. Significantly, all
strains of both species had the peak of alkaline—PNPPase
activities in the gel section number 4, or in between
1.5 - 2.0 cm from the starting point of electrophoresis
in the small pore gels. Table 16 and Figure 14 show
these results.

A Study of Alpha and Beta-PNPGases

andfiAlkaline-PNPPase Produced by H.

capsulatum Type "A" and "B"

When Grown on BHI Agar Plates

with 0.1% Cysteine for 21 Days
at 25 C

 

 

 

 

 

Three strains of E. capsulatum type "A" and "B"

 

when grown on BHI agar with 0.1% cysteine at 25 C for

20 days were checked for enzyme activity by the agar

 

77

TABLE l6.--The distribution oflalkaline-PNPPase in polyacrylamide
gels from disc electrophoresis of lyophilize culture filtrates
of 3 strains of H. cgpsulatum and H. duboisii in CPYG media

_ for 30 days at—25 C.

 

 

Organism Activity* of alk-PNPPase in section number**
1 2 3 4 5 6 7 8

 

H. capsulatum G—184B 70 170 450 490 200 20 20 10
H. capsulatum 222B 10 25 30 .75 45 15 15 10

H. capsulatum G-186B 40 65 180 1400 1050 115 20 15

 

H. duboisii MSU 65 140 310 780 250 15 5 5
H. duboisii B-937 20 150 500 1400 1100 140 10 10

H. duboisii B-939 20 35 250 850 510 50 10 10

4‘

*
O.D./gel section/hour x 1000

**
Each section = 5mm.

ENZYME ACTIVITY

78

...... H.capsulafum G 1848

8 . ___.. H.capsulatum 222 B
2 ......... H.capsulatum G 1868
o__ H.duboisii B 937
8 . ~. 0..-. Hduboisii B 939
g g 0 ...... H.duboisii MSU
o .'
o .'
9 5
i Q
C : \
8 s I?!“
ll}: '-. \
8 5 I a": \\ 1'.
~o .‘ I '3. ‘
, ~' I '1
...] '3 ‘1‘ '2
8 ’ .4, . ‘ 3
" =1 1. \ -.
.6! \\ '2
o "x /J_ \0 \
o //.,3 1 2
N z I 'o'. \ 1
Z \
~ ......... «7’ ,.--~ ~~~~~~~~ "'10.
I 2 3 4 5 6 7 8

PORTIONS or THE GELIO.5CM EACH)

FIGURE l4.--Graphic diagram showing the distribution
of alk—PNPPase in polyacrylamide gels.

 

 

79

plug method. Both types of E. capsulatum produced

 

high activity of alpha and beta-PNPGases and low
activity of alk-PNPPase. Significantly, type "A"

had 2-8 time higher activity than type "B" for alpha and beta-
PNPGases and 2-5 time highernactivity than type "B" for alk-

PNPPase. The results are listed in Table 17.

Study of Alpha and Beta-
PNPGase and Alkaline-PNPPase
Activities in 5 Strains of

H. capsulatum and H. 00501011
in CPYG Agar Plates After

9 Days at 37 C

 

 

 

 

 

 

High activity of'alkaline-PNPPase was found in the
plugs taken from near the colonies in CPYG agar medium
of cultures at 37 C of 5 strains of E. duboisii while

5 strains of E. capsulatum produced low activity of

 

the same enzyme. Histoplasma capsulatum strain B 913,

 

B 329, 105 and E. duboisii strain B 650, B 651, B 652
were received from Dr. L. Georg, Communication Disease
Center, Atlanta, Ga. All six isolates have the same
serotype based on fluorescent antibody technique

(Kaufman and Blumer, 1961). Histoplasma duboisii MSU

 

strain and E. capsulatum 222 A were the only ones

 

which produced high activities of alpha and beta-PNPGase.

The data are shown in Table 18.

 

80

 

 

 

m.m mm com omN m 0000 mmHo 0000H00000 .0
o.N cm oomH cocN 0 0000 mmHo 0000H00000 .0
o.m NH cmm cNm 0 0000 NNN 0000H00000 .0
m.m cm com ooNN 0 0000 NNN 0000H00000 .0
N.m 0N ocNH cmcH m 0000 oHN s00300000 .0
m.N cm ommN ccmN 0 0000 mHN 0000H00000 .0
Asoc
.000 .0H00 000002010H0 000002010000 00000201000H0

 

Accca x H:O£\msam HmmM\.o.oc >0a>auom wEmNcm

Emacmmno

 

.u mN 00 0000 HN 000 00000000 mH.o m003
mmumam H000 amm co 0m: com :4: mmmp 8:00a5mmmo .3 mo msamnpm m we cmoscoum

 

mma0a>auom mmmmmzmlmcaamxam pom mmmmwmzmlmumh com mgmam mo comaummfiov11.ma mamma

81

TABLE 18.--Activities of alpha and beta-PNPGases and alkaline
PNPPase of strains of E, capsulatum and E. duboisii on CPYG
agar plates for 9 days at 37 C.

 

 

Organism

Enzyme activity (O.D./agar plug/hour x 1000)

alpha—PNPGase

beta—PNPGase

alk-PNPPase

 

capsulatum B 913
capsulatum B 329
capsulatum 105
capsulatum MSU
capsulatum 222 A
duboisii B 650
duboisii B 651
duboisii B 652
duboisii B 939

duboisii MSU

5
O
5
20

170

290

10
0

5
10
130

10

250

45
4O
65
6O
75
330
560
375
340

185

 

 

  

82

Fine Structure in Young Hyphae of
Histoplasma duboisii and Histoplasma capsulatum

 

The mycelial phase of g. duboisii grown for 10

days in Sabouraud dextrose broth (Figure 15) showed fine

 

structure resembling fl. capsulatum. It is multinucleate
(N) in a hyphal cell with prominent nucleoli (NCL). A
small intracytoplasmic membrane system (IM) was found
close to the plasma membrane (PM) (Figure 16).

In the limited number of sections of g, duboisii

observed, Woronin bodies were not found associated in the area

 

of the septum (Figure 17). On the other hand, a section

of a hypha from E. capsulatum showed two Woronin body-

 

like structures (Figure 18) in the area near the septum

similar to those reported by Garrison (1970).

Attempts to Develop a Differential Medium
for Histoplasma capsulatum and H. duboisii

Yeast Phase

 

The MSU strain of 3° duboisii began to show a
color change from cream color to purple after 10 days
at 37 C on CPYG agar containing either the combination
of ferrous sulfate and sodium thiosulfate or ferrous
sulfate alone in all concentrations tested. The color
did not change in the colonies growing on the CPYG
medium that contained ferrous sulfate with the addition
of cysteine for up to 30 days at 37 C. The colonies

of all five strains of E. capsulatum remained cream

 

color until the end of 30 days at 37 C. The photograph

83

 

FIGURE.15.—-A segment of a hyphal cell of Histoplasma
duboisii showing three nuclei x 18,000.

 

84

FAA.

 

FIGURE l6.--A segment of a hyphal cell of Histo lasma
duboisii showing intracytoplasmic membrane system

(IM) near the plasma membrane (PM) x 102,000.

 

FIGURE l7.--A septum in a segment of a hypha of g.
duboisii, Woronin bodies were not found x 57,000.

85

 

 

 

FIGURE l8.-—A segment of a hyphal.cell of‘Histoplasma

ca‘sulatum showing a septum with two Woronin
Body-like structures (WB) x 54,000.

86

showing the difference in color of the colonies of

these two organisms is illustrated in Figure 19. However,
when additional strains of E. duboisii strains were

grown on this modified medium, the colonies failed to
change to the purple color characteristic of E. duboisii

MSU strain. When the mycelial phase of both E, capsulatum

 

and E, duboisii were used as inoculum in this

 

medium and incubated at 37 C, the colonies became purple

after a 2 or 3 subcultures on this medium.

Mycelial Phase

 

An indicator medium with phenol red was prepared
to investigate any changes in the pH in the media during

the growth of E. capsulatum strains 488, B-621, B-875,

 

and B-293 and E. duboisii strains B—937, B-938, B-939,
and MSU. All cultures inoculated with the mycelial
phase were grown in slants at 25 C. The color change

of the medium from yellow to red was noted during colony

development. More strains of E. capsulatum turned the

 

color of the medium red than E, duboisii. Histoplasma

capsulatum strains produced alkaline reactions, as

 

indicated by the medium turning red, in variable
lengths of time. After 9 days, all four strains of

E. capsulatum changed the color in the medium

 

red while the medium for 3 strains of E. duboisii

 

87

 

 

FIGURE l9.--A photograph of a purple colony of E. duboisii
MSU strain and a colony of E. ca sulatum strain
G-184A on a modified medium for E8 days at 37 C.

 

 

 

88

(B 937, B 938, B 939) was still yellow. Only the MSU
strain showed moderate degree of alkaline reaction.

In order to make this medium a better one for the
differentiation of these two species, the concentration
of the yeast extract in the medium was adjusted to vary

from 0.5 g/l to 1.5 g/l. Histoplasma capsulatum strains

 

488, B-293, B—621, B—875, B-913, 105 and B-329 and
E. duboisii strains B-937, B-938, B-939, B-650, B-651,

B-652 and MSU were grown on this medium. The best

 

results were obtained on the medium containing 1.5 g/l
yeast extract for 12 days at 25 C. ,All strains of E.

capsulatum changed the color in the medium red while

 

only 2 of the 7 strains of E. duboisii (B-652, MSU) showed

some red color.

 

 

DISCUSSION

Disc Electrophoresis of Soluble
Protein in Culture Filtrate

 

 

The method followed for determining the protein

fractions of Histoplasma capsulatum and E. duboisii was

 

similar to that used by Schechter SE El° (1966) in his
investigations of the electrophoretic patterns of

dermatophytes and Coccidioides immitis. In preliminary

 

experiments, the selection of an appropriate medium was
necessary so that the organisms would produce proteins

in the filtrate which would yield more distinct electro-
phoretic patterns in polyacrylamide gels. Different

liquid media including brain heart infusion broth plus

0.1% cysteine, Sabouraud dextrose broth, and CPYG medium
(Beneke, EE.E$"1969) were used. It was found that the
culture filtrate of Sabouraud dextrose broth and brain
heart infusion broth with 0.1% cysteine at 25 C and

37 C did not show good electrophoretic patterns even though
these filtrates had been concentrated by either saturated
ammonium sulphate or by lyophilization. The lyophilized
culture filtrates of both phases of the organisms in CPYG
medium produced the best results by disc electrophoresis.
Therefore CPYG medium was selected for studying the electro-

phretic patterns of various strains of E. capsulatum and

 

E. Duboisii.
89

 

90

The CPYG medium is highly enriched with amino acids
in the casein, vitamin B complex in yeast extract,
nitrogen in the neopeptone (Difco), and carbon in the
1% glucose. Thus, the medium will meet the nutritional

requirements of E, capsulatum which is know to require

 

certain vitamins (Pine, 1957; Salvin, 1949) and different
amino acids (Rowley and Pine, 1955a; Pine,l955b) for
growth. In such an enrichment medium as CYPG, the
organisms have sufficient nutrients to synthesize proteins,
which resulted in more fractions in disc electrophoresis.
In later experiments it was found that acetone
precipitation of proteins was superior to lyophiliza-

tion of the culture filtrate of Histoplasma species for

 

concentration of proteins used in disc electrophoresis.

On this basis, the acetone precipitation method was
employed for the later experiments. Since the inocula
were standardized, one would expect every subculture

of the same strain to grow at the same rate and to Produce
soluble proteins at the same stage of growth, and to

give the same uniform electrophoretic patterns. A

strain of E. duboisii (MSU) was used to determine the

 

effect of the amount of inoculum on protein fractions
by disc electrophoresis. An increase in the amount
of inoculum from l800xlO6 to 3600x106 cells per ml or
100% showed no difference in the electrophoretic

patterns after 30 days incubation. For this reason the

 

9l

amount of inoculum was not critical if the amount was
sufficient and the incubation period was long enough.
Although Shechter 23 El. (1966), in their experiment with
dermatophytes to determine phylogenetic relationship by
disc electrophoresis, did not standardize the inocula,
comparable results were obtained. Although the growth

curves of various strains of E. capsulatum and E.

 

duboisii in CPYG medium were not determined, the electro-
phoretic patterns of soluble proteins in the culture
filtrates at 30 day incubation should be comparable.

The mycelial phase incubated at 25 C for 10 days
showed some protein bands by disc electrophoresis. By
increasing the incubation period to 20 and 30 days, there
was an increased protein production, probably due to
greater permeability of the cell membrane or increased
autolysis. Surprisingly, strain 184G type A had fewer
protein fractions than type B of the same strain at 25 C
even after 30 day incubation. The results showed that

a strain of E. duboisii and E. capsulatum strain 184G

 

type A and B were all different in electrOphoretic
patterns after 30 day incubation at 25 C. For 10 day

incubation of cultures of E. capsulatum strain G-184

 

type A and type B at 25 C, the filtrate yielded only 1
homologous band, an indication that type A and type B
of the same strain produced the same proteins in the

younger cultures. Protein patterns changed after longer

 

 

 

92

incubation periods. These results agree with those
reported by Shechter 22.2i- (1967), who discovered that
proteins in the filtrates of 15 and 30 day cultures of

Q. immitis in asparagine medium were quantitatively and
qualitatively different. The difference in electrophoretic
patterns of type A and B of the same strain of E.

capsulatum was clearly demonstrated after 20 and 30

 

days of incubation at 25 c and 37 c, while at 10 days
incubation, they were the same. Histoplasma duboisii

showed common fractions with both E. capsulatum type A

 

and B indicating the close relationship of these two
species. When the results of additional strains of both
species at 30 day incubation at 25 C are compared, strains

of E. capsulatum yielded variable protein patterns. Some

 

were similar, different, or clearly related, indicating
the degree of strain differences. In addition, E.
duboisii strains B 937, B 939 did not yield identical
patterns. When a number of isolates of both species were
studied by this method, specific characteristics of
electrophoretic mobility of soluble protein in the species
could not be determined. Strains of E. capsulatum

showed different characteristic protein patterns when
compared with each other and with strains of E. duboisii.
The interpretation of the difference or similarity of

these two types of Histoplasma based on disc electro-

 

phoresis is rather difficult.

 

93

Shechter EE.§£° (1966, 1968) found that different
species of dermatophytes yielded different characteristic
protein bands. The number of electrophoretic mobilities
of the protein fractions of the 4 isolates of each
species were very similar. Their data indicated that the
qualitative electrophoretic patterns of these species
are relatively stable taxonomic characteristics.

Stipes (1970) also found variation in electro-
mobility of protein extracts from the mycelium of 4

strains in each of three species, Ceratocystis ulmi

 

and g. fagacearum, and g. fimbriata but observed greater

 

 

similarity in mycelial protein within the isolates of the
species. Logically the electrophoretic characteristics

of 13 strains of E. capsulatum should be more similar

 

than those of 3 strains E. duboisii if these two species

are really different. Stipes has already shown that

two species are morphologically different if protein

extracts from the two species give different character-

istic patterns in disc electrophoresis, on the other

hand the patterns of protein extracts should not be much
different if the two Species are similar morphologically.
These references along with our results support the work

of Drouhet (1967) and Andrieu EE.§£°(1969) that E. duboisii is

very closely related to E. capsulatum, due to the similarity

 

in fractions.

 

 

 

94

Membrane protein in the yeast-like phase of both

species of Histoplasma was applied according to Takayama

 

(1966), Razin and Rottem (1967) using phenol acetic

acid water reagent to dissolve the membrane protein in
the cells. This was followed by disc electrophoresis
with 5M urea in the polyacrylamide gels. The results
showed little variation in electrophoretic patterns among

strains of both species of Histoplasma and in the same

 

species. Lipman (1970) pointed out that disc electro-

phoresis of membrane protein used by Razin and Rottem

 

would not differentiate strains of Mycoplasma pgeumoniae.

 

This method therefore is useful to differentiate between
species rather than between strains in the same species.
This was a good tool to study the difference in the

electrophoretic mobility between E. capsulatum and E.

 

duboisii since the results of this research showed

 

essentially the same electrophoretic patterns between

strains of E. capsulatum and E. duboisii in the yeast

 

 

phase. This indicates these two are very closely related.
The culture filtrate of the yeast-like phase of

the organism from CPYG agar medium incubated at 37 C

for 30 days produced electrophoretic patterns that

also gave encouraging results. Although strains of H.

capsulatum yielded different protein patterns in the

 

yeast phase, common fractions among strains were clearly

evident. A strain of E. duboisii gave a protein band

 

95

which was not found in strains of E. capsulatum. This

 

protein band was later shown to be a possible PNPGase.
This marked difference in the yeast phases in comparison
to the mycelial phases of these organisms is significant
for the preliminary screening to find the difference in

E. capsulatum and E. duboisii that lead to extensive

 

 

investigation with a number of isolates of both species.
The protein patterns from the yeast and mycelial phases
of the same strain in the same medium were much different
indicating the unique metabolic patterns of the two
phases of the organism. This result supports Fine (1957)
who hypothesized that the different groups of metabolic
reactions occur for each form.

Survey of Enzyme Activities in Liquid

and Solid Culture Media of
H. Capsulatum and H. Duboisii

 

The survey of enzyme activities in E. capsulatum

 

and E. duboisii followed the method described by Beneke,
EE.Ei° (1969) for detecting the «diffusible enzymes in
different media, both liquid and solid by use of
paranitrophenyl substrates.

In the mycelial phase E. capsulatum and E. duboisii

 

 

 

when grown in CPYG and BHI with 0.1% cysteine, were not
significantly different in enzyme activities with the

substrates used. However, in Sabouraud dextrose broth

 

96

or agar, the mycelial phase of one strain (MSU) of E.

duboisii produced high activity of alpha and beta-PNPGase

 

when the incubation period was increased. In the yeast
phase at 37 C again only one strain of E. duboisii
produced high activities of alpha and beta-PNPGase in
CPYG and PHI with 0.1% cysteine agar media. All but

one strain of E. duboisii_produced PNP alkaline

 

phosphatase while none was produced by any of the E.

capsulatum strains. The strain of E. duboisii at 37 C

 

 

showed a marked difference in theseqphysiological

 

activities from strains of E. capsulatum. It is possible

 

that E. capsulatum strains in the yeast phase have differ-

 

ent metabolic requirements for synthesizing these enzymes,
since in the mycelial phase both species produced high
activities of alpha and beta-PNPGase in BHI with cysteine
and CPYG media. On the other hand the degree of

permeability of E. capsulatum specific for alpha and beta-

 

PNPGase in Sabouraud dextrose broth at 25 C and in CPYG
and BHI broth at 37 C might be lower than that of the

E. duboisii strain.

 

The organisms were grown on an enrichment medium
well supplied with vitamins and amino acids necessary for
growth and synthesis of proteins. On this basis the problem
of variation in enzyme production due to different

nutritional requirements of the organisms should be

97

minimized. By comparison of different enzymatic activities
in the same medium under the same environmental conditions,
the enzymatic activities of one organism can be identified
as differentiated from the others. The strains of both
species produced similar enzymatic patterns. In the

yeast phase of all but one strain of E. duboisii produced
much higher alpha and beta PNPGase and alkaline PNP-

phosphatase than the strains of E. capsulatum. Later,

 

it was found that 3 strains of Histoplasma duboisii

 

produced much higher activities of alpha and beta-
PNP-glucosidases in Sabouraud dextrose agar in 30 days
at 25 C in the mycelial phase and higher activities of
alkaline PNP-phosphatase in CPYG and BHI agar media in
20 days at 37 C in the yeast phase. Although the B-939
strain of E. duboisii did not produce high activities
of alpha and beta PNP-glucosidase in the yeast phase,
alkaline PNP-phosphatase was high. These differences
in enzyme activities of the two species in the yeast
and mycelial phase are indications of physiological

difference in E. cgpsulatum and E. duboisii. Previous

 

studies have shown differences in urease production
(Coremans, 1963), hydrolysis of amino acid (Rosenthal and
Sokolsky, 1965) and hydrolysis of gelatin (Berliner, 1967)
when certain strains were used, however recent studies
with additional strains have shown variable results

(Blumer and Kaufman, 1968).

 

 

98

In regard to physiological difference between

Type A and B of the 4 strains of E. capsulatum, Type A

 

of each strain was 2-3 times higher in activities for
alpha and beta PNPGase and alkaline PNP-phosphatase

than those in type B in BHI medium plus 0.1% cysteine
after 20 days at 25 C. These differences supplement

the others summarized by Berliner (1967) who was the
first to report type A and B occurred in the same strain

of E. capsulatum. White, Garrison and John (1970) found

 

chromatographic differences in histoplasmins derived
from type A and B of the same strain.

Alpha and beta-PNPGases produced by the MSU
strain of E. duboisii in the CPYG medium at 37 C are

not produced by E. capsulatum. A further study of

 

the nature of these enzymes was made to obtain a better
understanding of the physiology of this organism.

The enzymes alpha and beta-PNPGase were found to
be located in the same position in the polyacrylamide
gels so they might be similar proteins which are specific
for either alpha or beta PNP—glucosidase linkage. Other
evidences that supported this assumption were the optimal
pH and the activities at high temperature of both
enzymes were nearly identical. The optimal pHs for the
enzymes were 4.6 and 4.8. The optimal pH of apparently

these same enzymes produced by Polyporus annosus was

 

 

 

 

99

3.5 - 3.9 (Norkraus, 1957), by Schizophyllum commune

 

was 5.4 (Wilson, 1967), and by Collybia velutipes was

 

4.6 - 5.6 (Nonknans, 1957). However, in this investigation
the enzyme preparation was the culture filtrate instead

of using acetone extracted enzyme. Temperature maximum

for these enzymatic reactions was 69 C which was

relatively high when compared with 52 C for the same enzyme

produced extracellularly by Schizonphyllum commune (Wilson

 

and Niederpruem, 1967). Surprisingly, activity of

alpha and beta PNPGases from E. duboisii in CPYG medium

 

 

at 37 C was destroyed completely when held at 60 C for
20 minutes, which is almost similar to PNPGase produced

by ggllybia velutipes (Norkraus, 1957). This enzyme,

 

produced by Polyporus annosus, was destroyed completely

 

by heat at 80 C for 20 minutes (Norkraus, 1957). An

alkaline pH in the culture filtrate of E. duboisii might

 

cause a synergistic effect with the heat in deteriorating
enzyme activity, but in the frozen state (—20 C), the
alkaline pH showed no effect on enzyme activity after
storage of the culture filtrate in the freezer for 90
days, the enzyme activities were still the same as

before storage. In an attempt to determine the essential
factors for PNPGase production in CPYG medium at 37 C,
casamino acids (acid hydrolysate of casein) were sub-
stituted for the essential casein in the CPYG liquid

medium. None or much lower alpha and beta PNPGase

100

activity occurred. In the solid medium in which 1.5%
agar was added to CPYG medium and when casamino acids
were substituted for casein, the activity of alpha and
beta PNPGases were detected but lower than the same
medium with casein. This evidence indicated that the
important factors that induced alpha and beta PNPGase
were in the casein. When casein was acid hydrolysed

to casamino acids these factors were apparently
eliminated by acid hydrolysis. When 1.5% agar (Difco)
was added to this medium, some impurities in the agar
might function as co-factors for enzyme production as

in casein. Such factors could be mineral salts,

enzyme substrates, or even amino acids necessary for
enzyme synthesis, enzyme activator or a certain kind of
vitamin. Generally during acid hydrolysis of protein,
tryptophane is destroyed (Lehninger, 1970). Also, after
acid hydrolysis of casein, the amount of iron was
decreased to 5 micrograms per one gram of casamino acids
(Difco Manual 9th Ed., 1953). These substances were
thought to be essential for PNPGase production: iron
salts such as ferric phosphate, ferric citrate, and
ferrous sulphate; amino acids as tryptophan and serine;
the enzyme substrate, dextrine, and the salt sodium
phosphate. Each was added separately in 1 mg/ml amounts
to CPYG liquid medium in which casamino acids were

substituted for casein. Standardized inocula were used

 

101

in each culture to produce the same amount of the organism.
No differences in enzyme activities were detected in
the filtrate of the media for PNPGases after 15 days
incubation at 37 C. No further attempts were made to
determine the essential factors in the medium for
enzyme production.

D-cellobiose was found to be the carbohydrate
next to glucose that promoted good growth of E. duboisii
and was among the best for production of PNPGases in

culture. It is a beta glucosidic linkage compound which

 

is possibly a natural substate of PNPGase that is

produced by E. duboisii at 37 C. Cellobiose is known

to enhance alpha and beta PNPGase of several fungi in

the Ascomycetes and the Basidiomycetes (Wilson, 1970).
Glucose is an inhibitor for this enzyme production

as the concentration of glucose affected the amount of

inhibition when glucose was mixed with PNPGase. This

agreed with Wilson and Niederpruem (1967) who found that

glucose was the competitive inhibitor of beta-PNPGase

produced by Schizophyllum commune. When varying amounts

 

of glucose were added in CPYG medium and the activities
of alpha and beta PNPGase were assayed, the highest
activities were found when 1.5% glucose was incorporated
in the medium. With either increasing or decreasing
concentration of glucose, the activities of alpha and

beta PNPGases were lower. The concentration of glucose

102

in the medium controls or regulates the production of these
two enzymes. Hauge, MacQuillan and Halvorson (l9 ) also

found that when yeasts (Saccharomyces fragilis X E.

 

dobzamskii) were grown in a synthetic medium with 2%

 

glucose, the production of soluble beta glucosidase was
repressed over 90% when compared with that in synthetic
medium with succinate.

From these results it appears that 1.5% glucose
in the medium provided a suitable energy source for the
E. duboisii to grow more luxurantly than in the medium
containing no glucose or with glucose below 1.5%. If
the medium had above 1.5% glucose, the organisms grew
abundantly but the excess amount of glucose appeared to
repress PNPGase activity and later inhibit PNPGase
activity.

In the limited number of ultra thin sections made

of the mycelial phase of Histoplasma cgpsulatum and

 

E. duboisii, the organelles appeared similar except
for the Woronin bodies in the former. With a more
intensive electron microscopic study of E. duboisii
these bodies may be found.

A Possible Differential Medium for
Histoplasma capsulatum and H. dubOISii

 

 

Comparative study of these two organisms led to

an attempt to develop a medium in which an observable

 

103

change, resulting from a biochemical reaction in the cultures

could be used to differentiate E. duboisii from E. capsulatum.

 

Such a medium would be very useful since there is no way
to differentiate the two organisms $2.2EEEEJ This type
of medium is relatively difficult as considerable variation
occurs among strains of both species.

In the enzyme experiments an unusual reaction
occurred in the MSU strain of E. duboisii. Every time
the iron salts were added to CPYG agar, the culture of
this E. duboisii strain turned a dark purple color after
10 days incubation at 37 C. An assumption then was made
that the iron salt in the medium was reduced to form a
compound of iron and sulphur, such as ferrous sulfide
which was dark purple in color. A reducing agent such as
hydrogen sulfide could be produced by the organism in
CPYG agar. The combination of ferrous sulphate and sodium
thiosulphate were found to be the best hydrogen sulfide
detecting agent in bacterial cultures according to
Sulkin and Willet (1940). They found incorporation of
these agents into CPYG medium which was very good for

supporting the growth of E. capsulatum and Blastomyces

 

 

dermatitidis, should be a useful medium for detecting

 

hydrogen sulfide produced by both organisms. Hydrogen
sulfide blackens the agar medium around or under the colonies,

since hydrogen sulfide reduces ferrous sulfate producing

 

 

104

a dark color from the sulfur-containing compound. Sodium
thiosulphate is the available sulfur source for hydrogen
sulfide production (Sulkim and Willet, 1940). The results
proved that thio medium was usable. Only the colonies of
one strains of E. duboisii (MSU strain) turned purple

color while 5 strains of E. capsulatum did not change in

 

color after 14 days incubation at 37 C. The color of

colonies of E. capsulatum remained white until the end

 

of the experiment (30 days). It was found later that only

ferrous sulfate, added to CPYG agar, was sufficient to

 

develop a purple color in the yeast phase of E. duboisii.
On the other hand ferrous sulfate alone, or a combination
of ferrous sulfate and sodium thiosulfate, added to BHI
agar produced no color change after 30 days incubation

at 37 C as occurred in CPYG medium after 10 day incubation.
This results indicated that sodium thiosulfate was not
the source of sulfur that this fungus used to produce
hydrogen sulfide as is the case in some bacterial
cultures. The reducing agent such as hydrogen sulfide,
if it really existed, probably came from catabolism of
organic sulfur in the CPYG medium. Sulfur amino acids

as cystine or cysteine when oxidized to form pyruvic
acid, one of the pathways is catalized by the enzyme
desulfhydrase (Lehninger, 1970). Thus the free SH groups
might form hydrogen sulfide in the medium. The growth

of E. duboisii at 37 C in BHI agar with either ferrous

 

 

105

sulfate alone or a combination of ferrous sulfate and
sodium thiosulfate could not reduce ferrous sulfate.
This might be due to a deficiency in the sulfur source
of this medium. If this assumption was right the
addition of cysteine, one of the essential sources of

nitrogen for E. capsulatum (Salvin, 19 ) and ferrous

 

sulfate in BHI or CPYG agar should induce the organism

to produce hydrogen sulfide. Escherichia coli was able

 

to produce hydrogen sulfide from cysteine by means of
cysteine sulfhydrase from washed cells and glucose as
a stimulating agent (Anderson 25 El. 1963). For this

reason, colonies of Histoplasma strains that produced

 

hydrogen sulfide on both media should turn a purple
color after a suitable period of incubation. This
results did not occur. All three strains of E. duboisii
did not become purple in color in both media after 10

days incubation while all 5 strains of H. capsulatum like—

 

wise had no color development at the end of 30 days
incubation at 24 and 37 C. This indicated that with
excess cysteine in the medium, production of hydrogen

sulfide is inhibited. The reason E. capsulatum cannot

 

reduce ferrous sulfate has not been determined, however,
there are three pathways in which the process of oxidation

of cystine or cysteine to pyruvic acid occurs. One involves

 

106

the transfer of SH groups to some other compound by the
enzyme transulfhydrase (Lehninger, 1970) so there is
never a chance to form hydrogen sulfide. The oxidation

of these amino acids by E. capsulatum and some strains

 

of E. duboisii might be similar to the thio pathway.

 

Most strains of E. capsulatum changed the color

 

of the casamino acid-yeast extract-cellobiose color
indicator medium to red more rapidly than strains of

E. duboisii in the mycelial phase. It appears that E.

 

capsulatum is able to break down the nitrogenous compounds

 

more readily, releasing ammonia, giving the alkaline
reaction in the medium. This reaction is similar to the
reactions reported by Taplan 35 3;. (1969) on dermatophytes
which produce an alkaline pH in the medium containing
soytone while most contaminant fungi do not. When
cellobiose was replaced with glucose in the medium, the

alkaline pH change occurred more slowly in E. capsulatum

 

for an undertermined reason.
When this medium was used for the yeast phase of
both species, alkaline pH changes in the medium was

evident for strains of E. capsulatum after 24 hours while

 

most strains of E. duboisii required 48 to 72 hours.

 

On the basis of the electrophoretic protein patterns

between strains of E. capsulatum and E. duboisii, the two

 

species appear very closely related. However, alkaline

PNP—phosphatase was consistantly higher in the yeast phase

 

107

of E. duboisii with one exception. Even in this exception,
the strain produced the typical large sized yeast cells

of E. duboisii on CPYG medium at 37 C. Wang EE.il' (1958)
and Pine 33 20° (1964) indicated presence of larger yeast
cells in culture or tissue was significant in E. duboisii.
The enzyme studies and electrophoretic protein patterns

of a number of strains of E. capsulatum and E. duboisii

 

support the suggestion made by Ajello (1968) that the
large yeast cell form of histoplasmosis should be consid-

ered E. capsulatum Darling, 1906 var duboisii (Vanbreuseghem,

 

1952) Ciferri 1960. Additional studies on the enzyme
systems, other biochemical activities, and the discovery

of the sexual reproduction of E. capsulatEm and E.

 

capsulatum var duboisii may lead to a better means of

 

£E_vitro differentiation.

 

 

 

SUMMARY

1. A comparison was made between the soluble

proteins in the culture filtrates of strains of Histoplasma

 

capsulatum and E. duboisii in CPYG, BHI and Sabouraud

 

dextrose liquid and solid media by the disc electro-
phoresis method. The protein bands showed close

relationships between E. capsulatum and E. duboisii, with

 

some variation in patterns among strains of both species.
2. Disc electrophoresis of soluble proteins
extracted from culture filtrates after 20 days at-25 C

from E. capsulatum strain G 184 type "A" and "B", and

 

E. duboisii MSU strain, produced 1, 8, and 4 fractions

 

respectively. After 30 days, type "A" had 3, type "B",
7, and E. duboisii had 5 fractions. Three fractions of
A were common with B, while 3 of the fractions of E.
duboisii were common with B, but not homologous to A.
3. Disc electrophoresis of culture filtrates

at 37 C of E. capsulatum type A produced only 5 fractions,

 

type B, 2, both of which were homologous with those in

type A, while E. duboisii produced 3 fractions, 1 of

 

which was common for type A and B.

108

 

 

 

109

4. Disc electrophoresis of phenol acetic acid
water cell extract of 2 species grown on CPYG medium at
37 C showed the band patterns were very similar for 9

strains of E. capsulatum and 5 strains of E. duboisii.

 

Histoplasma capsulatum type A and type B had almost

 

identical patterns with a range of 8 to 10 bands.
5. Paranitrophenol substrates were used to assay
the culture filtrates and agar media for enzyme activities

form E. gapsulatum and E. duboisii. In the mycelial phase

 

both species produced high enzyme activities in PNP-
alpha- and PNP-beta—D-glucoside substrates. Significantly,

E. capsulatum type A had 2 - 8 times higher activity in

 

BHI agar than type B for alpha-and beta-PNPGases. In
the yeast phase at 37 C only E. duboisii had high enzyme
activities for PNP-alpha and PNP—beta-D-glucoside.

6. The kinetics, inhibitors, optimal temperature,
and pH were studied for alpha- and beta-PNP-glucosidases.
These two enzymes were found to be located in the same
fraction in acrylamide gels.

7. High PNP-alkaline phosphatase was produced
by E. duboisii strains in CPYG agar at 37 C while it was

low in E. capsulatum strains.

 

8. The study of the fine structure of the young

hyphae of E. duboisii showed similar organelles to those

 

in E. capsulatum as reported in the literature. Woronin

 

 

110

bodies were found near the septa in the hyphae of E.

capsulatum, but none in the limited number of sections

 

made for E. duboisii.

 

9. Many strains £0 E. capsulatum changed the pH

 

in the medium more rapidly than most strains of E. duboisii.

 

With a phenol red indicator in the medium, E. capsulatum

 

changed the color of the modified CPYG medium to red

while most strains of E. duboisii did not change the yellow

 

color in the medium.

10. The similarities in enzyme studies and electro-
phoretic protein patterns in the two species appears to
support the proposal that the large yeast cell of

histoplasmosis should be considered E. capsulatum Darling,

 

1906 var. duboisii (Vanbreuseghem, 1950) Ciferri 1960.

 

 

 

BIBLIOGRAPHY

111

 

 

BIBLIOGRAPHY

Ajello, L. 1968. Comparative morphology and immunology
of members of the genus Histoplasma. Mykosen,
ll(7):507—514.

 

Anderson, D. C. and K. R. Johanson. 1963. Effect of
glucose upon formation of hydrogen sulfide from
cysteine by Escherichia coli. J. Gen. Microbiol.
30:485-495.

 

Andrieu, S.; J. Biguet; L. Dujardin and Vaucelle. 1969.
I. Histoplasma capsulatum et H. duboisii
relations aVec H. farciminosumT Gymnoascus
demonbreunii, BIastomyces dermatitidis et
Paracoccidioides brasiliensis. Mycopathologia,
39:97-108.

 

 

 

 

 

 

 

 

Berliner, M. D. 1967. Gelatin hydrolysis for identifi-
cation of the filamentous phase of Histoplasma,
Blastomyces, and Chrysosporium. Sabouraudia, 5:
274—277.

 

 

 

 

1968. Primary subcultures of Histoplasma
capsulatum, I. Macro and micro-morphology of
the mycelial phase. Sabouraudia, 6:111-118.

 

 

Beneke, E. S.; R. W. Wilson and A. L. Rogers. 1969.
Extracellular enzymes and Blastomyces dermatitidis.
Mycopathologia, 39:325—328.

 

 

Binford, C. H. 1955. Tissue reactions and morphologic
variations of the fungus. American and Clin.
Path. 25:25-36.

 

 

 

 

 

 

 

Blumer, S. and L. Kaufman. 1968. Variation in enzymatic
activities among isolates of Histoplasma
capsulatum and Histoplasma duboiSii. Sabouraudia,
6:203-206.

Chang, L. O.; A. M. Srb and F. C. Steward. 1962.
Electropharetic separations of the soluble

proteins of Neurospora. Nature (Lond.)l93:
756—759.

 

112

113

Clare, B. G. 1963. Starch-gel electrophoresis of
proteins as an aid in identifying fungi. Nature.
200:803-804.

Conant, N. F. 1941. A cultural study of the life
cycle of Histoplasma capsulatum. Darling. J.
Bact., 41:563-579.

 

 

Coremans, J. 1963. Biochemical test for differentiation
of Histoplasma duboisii Vanbrueseghem 1952 from
Histoplasma capsulatum Darling 1906. C. R. Soc.
Biol. (Paris) 157:1130-1132.

 

 

 

and J. F. Kessel. 1931. Histoplasmosis
(Darling) without splenomengaly. Am. J. Trop.
Med. 11:435—449.

 

Daniels, L. S.; M. D. Berliner and C. C. Campbell. 1968.
Varying virulence in rabbits infected with
different filamentous types of Histoplasma
capsulatum. J. Bact., 96:1535-1539.

 

 

Darling, S. T. 1906. A protozoon general infection
producing pseudotubercles in the liver, spleen
and lymphnodes. J.A.M.A., 46:1283-1285.

Davis, B. J. 1964. Disc electrophoresis. II. Methods
and applications to human serum proteins. Ann.
N.Y. Acad. Sci., 121:404-427.

 

DeMonbreun, W. A. 1934. The cultivation and cultural
characteristics of Darling's Histoplasma
capsulatum. Amer. J. Trop. Med., 14:93-125.

 

 

 

Difco Manual of Dehydrated Culture Media and Reagents.
9th ed., p. 265, Difco Laboratory, Detroit,
Michigan.

Dowding, E. S. 1948. The spores of Histoplasma.
Canad. J. Res., 26:265—273.

 

. 1967. The use of immuno-electrophoresis
analysis in the standardization of fungal
allegens. Standard., 4:169-176.

Drouhet, E. and J. Schwarz. 1956. Comparative studies
with 18 strains of Histoplasma. J. Lab. Clin.
Med., 47:128—139.

 

 

114

Duncan, J. T. 1958. Tropical African histoplasmosis.
Trans. Roy. Soc. Trop. Med. Hyg., 52:468-474.

 

Durbin, R. D. 1966. Comparative gel-electrophoretic
investigation of the protein patterns of
Septoria species. Nature, 210:1186-1187.

Edwards, M. R.; E. L. Hazen and G. A. Edwards. 1959.
The fine structure of the yeast-like cells of

Histoplasma in culture. J. Gen. Microbiol.,
20:496—503.

 

. . and . 1960. The micromorphology
of the tuberculate spores of Histoplasma capsulatum.
Canad. J. Microbiol. 6. 65- 69.

 

 

Garrison, R. G.; J. W. Lane and M. F. Field. 1970.
Ultrastructural changes during the yeast-like
to mycelial phase conversion of Blastomyces
dermatitidis and Histoplasma capsulatum.

J. Bact., 101:628—635.

 

 

 

 

Goos, R. D. 1964. Germination of the macroconidium of
Histoplasma capsulatum. Mycologia, 56:662—671.

 

 

Hauge, J. G.; A. M. MacQuillan; A. L. Cline and H. O.
Halvorson. 1961. The effect of glucose repression
on the level of ribosomal—bound B glucosidase.
Biochem. Biophys. Res. Comm" 5:267—269.

 

Howell, A., Jr. 1939. Studies on Histoplasm§_capsulatum
and similar form species. I. Morphology and
development. Mycologia, 31:191-216.

 

 

Kaufman, L. and S. Blumer. 1966. Occurrence of serotypes
among Histoplasma capsulatum strains. J. Bact.,
91:1434-1439.

 

and . 1968. Development and use of
a polyvalent conjugate to differentiate Histoplasma
capsulatum and Histoplasma duboisii from other
pathogen. J. Bact., 95:1243-1246.

 

 

 

Korn, E. D. 1966. Structure of biological membranes.
Science, 153:1491—1498.

 

 

115

Lehninger, A. L. 1970. Pathways leading to acetyl co A,
p. 440. In A. L. Lehninger, Biochemistry. New
York, N. Y. 10010.

Leone, C. A. (ed.) 1964. Taxonomic Biochemistry and
Serology. The Ronald Press Co., New York.

Lipman, R. P.; W. A. Clyde, Jr. and F. W. Denny. 1969.
Characteristics of virulent attenuated, and
avirulent Mycoplasma pneumonial strains. E.
Bact.,100:1037-1043.

Lowry, O. H.; N. J. Rosebrough; A. L. Farr and R. J.
Randall. 1951. Protein measurement with the
Folin phenol reagent. J. Biol. Chem., 193:265-275.

 

Norkrans, B. 1957. Studies of B-glucoside and cellulos-
splitting enzymes from different strains of
Collybia velutipes. Physiol. Plant., 10:454-465.

 

 

 

. 1957. Studies of B-glucoside and cellulose
splitting enzymes from Polyporus amnosus Fr.
Physiol. Plant., 10:198-213.

 

 

Olurin, O.; A. O. Lucas and A. B. O. Oyediran. 1969.
Orbital histoplasmosis due to Histoplasma
duboisii. Am. J. Ophth., 68:14-18.

 

 

Ornstein, L. and B. J. Davis. 1962. Disc electrophoresis.
Rochester, N. Y., Distillation Products Industries,
Eastman Kodak Co.

Pine, L. 1970. Physiological and chemical character-
istics of strains of Histoplasma capsulatum
and Histoplasma duboisii. X. Internat. Congress
Microbiol. Id:6:155. (Abstr.)

 

Pine, L.; E. Drouhet and G. Reynold. 1964. A Comparative
morphological study of the yeast phase of
Histoplasma capsulatum and Histoplasg§_duboisii.
Sabrouadia, 3:211-224.

 

. 1957. Studies on the growth of Histoplasma
capsulatum. III. Effect of Thiamine and
other Vitamins on the growth of the yeast and
mycelial phases of Histoplasma capsulatum.

J. Bact., 74:239-245.

 

 

 

1954. Studies on the growth of HistOplasma
capsulatum. I. Growth of the yeast phase in
liquid media. J. Bact., 68:671-679.

 

 

111i

 

116

Razin, S. and S. Rottem. 1967. Identification of
Mycoplasma and other microorganisms by polyacrylamide-
gel electrophoresis of cell proteins. J. Bact.,
94:1807—1810.

Reichle, R. E. and J. V. Alexander. 1965. Multiperforate
septations, Woronin bodies, and septal plugs in
Fusarium. J. Cell. Biol., 24:489-496.

Rosenthal, S. A. and H. Sokolsky. 1965. Enzymatic
studies with pathogenic fungi. Dermat. Int.,
4:72-79.

Rottem, S. and S. Razin. 1967. Electrophoretic patterns
of membrane proteins of Mycoplasma. J. Bact.,
94:359-364.

Rowley, D. A. and L. Pine. 1955. Some nutritional
factors influencing growth of yeast cells of
Histoplasma capsulatum to mycelial colonies.
J. Bact., 69:695-700.

 

 

Salvin, S. B. 1949. Cysteine and related compounds
in the growth of the yeast-like phase of
Histoplasma capsulatum. J. Infect. Diseases.,
84:275-283.

Schwarz, J. 1953. Giant forms of Histoplasma capsulatum
in tissue explants. Am. J. Clin. Path., 23:898-

903.

Shechter, Y.; J. W. Landau; N. Dabrowa and V. D. Newcomer.
1968. Disc electrophoretic studies of intraspecific
variability of protein from dermatophytes.
Sabouraudia, 6:133-137.

 

.; J. W. Landau and V. D. Newcomer. 1967. Disc
electrophoretic studies of proteins from
Coccidioides immitis. J. Invest. Derm., 48:119-
123.

 

.; ; N. Dabrowa and V. D. Newcomer. 1966.
Comparative disc electrophoretic studies of
proteins from dermatophytes. Sabouraudig, 5:
144-169.

 

Stipes, R. J. 1970. Comparative mycelial protein and
enzyme patterns in four species of Ceratocystis.
Mycologia, 62:987-995.

 

117

Sulkin, S. E. and J. C. Willett. 1939. A triple sugar
ferrous sulfate medium for use in identification
of enteric organisms. J. Lab. and Clin Med.,
25:649-653.

 

Takayama, K.; D. H. MacLennan; A. Tzagoloff and C. D.
Stoner. 1966. Studies on the electron transfer
system LXVII. Polyacrylamide gel electrophoresis
of the mitochondrial transfer complexes. Arch.
Biochem. Biophys., 114:223-230.

 

Taplin, D.; N. Zaias; G. Rebell and H. Blank. 1969.
Isolation and recognition of dermatophytes on a
new medium (D.T.M.), Arch. Derm".99:203-206.

 

Vanbreuseghem, R. 1953. Histoplasma duboisii and African
histoplasmosis. Mycologia., 45:80 - l6.

 

 

Wang, C. J. K.; J. Schwarz and G. L. Baum. 1958. Large
forms of Histoplasma. Mycopathologia, 10:53-70.

 

 

White, T. G.; R. G. Garrison and L. E. Johns. 1970.
Chromatography of Histoplasmin derived from the
albino and brown filamentous varieties of
Histoplasma capsulatum. Bacteriolog. Proc.

The 70th Annual meeting Amer. Soc. of Microbiol.

Whipple, H. E. (ed.) 1964. Gel electrophosesis. Ann.
N. Y. Acad. Sci., 121:305-650.

Wilson, R. W. 1970. Influence of cellabiose on the
colonial morphology and glycosidases of
several fungi. Can. J. Microbiol., 16:629-634.

 

. and D. J. Niederpruem. 1967. Control of
-glucosidases in Schizoph llum commune. Can.
J. Microbiol., 13:1009-1020.

 

 

 

 

 

 

23

3 03062 2

 

(111111