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This is to certify that the dissertation entitled Protein Expression During the low Calcium Response in Yersiniae presented by Richard Joseph benign has been accepted towards fulfillment of the requirements for I h .13- . degree in May flaw/6W Major professor I Dateé/p/7/ MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 F LIBRARY Michigan State 1 University ~— J fi. PLACE IN RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or before dde due. =_——_——_1 DATE DUE DATE DUE DATE DUE MSU lo An Nflrmetlve ActiorVEquel Opportunlly Institution ammo-91 PROTEIN EXPRESSION DURING THE LOW CALCIUM RESPONSE IN YERSINIAE BY Richard Joseph Mehigh A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1991 ABSTRACT PROTEIN EXPRESSION DURING THE LOW CALCIUM RESPONSE IN YERSINAE BY Richard Joseph Mehigh The plasmid-mediated low calcium response (Lcr) occurs when pathogenic yersiniae are grown in yitzg at 37'C in Ca2+-deficient media and is characterized by a stepdown in vegetative growth accompanied by induction of Lcr-specific virulence factors. The latter include Lcr plasmid-encoded outer membrane peptides (Yops) that undergo hydrolysis in legginia pestig due to the plasminogen activator/coagulase (PAC) activity encoded on the pesticin plasmid which is not found in the enteropathogenic strains. In this report, protein expression was examined to determine the production and degradation of important Lcr-mediated peptides and also to identify the production of other virulence factors or auxiliary proteins during the Lcr. Using [3581-methionine in pulse-chase experiments, important Yops of 1‘ pestis were found to be maintained in the steady state or exist as stable degradation products. Using the above method, a mania Wis strain carrying the pesticin plasmid exhibited a similar Yop degradation pattern as I; pestifi. Differences in this x&,p§gggg§gpergnlg§1§ strain include the presence of a major Yop that was only partially degraded and a species specific adhesion protein (YOp A) that exhibited a discrete, stepwise degradation pattern. Pulse-chase studies of x; pestig revealed the production of two major heat-shock protein occurs before onset of Lcr. Radiolabeling of X; pgstis during the Lcr revealed the production of several major stable peptides. These cytoplasmic peptides were labeled and then fractionated on various columns for identification of the peptides produced during the Lcr. Correlation of the peptides produced during the Lcr with known products produced by X; pestig was done mainly by immunoblotting. Products identified include the Lcr plasmid-encoded V antigen and an unknown 20 kilodalton peptide, the pesticin plasmid-encoded pesticin and a 35 kilodalton degradation product of the PAC, and the 110 kb cryptic plasmid-encoded capsular antigen (fraction 1) and the plague murine exotoxin. Chromosomally-encoded peptides of 70 and 56 kilodaltons were found to be a novel basic peptide with catalase activity and the yersiniae equivalent to the E; ggli_Gro EL heat shock peptide, respectively. To my Mom and Dad who brought me into the world and helped me whenever I needed it and to my lovely wife, Chris, whose love and support made me a complete person. iv ACKNOWLEDGMENTS I would like to thank the members of my committee: Dr Wendy Champness, Dr. Julius Jackson, Dr. Richard Schwartz, and Dr. Barbara Styrt for all of their help and understanding. I would also like to thank my advisor, Dr. Robert R. Brubaker for his help through the years and for all the countless tales told by him. TABLE OF CONTENTS Page LIST OF TABLES O O ....... O O O O O C O O C O I I O OOOOOOOOOOOOOOOOOOO v i i 1 LIST OF FIGURES . O O C O O O I O O O O O O O O C O O O O O O O O O O O O O ......... O O 0 ix INTRODUCTION 00.000.00.000.000000000000000000000......O....1 CHAPTER 1 LITERATURE REVIEW .........................................3 Purine Biosynthesis ....................................5 Fraction 1 .............................................7 Pigmentation ...........................................8 Pesticin Plasmid ......................................11 Lcr plasmid .................................... ....... 13 References ....................................... ..... 20 CHAPTER 2 ARTICLE: Expression of the low calcium response in Yersinia DQSLIS ...................................................28 Abstract ........................... ......... . ......... 29 Introduction .. ........ ........ ...... ..................30 Materials and Methods .................................32 Results ...............................................35 Discussion ............................ ....... . ..... ...51 References .................................. ..... .....58 CHAPTER 3 ARTICLE: Lcr Plasmid-mediated Protein Expression and Processing in a Pesticinogenic legsinig pesgdotuberculosis Strain OOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOO0.0.064 Abstract ..............................................65 Introduction ..........................................66 Materials and Methods .................................69 Results ...............................................72 Discussion ............................................84 References ............................................90 vi CHAPTER 4 ARTICLE: Major stable peptides of legginia pestis produced during the low calcium response ..........................96 Abstract ..............................................97 Introduction .................................. ........ 98 Materials and Methods ................................100 Results ..............................................105 Discussion ...........................................123 References ...........................................129 SUMMARY AND CONCLUSIONS ............. ........ . ........... 134 vii LIST OF TABLES Tahlei mg CHAPTER 1 1 Virulence factors of Y. pestis....................6 CHAPTER 2 1 L050 of yng and ygpq mutant x; pestis strains....44 CHAPTER 3 1 Assays Pesticin Plasmid associated activities.....74 CHAPTER 4 1 Purification of p70 catalase......... ......... ...121 viii LIST OF FIGURES EIQHBE_1 BAQE CHAPTER 2 1 Long term pulse-chase of Lcr+, Pst+ x; pg§;1§.. ..... 37 2 Long term pulse-chase of Lcr+, Pst’ I; pggtis.......38 3 Pulse-chase of Pst+ ygpfi and 209; mutant strains....41 4 Pulse—chase of Pst' yopE and ygpg mutant strains....42 5 Pulse-chase of 1; pestis into restriction...... ..... 48 6 Immunoblot and autoradiogram of p56 and p70 ........ .50 CHAPTER 3 1 Pesticin plasmid map................................73 2 Stained gel of isolates of 1&pgegggtgbgrgglgsis and hmstrainSOOOOOOOOOO...IOOOOOOOOOOOIOOO0.0.0.76 3 Yop immunoblot of isolates of 1&pgguggtubergulgsis and 1; pggtis strains...............................78 Pulse-chase of I; pseggoggberculosis................81 Pulse-chase of x; pestis.................. ...... ....82 01:5 CHAPTER 4 Pulse-chase of Lcr+ and Lcr' 1; pesti .... .......... 106 Column and radioactivity profiles............ ..... ..109 Protein profile of Sepharose A-1.S..................111 Protein profile of DEAE cellulose...................112 Protein profile of calcium hydroxyapatite...........113 Immunoblot of x; pestis cytoplasm for known virulence factors.............................................117 Immunoblot with anti-p56............................116 2-D gels and blot of p56 and W antigen..............118 Protein purification profile of p70.... ........ .....120 0 Cytoplasmic catalase column profile........ ....... ..122 GUIbthH 0"me ix INTRODUCTION There are three Yersinia species pathogenic for humans; legginig pggtig, the causative agent of bubonic plague, and the enteropathogenic yersinia, x; gntgxggglitiga and 1; pgguggtubezgglggig. These three gram-negative organisms are facultative intracellular parasites. An important virulence factor common to all three is the carriage of a approximate 70 kb plasmid that mediates the low calcium response (Lcr). The Lcr occurs when the cells are grown at 37’C in media lacking Ca2+, whereby the organisms undergo a slow down or a restriction of growth and the induction of Lcr plasmid- mediated virulence factors. During this restriction of growth in 1.2estis, only the Lcr-mediated peptides are produced while most vegetative protein production is shutdown. This plasmid encodes several virulence factors that are necessary for virulence of this organism, since loss of this plasmid results in avirulence of these pathogens. A subset of these Lcr-mediated virulence factors termed Yops (yersiniae guter membrane peptides) undergo hydrolysis only in X‘,p§§§1§ due to the presence of a second plasmid, the 1 pesticin plasmid. This pesticin plasmid encodes a plasminogen activator/coagulase (PAC) that has fibronlytic activity and it degrades the Yops. Most of the research involving the low calcium response has been to identify the role of these Yops and the regulation of expression of the Lcr. This dissertation continues to examine the production and degradation of Yops in I; pegtis and also examines Yop degradation in a second strain of yersiniae, 1; seu e s' . In addition to the Lcr, 1; pestis has other discrete virulence factors the are necessary for virulence of the organism. Since most protein production ceases during the low calcium response, it was of interest to see what other kinds of proteins are produced during the Lcr. Major stable proteins of x; pestis were examined and identified to see if they were virulence factors or if they were proteins necessary for survival of the organism. CHAPTER 1 LITERATURE REVIEW legginia pestig, the etiological agent of bubonic plague, is a gram negative bacterium that is capable of causing a fatal infection in mice when only about ten organisms are injected subcutanously (68). X; pestis is a facultative intracellular parasite that is able to multiply within mammalian phagocytic cells, (24) usually macrophages. Alexander Yersin was the first person to correctly identify this bacterium as the causative agent of bubonic plague during an epidemic in Hong Kong in 1894 (76). This organism was assigned to a number of different genera until 1970 when it was assigned its own genus, Xersinia, to honor the discovery by Dr. Yersin and that the organism is unique to other known pathogens. The primary transmission of the plaque to man is through a flea bite. The fleas feed on an infected host which is carrying the organism in its blood. The blood coagulates in the flea and blocks its ability to ingest more blood. This distressed flea abandons its normal host and attempts to feed on other hosts. The flea regurgitates the bacteria into the bloodstream of the new host where they travel to the lymphatic system. They gather in lymph nodes and begin 3 4 to multiply intracellularly until the lymph nodes are tender and swollen. Eventually the bacteria swamp the lymph system and spill into the blood creating a septicemia. Death usually follows within a few days if the infection remains untreated due to the large number of bacteria present in the host. Another form of the disease is pneumonic plague which is caused by airborne bacteria that infect the lungs. This results in a rapid pulmonary infection that is usually fatal. There are two other yersiniae species that are pathogenic for man: Xersinia enterggglitiga and Yezsinia psgyggtubergglggis. Both of these species are closely related to X; pgstis but they are much less virulent (68). The normal route of infection of these organism is through contaminated food or water thus eliminating the need for the highly invasive properties found in x; pestis. They cause prolonged enteric diseases with fever and diarrhea or an appendicitis-like syndrome. Yersiniae are susceptible to a number of antibiotics, thus these types of infections can be controlled, but these organisms serve as an excellent model system. There have been many important discoveries made in bacterial pathogenesis using the yersiniae system. Plasmid borne invasive functions being genetically linked to bacteriocin production was first found in x; pestis (12). Iron uptake by a non-siderophore mechanism and the ability to utilize 5 exogenous hemin as a sole source of iron were both initially reported in I; pestig (47) as well as the importance of iron privation as a non-specific mechanism of host defense (6). Other important discoveries include: (i) plasmids can mediate bacterial invasiveness (11,30,42), (ii) mutations in purine biosynthesis but not other metabolites can cause avirulence (13,17), and (iii) the ability to absorb exogenous hemin or congo red serves a virulence function (38,62). legginia pestis serves as an important model for facultative intracellular parasites. Avirulence can result from the loss of any one of several distinct virulence factors as shown in Table 1. The different virulence factors give different degrees of avirulence as measured by the lethal dose needed to kill half of the experimental animals (LD50 values). Each of these virulence factors is explained in more detail below. PURINB BIOBYNTHEBIB The ability to synthesize purines (Pur+) is a requirement of all pathogens. This virulence factor has been reported for many pathogens including 11 pestis (17). It is assumed that free purines are not available in the mammalian host cell and that they must be manufactured ge 3939 by the pathogen in order to proliferate (1). Thus, the loss of the ability to produce them results in avirulence of Table 1 Virulence factors of 1g3§inig peggig and their effect on the LD50 value for guinea pigs and mice Virulence Factora LD50" Lcr Pst Pgm Fra Pur mouse guinea pig mouse + p63 + c + + + + + < 10 < 10 < 10 0 + + + + >107 >108 >107 4- O + + + -105 - 106 < 10 + + O + + > 107 > 103 < 10 + + + O + < 10 -104 <10 + + + + od «102 ~104 ~102 1‘ + 1’ + 0c > 107 > 108 > 10‘7 3abbreviations for Virulence Factors Lcr= Carriage of the Lcr Plasmid Pst= Carriage of the Pesticin Plasmid Pgm = ability to absorb exogenous hemin (pigmentation) Fra = Fraction 1 (capsular antigen) Pur= Purine Biosynthesis bintraperitoneal injection cSufficient iron injected to saturate serum transferrin dMutation blocks Purine biosynthesis before Inosine monophosphate eMutation blocks Purine biosynthesis after Inosine monophosphate .7 the organism. Evidence to support this idea came from in yitrg infection of cultured macrophages with 1; pggtis. Purine auxotrophs were unable to survive or replicate unless hypoxanthine or guanosine were added to the media while Pur+ strains were able to grow in the macrophage (63). The location of the mutation in the purine biosynthetic pathway is important in the loss of virulence (Table 1). If the block occurs before the synthesis of inosine monophosphate (IMP), an early step in purine biosynthesis, then there is only a slight reduction in the pathogencity of the organism for mice. However, If the block occurs in the conversion of IMP to guanosine monophosphate, then the strain becomes completely avirulent in mice (13). PRACTICN 1 Fraction 1 (Fra+) represents the highly immunogenic capsular antigen of x; pestis which has been purified and described as a protein-carbohydrate complex (2,5). The subunits are approximately 15 kdal which consists of a protein bound to a small carbohydrate described as an oligomeric galactan (5). These subunits form structures up to 300 Kdal in size and can be dissociated into subunits with SDS and upon removal of SDS will spontaneously reform large structures (5). Its production is temperature dependent with maximum production at 37'C and only small 8 levels detected at lower temperatures (5). Mutation to Fra reportedly occurs at a high frequency both in 2129 and in yigzg (20,50) which may verify its reported existence on the 110 kb cryptic plasmid in 1‘ pggtis (30,51). There is a second class of mutations designated F1: (21) which produces fraction 1 but fails to incorporate the antigen into an extracellular capsular structure. The exact role of fraction 1 during an infection is unclear. When Fra‘ strains of 1; pestis are injected intraperitoneally into guinea pigs there was reduced virulence (20) but the organism remained fully virulent for mice(14) (Table 1). Visible encapsulation of X; pestig had no effect on resistance to ingestion by murine phagocytes ip yiyg (19). It has also been reported that Fra' strains were able to cause chronic and sometimes lethal infections in laboratory rats(73,74). There has also been a F11 1; pgstis strain isolated from a plague patient (75). All of this evidence casts a doubt on whether or not fraction 1 is an important virulence factor in humans. PIGMBNTATION Virulent strains of I; pggtig usually form pigmented colonies (Pgm+) when grown on semi-solid media containing hemin (38) or Congo red(66). The importance of this observation was not known until nonpigmented isolates were 9 found to be avirulent by subcutaneous or intraperitoneal routes of injection (39,68) but were still fully virulent when injected intravenously (68). These nonpigmented strains could become fully virulent by peripheral routes if sufficient iron was injected to saturate serum transferrin of the host (39) (Table 1). Hence, pigmentation was then thought to play a role in iron acquisition in the mammalian host. Iron is found in very minute amounts in the extracellular environment of a mammal, so for invading organisms to proliferate in this niche they must have some type of high affinity iron uptake system. Many organisms use excreted low molecular weight peptides, termed siderophores, that chelate iron and then are taken up by the pathogen. In iron limiting conditions, yersiniae do not produce siderophores and only the enteropathogenic strains are capable of using them as an iron source (49). I; pestis is capable of growth with ferritin, hemin, and hemin- containing proteins but not transferrin or lactoferrin (57). When strains of XL pestis are grown in iron limiting conditions, nonpigmented strains can only grow for a few generations while the pigmented strains grow for at least 16 generations (56). This evidence has led to the theory that yersiniae have some type of cell-bound high-affinity iron uptake system that is lost upon mutation to nonpigmented. 10 This mutation may be part of the uptake system or found in a iron storage function. Recent work in this area has revealed that a deletion occurs in the chromosome (unpublished data) upon mutation to the nonpigmented phenotype which results in the loss of 5 outer membrane peptides (57). The peptides that are lost are only produced under iron privation and are repressed when sufficient quantities of iron is present (57). This would be expected if the peptides were needed for iron uptake under iron limiting conditions. A mutant strain that contains one of these peptides, but not the other four, has been correlated with the pigmentation phenotype, but this peptide alone does not confer growth in iron limiting conditions (57). The enteropathogenic yersiniae were not found to be pigmented (66), on a modified Congo red medium except for some clinical isolates of Xersinia enterggglitiga (52). This was a different type of pigmentation which correlated with the presence of the Lcr plasmid and was limited to only virulent isolates of 1; gntgzggglitiga (52). Pigmentation has also been found in virulent strains of E; coli, Shiggllg species and neiggeria meningitigig and may play a similar role in these organisms (48). 11 PEBTICIN PLABMID The pesticin plasmid is a 9.5 kb plasmid named after the first product to be associated with the plasmid: the bacteriocin pesticin. Pesticin is a 44 kilodalton protein that has N-acetylglucosaminidase activity (29) and it has been shown to inhibit the in 2129 incorporation of diaminopimelic acid into the peptidogylcan of E; ggli 0. It has been shown to promote the in yitrg and in 2119 release of diaminopimelic acid from peptidoglycan. Strains of 11 pestig that produce pesticin also produce an inhibitor of pesticin termed immunity protein, which prevents self-destruction (9). Strains of 1; pestig lacking the pesticin plasmid may or may not be sensitive to pesticin. A majority of cells resistant to pesticin were also found to be nonpigmented. One of the outer membrane peptides that is lost upon mutation to nonpigmented apparently acts as the uptake protein for pesticin (57). Coagulase and fibrinolysin activities were also linked to the production of pesticin (4,22) and eventually to the pesticin plasmid (62). These two activities were found to be accomplished by the same protein (59). This outer membrane peptide is found as a 37 kilodalton protein and a 35 kilodalton degradation product (59). It is not known if only one or both of these peptides has the activities or if the activity is different for each of the peptides. It has been shown that coagulase activity is predominate at 12 temperatures below 30°C while the fibrinolytic activity is greater at higher temperatures (45). This temperature dependence may cause the blockage in the flea whose body temperature is lower than that of mammals. X; pestifi strains that lack the pesticin plasmid show a drop in virulence (Table 1), especially by the peripheral routes, which may indicate that the pesticin plasmid is responsible for the invasiveness of the organism (11,67). The LD50 for mice of a wild type strain of 1‘ pestis is less than ten organisms by any method of injection. An isogenic strain lacking the pesticin plasmid retains a similar LD50 (71 organisms) by the intravenous route of injection, an intermediate LD50 (3.8x105) by intraperitoneal injection and is completely avirulent by the subcuteneous route. The virulence of this mutant strain could be restored by the intraperitoneal route but not the subcutenous by injecting the mice with an excess of iron before the challenge (11). This effect of iron is similar to that reported for the non- pigmented mutation (39) but there are no iron regulated proteins found on the pesticin plasmid (S7). The effect in this case may reflect suppression of non-specific mechanisms of host defense rather than a loss of iron acquisition ability (56,70,72). The loss of these mechanisms may give the pathogen enough time to find a suitable host cell in which to replicate. The only function specific to the pesticin plasmid that may be responsible for the 13 invasiveness is the fibrolytic activity. More work is needed to determine the exact function the pesticin plasmid plays in the mammalian host during infection. LCR PLABMID Early in yitrg research on X; pegtis was hampered by an unusual switch to avirulence of the cultures when the organisms were grown at 37'C in standard culture media. The virulent organisms failed to replicate and lysed under these conditions and were quickly overgrown by strains lacking the Lcr plasmid. Addition of 22 mM Mg2+ could prevent lysis but it was eventually found that 2-4 mM Ca2+ would allow the virulent strains to grow at this temperature (37). What made this problem so baffling was both strains grew equally well at 26'C irregardless of the calcium concentration. It was later found that zinc and strontium also allowed the virulent cells to grow at 37'C. The severity of the growth restriction was also found to depend on the Mg2+ concentration. The lower the level of Mg2+ the lower the levels of Ca2+ needed to allow the cells to grow (10). Using this information, a medium was developed to mimic mammalian intracellular fluid with respect to divalent cations (20 mM Mg2+, 0 mM Ca2+). When virulent I; pesgis are grown in this medium at 37°C, they undergo restriction of vegetative growth and begin synthesizing a set of 14 virulence factors. This change is referred to as the low calcium response (LCR) (34). The LCR is mediated by an approximate 75 kilobase plasmid (lcr+) common to all pathogenic yersinae. When 1cr+ cells, growing logarithmically at 26°C in calcium-deficient media, are switched to 37°C the LCR is induced. The first known change in the cells is a termination of stable ribonucleic acid (rRNA) synthesis but not necessarily messenger RNA (25). There is also a reduction in adenylate energy charge and a blockage in DNA synthesis initiation (79). Addition of exogenous adenine triphosphate (ATP) could relax the restriction of growth but the ATP was not metabolized. It is believed that the ATP acts as a chelator of the magnesium thus lowering the stringency of the restriction (80). The LCR appears to be an ordered metabolic shutdown resulting from a block in stable RNA synthesis (25). The exact mechanism of how this plasmid restricts growth and begins producing virulence factors is not known. There is a 17-kb region on the 1cr plasmid which seems to control the regulation of temperature and calcium. Transposon insertions in this region result in Ca2+-independent mutants which do not require Ca2+ for growth at 37°C (26,27,34,77). These mutants also lose their ability to produce Lcr+- specific virulence factors and the resulting avirulent strains are similar to Lcr' strains. There are several 15 different loci identified with this region termed lgrA to minhnestiewm andxirAtoxirEinLentemseLitiga (26,27). The 1925 locus was further divided into 1919 and 1923 loci(78). There were mutants found within the lgrg locus that were termed Ca2+-blind (78). This mutant showed a constitutive lcr phenotype and its growth is restricted at 37°C even in the presence of 2.5 mM Ca2+ (78). A 3.8-kb fragment of this locus was able to complement this mutation and was designated 19:3. In the lgrE locus the mutation was mapped to the same location as the structural gene for an 1cr encoded protein termed Yop N (71). Upon further investigation it was found that Yop N (renamed LEIE for its role in regulation of the LCR) was the first gene in an operon of six genes (71). The other five genes have no known function but are able to produce proteins(71). The 213E gene has been shown to be a transcriptional activator controlling the lcr virulence genes (28). The expression of this protein is increased at 37°C and is probably responsible for the production of Yops, while Ca2+ concentration has no effect on the expression of this gene(28). In addition to this regulatory area, at least one important gene in virulence, Yop E, has a regulatory protein located just upstream from its coding sequence (33). Also one of the Lcr proteins, encoded on the plasmid, appears to be needed for export of the outer membrane proteins that are 16 produced (53). Therefore, the regulation of the LCR is very complex and will take a long time to decipher. In addition to the restriction of growth there is a set of virulence proteins that is only made under these restrictive conditions (37°C, No Ca3+). These virulence factors are important, since loss of the plasmid results in complete avirulence of the organism (Table 1). V and W antigens were shown to be produced only by virulent strains of I; pgstis both in yitzg and in infected laboratory animals (18). These proteins were eventually found to be produced only under the conditions needed to induce the LCR and production was elevated when high levels (>20mM) of magnesium were present (10,43). It was shown that antibodies specific for V antigen could passively protect mice from intraperitoneal challenge with a lethal dose of 1; pgstis. However, this did not hold true for antibodies specific for W antigen(43). Both V and W antigens were shown to be produced by the enteropathogenic yersiniae during growth at 37°C in calcium deficient media (22,23). The first protein to be characterized was V antigen. This protein is a 90 kdal protein that is found both in the bacterial cytoplasm and its culture fluid (15). V antigen has been shown to prevent granuloma formation at the foci of infection in liver tissue (69). Monoclonal or polyclonal antisera to V antigen injected along with the organisms restores granuloma formation and limits the invading 17 organisms (69). This lack of neutrophil and mononuclear cell response indicates that V antigen may act as a anti- chemotactic agent. V antigen has been purified but this protein undergoes rapid proteolysis in the purified form (15). There is not much information on W antigen but it has been reported to be a lipid-protein complex (22). In addition to V and W antigens, the lcr plasmid also encodes a set of yersiniae outer membrane peptides termed Yops. These proteins are only produced during the low calcium response (6,61). There have been at least 13 different Yops described and they have been given the nomenclature of YopA through YopM (64). Yop A is an adhesion protein and is only found in the enteropathogenic yersiniae. It seems to have many roles including such things as autoagglutination (58), mannose-resistant haemagglutination of guinea-pig erythrocytes (40,41), and inhibition of the anti-invasive effect of interferon (16). In XL gntgzggglitiga Yop A has been associated with serum resistance (3), adherence and inhibition of internalization of the bacterium in HEp-2 cells (36), and expression of fibrils on the surface of the bacteria (41). This Yop is different from the other Yops in that it is induced by the higher temperature (37°C) and its production is not regulated by calcium (7,8,31). Yops G and I are only found in X; pfiguggtgbergglgfiig and their function remains unknown (65). Yops K, L and M are only found in x; pestis (65). 18 Yop M has been shown to inhibit platelet aggregation and may be necessary for virulence (44). The two most important Yops are Yop E and Yop H (32,64). These Yops are highly conserved between the three species and avirulence results upon mutation of these genes. Yop H is a protein tyrosine phosphatase (PTPase) related to the eukaryote PTPases such as CD45 and leukocyte antigen-related protein (LAR) (35). How it effects the host still remains unknown. Yop E is cytotoxic for HeLa cells but only if the bacteria are bound to them (53). It is also cytotoxic to mouse macrophages and it may also influence the ability of the pathogen to resist phagocytosis (53). Insertional mutation of this gene results in avirulence (32,64) but this can be restored by injection with iron (47). Large quantities of Yops were originally only found on the enteropathogenic strains. 1; pgstis did not appear to make Yops in yitrg although immune sera from convalescent plague patients contained antibodies to Yops which recognized the Yops in the enteropathogenic strains (8,46) thus indicating Yop production in yiyg. Transfer of the Lcr plasmid from 1; pestis to a Lcr' strain of I; psegggtupgxgulgsis allowed full expression of Yops indicating that 1; pegtig had the coding capacity for Yops (64). The controversy was resolved when Sample et. al. showed that curing the organism of the pesticin plasmid resulted in the full production of Yops (54). Pulse-chase 19 studies of protein expression later showed that the Yops were being produced but were then rapidly degraded (55). This proteolysis was later found to be the fibrinolytic activity that is encoded on the pesticin plasmid (60). The low calcium response is an important virulence factor in all of the yersiniae strains but there is still a lot of information to be gathered to truly decipher the exact role in pathogenesis. In this study, the general protein expression during the LCR is investigated with most of the emphasis on x§1§inia pestig. 20 REFERENCES Bacon, G.A., T.W. Burrows, and M. Yates. 1951. The effects of biochemical mutation on the virulence of W W: the loss of virulence of certain mutants. Brit. J. Exp. Pathol. 32:85-96 Baker, E. E., H. Sommer, L. E. Foster, E. Meyer, and K. F. Meyer. 1952. Studies on_ immunization against plague. I. The isolation and characterization of the soluble antigen of Willa Degas. J. Immunol. 68:131-145. Balligand, G., Y. Laroche, and G. Cornelis. 1985. Genetic analysis of virulence from a serogroup 9 Xersinig gntgrggglitiga strain: role of outer membrane protein P1 in resistance to human serum and autoagglutination. Infect. Immun. 48:782-786. Beesley, E. D., R. R. Brubaker, W. A. Janssen, and M. J. Surgalla. 1967. Pesticins. III. Expression. of coagulase and mechanism of fibrinolysis. J. Bacteriol. 94:19-26. Bennett , L. G . and T . G . Tornabene . 1974 . Characterization of the antigenic subunits of the envelope protein of ,1gzsin1§_‘pe§ti§. J. Bacteriol. 117:48-55. Bolin, I., L. Norlander, and H. Wolf-Watz. 1982. Temperature-inducible outer membrane protein of 22.8.1111; Wendot c ° and Termini: Mantis: is associated with the virulence plasmid. Infect. Immun. 37:506-512 Bélin I, A., Forsberg, L. Norlander, M. Skurnik, and H. Wolf-Watz. 1988. Identification and mapping of the temperature-inducible plasmid-encoded proteins of Xersinia spp. Infect. Immun. 56:343-348. Bolin, I., and H. Wolf-Watz. 1988. The virulence plasmid-encoded ‘10pr protein of mg psggggtupgzgglgsis is a virulence determinant regulated by calcium and temperature at transcriptional level. M01. Microbial. 28237-245. Brubaker, R. R., and M. J. Surgalla. 1961. Pesticins I. Pesticin- bacterium interrelationships, and environmental factors influencing activity. J. Bacteriol. 82:940-949. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 Brubaker, R. R., and M. J. Surgalla. 1964. The effect of Ca++ and Mg++ on lysis, growth, and production of virulence antigens by W m. J. Infect. Dis. 114313-25. Brubaker, R. R., E. D. Beesley, and M. J. Surgalla. 1965. 2gg§guzellg_pe§ti§: role of pesticin I and iron in experimental plague. Science 149:422-424. Brubaker, R. R., M. J. Surgalla, and E. D. Beesley. 1965. Pesticinogeny and bacterial virulence. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig. 196:302-315. Brubaker, Rn R, 1970. Interconversion of purine mononucleotides in Eastegzglla pegti_. Infect. Immun. 1:446-454. Brubaker, R. R. 1972. The genus Legging: biochemistry and genetics of virulence. Curr. Top. Microbial. 57:111-158. Brubaker, R. R., A. K. Sample, D.-z. Yu, R. J. Zahorchak, P. C. Hu, and J. M. Fowler. 1987. Proteolysis of V antigen from W ML. Microbial Path. 2:49-62. Bukholm, G., G. Kapperud, and M. Skurnik. 1990. Genetic evidence that the 2925 gene-encoded .Iersinie. outer membrane protein Yop1 mediates inhibition of the anti- invasive effect of interferon. Infect. Immun. 58:2245- 2251. Burrows, T. W. 1955. The basis of virulence for mice of Ragtggzgllg pestis, pp. 152-175. In J. W. Howie and A. J. O'Hea (eds.), Mechanisms of microbial pathogenicity. Fifth Symp. Soc. Gen. Microbiol. Cambridge University Press, Cambridge. Burrows, T. W., and G. A. Bacon. 1956. The basis of virulence in Pasteurgllg pestis: an antigen determining virulence. Brit. J. Exp. Pathol. 37:481- 493. Burrows, T. W., and G. A. Bacon. 1956. The basis of virulence in W m: The development of resistance to phagocytosis in M. Brit. J. Exp. Pathol. 37:286-299. Burrows, T. W. 1957. Virulence of Pasteurella pgstis. Nature (London) 179:1246-1247. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 22 Burrows, T. W. 1960. Biochemical properties of virulent and avirulent strains of bacteria: Sglmgpgllg 32221125: and Remus pestle- Ann. N-Y. Acad- Sci. 93:1125-1135. Burrows, T. W., and G. A. Bacon. 1960. V and W antigens in strains of Pasteurellg pseuggtgberculasis. Carter, P. B., R. J. Zahorchak, and R. R. Brubaker. 1980. Plague virulence antigens from zersinia enteragglitiga. Infect. Immun. 28:638-640. Cavanaugh, D. C., and R. Randall. 1959. The role of multiplication of (Easteuxgllg, pestis, in. mononuclear phagocytes in the pathogenesis of fleaborne plague. J. Immunol. 85:348-363. Charnetzky, W. T., and R. R. Brubaker. 1982. RNA synthesis in Xersinia pestis during growth restriction in calcium deficient medium. J. Bacterial. 149:1089- 1095. Cornelis, G., M. P. Sory, Y. Laroche, and I. Derclaye. 1986. Genetic analysis of the plasmid region controlling virulence in 1g1§1nia ggtgxggglitigg 0:9 by Mini-Mu insertions and lac gene fusions. Microbial. Path. 3349-359. Cornelis, G., J.-C. Vanootegem, and C. Sluiters. 1987. Transcription of the yap regulon from X- gntgzggglitigg requires trans-acting pYV and chromosomal genes. Microbial . Path . 2 : 367-379 . Cornelis, G., C. Sluiters, C. Lambert, de Rouvroit, and T. Michiels. 1989. Homology between VirF, the transcriptional activator’ of the Xersinia virulence regullon, and AraC, the {Eggnerign13, ggli_ arabinose operon regulator. J. Bacterial. 171:254-262. Ferber, D. M., and R. R. Brubaker. 1979. Made of action of pesticin: N-acetylglucasaminidase activity. J. Bacterial. 139:495-501. Ferber, D. M., and R. R. Brubaker. 1981. Plasmids in Igrginia pestig. Infect. Immun. 31:839-841. Forsberg A., I. Balin, L. Norlander, and H. Walf-Watz. 1987. Molecular cloning and expression of calcium- regulated, plasmid-coded proteins of X- m tubezgglggis. Microb. Pathogen. 2:123-137. 32. 33. 34. 35. 36 37. 38. 39. 40. 41. 23 Farsberg A., and H. Wolf-Watz. 1988. The virulence protein YopS of lensinia W is regulated at transcriptional level by a repressar and an activator controlled by temperature and calcium. Mal. Microbial. 2:121-133. Forsberg, A. , and H. Walf-Watz. 1990. Genetic analysis of the 1923 region of mg; spp.: identification of a novel conserved locus, m, regulating 1925 expression. J. Bacterial. 172:1547- 1555. Goguen, J. D., J. Yother, and S. C. Straley. 1984. Genetic analysis of the low calcium response in Yersinia pestis Mu d1 (Ap lac) insertion mutants. J. Bacterial. 160:842-848. Guan, R., and J.E. Dixon. 1990. Protein Tyrosine Phosphatase activity of an essential virulence determinant in legginig. Science 249:553-556. Heesemann, J., and L. Gruter 1987. Genetic evidence that the outer membrane Yapl of Yezsinig entezgcglitiga mediates adherence and phagocytosis resistance to human epithelial cells. FEMS Microbial. Lett 40:37-41. Higuchi, R., L. L. Kupferberg, and J. L. Smith. 1959. Studies on the nutrition and physiology of Pasteurella pesti_: III. Effects of calcium ions on the growth of virulent and avirulent strains of Eastegzella pesti . J. Bacteriol. 77:317-321. Jackson, S., and T. W. Burrows. 1956. The pigmentation of W pestis on a defined medium containing haemin. Brit. J. Exp. Pathol. 37:570-576. Jackson, S., and T. W. Burrows. 1956. The virulence enhancing effect of iron on non-pigmented mutants of virulent strains of Pasteurella pestis. Brit. J. Exp. Pathol. 37:577-583. Kapperud, G., E. Namark, and H.-J. Skarpeid. 1985. Temperature- inducible surface fibrillae associated with the virulence plasmid of W W and W pse euggtubergglgsis. Infect. Immun. 45:561-566. Kapperud, G., E. Namark, M. Skurnik, and T. Nesbakken. 1987. Plasmid-mediated surface fibrillae of zersinia pseudotubercglosis and mini; enteracglitica: relationship to the outer membrane protein YOP1 and possible importance for pathogenesis. Infect. Immun. 55:2247-2254. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 24 Kol'tsava, E. G., V. G. Suchkav, and S. A. Legedeva. 1973. Transmission of a bacteriocinogenic factor in Pasteurglla pestis. Sav. Genet. 7:507-510. Lawton, W. D., R. L. Erdman, and M. J. Surgalla. 1963. Biosynthesis and purification of V and W antigen in £§§§gnr§11a pestis. J. Immunol. 91:179-184. Leung,K. Y., B. S. Reisner, and S. C. Straley. 1990. YopM inhibits platelet aggregation and is necessary for virulence of W BELLE in mice. Infect. Immun. 58:3262- 3271. McDonough, K.A., and S. Falkow. 1989. A Xersinia pestis-specific fragment encodes temperature dependent coagulase and fibrinolysin-associated phenotypes. Mol. Microbiol. 3:767-775. Mazza, G., A. E. Karu, and D. T. Kingsbury. 1985. Immune response to plasmid- and chromosome-encoded Yersinia antigens. Infect. Immun. 48:676-685. Mehigh, R. J., A. K. Sample, and R. R. Brubaker. 1989. Expression of the low-calcium response in ,1g;_ini_ pestis. Microbial Path. 6:203-217. Payne, 8. M., and R. A. Finklestein. 1977. Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infect. Immun. 18:94- 98. Perry, R. D., and R. R. Brubaker. 1979. Accumulation of iron by yersiniae. Infect. Immun. 137:1290-1298. Pirt, S.J., E.J. Thackeray, and R. Harris-Smith. 1961. The influence of environment an antigen production by W pestis studied by means of the continuous flow culture technique. J. Gen. Microbiol. 25:119-130. Protsenko, 0. A., P. I. Anisimav, 0. T. Mosarov, N. P. Donnov, Y. A. Popov, and. A. M. Kokushkin. 1983. Detection and characterization of 1g1§inig pestis plasmids determining pesticin I, fraction I antigen and mouse toxin synthesis. Genetika 19:1081-1090. Prpic, J. R., R. M. Robins-Browne, and R. B. Davey. 1983. Differentiation between virulent and avirulent W isolates by using Congo red tension agar. J. Clin. Microbial. 18:486-490. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 25 Rosqvist, R., A. Forsberg, M. Rimpiléinen, T. Bergman, and H. WOlf-Watz. 1990. The cytotoxic protein YopE of Xersinig obstructs the primary host defence. Mal. Microl. 4:657-667. Sample, A. R., J. M. Fowler, and R. R. Brubaker. 1987. Modulation of the low calcium rsponse in legginia pestis by plasmid-plasmid interaction. Microbial Path. 2:443-453. Sample A. R., and R. R. Brubaker. 1987. Pasttranslational regulation. of Lcr’ plasmid-mediated peptides in pesticinogenic 2g1§ini§ peggis. Microbial Sikkema, D. J., and R. R. Brubaker. 1987. Resistance to pesticin, storage of iron and invasion of HeLa cells by yersiniae. Infect. Immun. 55:572-578. Sikkema, D. J ., and R. R. Brubaker. 1989. Outer- membrane peptides of 1e:ginia_ pe§t1§_ mediating siderophore-independent assimilation of iron. Biol. Metals 2:174-184. Skurnik, M., I. Bolin, H. Heikkinen, S. Piha, and H. Wolf-Watz. 1984. Virulence plasmid-associated autoagglutination in legginia spp. J. Bacteriol. 158:1033. Sodeinde 0. A., and J. D. Gaguen. 1988. Genetic analysis of the 9.5- kilobase virulence plasmid of Xgrginia pestis. Infect. Immun. 56:2743-2748. Sodeinde, 0. A., A. K. Sample, R. R. Brubaker, and J. D. Gaguen. 1988. Plasminogen activator/coagulase gene of km pestis is responsible for degradation of plasmid-encoded outer membrane proteins. Infect. Immun. 56:2749-2752. Straley, S. C., and R. R. Brubaker. 1981. Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment. Proc. Natl. Acad. Sci. (Wash.) 78:1224- 1228. Straley, S. C., and R. R. Brubaker. 1982. Localization in Xersinig pgstis of peptides associated with virulence. Infect. Immun. 36:129-135. Straley, S. C., and P. A. Harmon. 1984. Growth in mouse peritoneal macrophages of gegginig pestis lacking established. virulence determinants. Infect. Immun. 45:649-654. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 26 Straley, S. C., and W. S. Bowmer. 1986. Virulence genes regulated at the transcriptional level by Ca2+ in pestis include structural genes for outer membrane proteins. Infect. Immun. 51:445-454. Straley, S. C. 1988. The plasmid-encoded outer- membrane proteins of mania pestis. Rev. Infect. Dis. 10:5323-5326. Surgalla, M. J., and E. D. Beesley. 1969. Congo red- agar plating medium for detecting pigmentation in Pastenrella pestis. Appl. Microbiol. 18:834-837. Takeuchi , A. 1971 . Penetration of the intestinal epithelium by various microorganisms. Curr. Top. Pathol. 54:1-27. Une, T., and R. R. Brubaker. 1984. In vivo comparison of avirulent Vwa- and Pgm- or Pstr phenotypes of yersiniae. Infect. Immun. 43:895-900. Une, T., R. Nakajima, and R. R. Brubaker. 1986. Roles of V antigen in promoting virulence in Xezsini_. Contrib. Microbiol. Immunol. 9:179-185. van Asbeck, B. S., and J. Verhoef. 1983. Iron and host defense. Eur. J. Clin. Microbiol. 2:6-10. Viitanen, A. -M., P. Toivanen, and M. Skurnik. 1990. The 19:3 gene is part of an operon in the 19; region Xerainia gntgzggglitiga 0:3. J. Bacteriol. 172:3152- 3162. Weinberg, E. D. 1974. Iron and susceptibility to infectious disease. Science 184:952-956. Williams, J.D., and D.C. Cavanaugh. 1983. Chronic infections in laboratory rodents from inoculation of nonencapsulated plague bacilli (mini; M) . Experientia 39:408-409. Williams, E.D., D.N. Harrison, and D.C. Cavanaugh. 1975. Cryptic infections of rats with non-encapsulated variants of yersinia pestis. Trans. R. Soc. Trop. Med. Hyg. 69:171-172. Winter, C.C., W.B. Cherry, and M.D. Moody. 1960. An unusual strain of mm pestis isloated from a fatal human case of plaque. Bull. Wld. Hlth. Org. 23:408-409. 76. 77. 78. 79. 80. 27 Yersin, A. 1894. La Peste bubonique a Hang-Kong. Ann. Inst. Pasteur. 8:662-667. Yother, J., L. W. Chamness, and J. D. Gaguen. 1986. Temperature-controlled plasmid regulon associated with low calcium response in 12min}; pest; . J. Bacteriol. 165:443-447. Yother, J., and J. D. Gaguen. 1985. Isolation and characterization of Ca2+-blind mutants of Xergigia pestis. J. Bacteriol. 164:704-711. Zahorchak, R. J., W. T. Charnetzky, R. V. Little, and R. R. Brubaker. 1979. Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in legginia pgsti . J. Bacteriol. 139:792-799. Zahorchak, R. J., and R. R. Brubaker. 1982. Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in 2g1§131§ pestis. Infect. Immun. 38:953- 959. CHAPTER 2 Expression of the low calcium response in Xersinig pestis by Richard J. Mehigh, Allen K Sample, and Robert R. Brubaker (Microbial Pathogenesis 1989. 6:203-217) 29 Abstract Pathogenic yersiniae undergo an established low calcium response (LCR) at 37°C in Ca2+-deficient media characterized by restricted growth with synthesis of Lcr plasmid-encoded virulence functions. The latter include outer membrane peptides (Yops) known to undergo Pst plasmid-mediated post- translational degradation in ngsinia pestis but not in the enteropathogenic yersiniae lacking this plasmid. Salient Yops of x; pestis are shown here to be either maintained in the steady state or exist as a stable degradation product (p24 of YopE). Processing of plague plasminogen activator (p36 to p33), responsible for the hydrolysis of Yops, required 2 hours. Avirulence of mutants with inserted Mu dll (Aprlac) in ygpfi was verified and shown to occur independently of introduced fusion-dependent peptides. However, avirulence of such ygpfi mutants but not that of isolates lacking the Lcr plasmid was phenotypically suppressed in mice injected with iron. Appearance of 20,500 and 40,55 Da heat-shock peptides preceded onset of the LCR. Lcr plasmid mediated V antigen (p38) and p20, Pst plasmid- encoded p36, and chromosomally promoted p56 and p70 were synthesized throughout the LCR. Classical antigen 5 was equated with p70 which was shared by 2gz§inia was but not lensing Wise- 30 INTRODUCTION Xerginig pestis, the causative agent of bubonic plaque, and the enteropathogenic yersiniae (Xersinia What: and uremia W) are generalized facultative intracellular parasites capable of growth in either Ca2+-enriched blood and lymph or Ca2+- deficient intraleukocytic fluids (11). However, at 37°C in typical culture media lacking Ca2+ these organisms undergo a unique metabolic stepdown, termed the low calcium response (LCR)(21), characterized by restriction of vegetative growth accompanied by selective synthesis of putative and established virulence factors (lcr+). An approximate 70 kb shared Lcr plasmid mediates the LCR which does not occur at 26°C in Ca2+-deficient media or at 37°C in the presence of at least 2.5 mM Ca2+. Lcr‘ mutants cured of this plasmid are avirulent and do not express the LCR regardless of temperature or concentration of Ca2+(10,16,34) Regulatory mechanisms of the LCR probably serve to identify the intracellular or extracellular environments occupied by invading yersiniae (6,30) thereby permitting induction of an appropriate attack against the distinct nonspecific mechanisms of host defense resident to these niches (11). An approximate 20 kb segment of the Lcr plasmid serves to promote the temperature-dependent response to Ca2+ (4,15,21,29,32,33,52). Mutations within this region (3,6,15,21,51) and a linked operon (29) typically permit 31 Ca2+-independent growth with loss of ability to express Lcr+-specific virulence functions, thereby conferring an avirulent phenotype similar to that resulting from cure of the Lcr plasmid. Products of genes within these regions were assigned diverse regulatory roles including an activator and repressor of virulence factor synthesis and possible sensors of Ca2+ and temperature (20,52). Lcr+-specific virulence functions shown to be induced during expression of the LCR and repressed by Ca2+ (3,4,6,15,19,20,29-33,37,51,52,54) include the soluble V and W antigens (13,26) and certain yersiniae outer membrane peptides (30,43) termed Yops (3). Structural genes for these determinants are usually located outside of the 20 kb regulatory region. Mutation in many such genes, especially yQpE and ygpfi (3,5,36,43,45) and possibly that for V antigen (29), lead to avirulence. A full component of yops is only expressed in 21:19 by Lcr+ cells of nonpesticinogenic X; pestis (37,38) lacking the 10 kb pesticin or Pst plasmid unique to this species (2,18). These structures do not accumulate in typical pesticinogenic (Pst+) isolates (37,43) where they are synthesized but undergo rapid post- translatianal degradation (38) catalyzed by a Pst plasmid- mediated (7,8) outer membrane plasminogen activator/coagulase activity (PAC) (1,41,42,44). PAC exhibits a molecular weight of 38,000 Da but undergoes rapid processing to 37,000 Da during secretion followed by slow 32 conversion (in minicells) to 35,000 Da (41). It is an enigma that Yops are not expressed in Lcr+o Pst+ cells of X; pestis since mutational loss of Yap E and H in this species results in avirulence (45). One objective of this paper is to reconcile this discrepancy by showing that significant concentrations of salient Yops are maintained in 21:19 bleL pestis in the steady state or exist as stable degradation products. We also demonstrate that avirulence caused by mutational loss of Yap E can be phenotypically suppressed in mice by injection of iron as opposed to that resulting from the cure of the Lcr plasmid. Finally, previously described (38) evident chromosomal and Pst plasmid-encoded peptides are defined which are regulated by a mechanism that abrogates the LCR. MATERIAL AND METHODS Bacteria Origins of isolates used in this study have been described (18,37,38,48). The parent of all derivatives of x; pestis was strain KIM (9). This Lcr+, Pst+ isolate was non-pigmented thus it and its mutants are avirulent in mice unless injected intravenously (46) or intraperitoneally with exogenous iron (24). Other yersiniae used possessed insertions of Mu dll (Aprlac) in the Lcr plasmid in yng or 229i: construction of these isolates has been described 33 (45). Lcr’ and Pst‘ mutants, lacking the Lcr or Pst plasmid (18), were selected at 37°C on magnesium oxalate agar (22) or by cold cure (37). I; pestis strain EV76, x; DSQQQQLQQQIQHIQSLS P81, and 2; gntgrggglitigg WA have been described (37,38,43,45). Where necessary, methionine meiotrophs of X; pestis strain KIM were isolated as previously reported (38). Bacteria were cultivated in chemically defined medium (54) lacking methionine as already described (38). All cultures contained 20 mM Mg2+ and no added Ca2+. To assure balanced growth prior to pulse with 35S-methionine, the organisms were subcultured twice at 26°C and then shifted to 37°C to induce the LCR (38). Pulsezehase 13231129 21 DAQESIIQ Procedures used for pulse-chase experiments were identical to those reported earlier (38). A typical experiment involved pulsing methionine meiotrophs, starved for Ca2+ in methionine-free medium, with 35S-methionine and then chasing with unlabeled methionine at great excess. Samples of whole cultures were removed at appropriate intervals, precipitated with cold 10% TCA and then prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resulting gels were 34 stained with Coamassie Brilliant Blue G prior to autoradiography as previously described (38). In experiments concerned with stable peptides expressed during the LCR, the organisms were pulsed and chased as already performed (38) to assure degradation of Yops and then centrifuged at 10,000 x g for 15 min at 4°C, washed twice in chilled 0.033 M potassium phosphate buffer, pH 7.0 (phosphate buffer) and then prepared for SDS-PAGE as described for TCA-precipitated samples. This process assured removal of radioactivity loosely associated with the outer membrane (primarily p24). SDS-PAGE and autoradiography were undertaken as defined previously (38). Qeterminatien ef ziruleneel Organisms were injected either intraperitoneally or intravenously in 0.1 m1 of phosphate buffer. Iron, if administered, was injected intraperitoneally as FeC12 (40 pg of iron per mouse) suspended in arachis oil (24). Doses of about 107, 105, 105, 104, 103, 102, and 101 organisms were injected into seven mice apiece in determinations of LD50 (calculated by the method of Reed and Meunch) (35). Mice were maintained as already defined (47) for 2 weeks. 35 Imunetzletting Immunoblots of antigen 5 were prepared as previously described (12). The primary antibody was the same monospecific polyclonal rabbit anti-E serum used as a temperature-dependent control in initially defining the LCR (6). The precipitin formed upon diffusion of this serum against cell-free extracts of yersiniae was previously shown (25) to be similar or identical to antigen 5 (17). RESULTS Peptides treneleted QQIinQ the LEE We have shown previously that Yops of LCR+, Pst+ Y; pestis undergo immediate post-translational proteolysis in xitrg (38). To detect putative long-term degradation products and to further define the kinetics of Pst plasmid- dependent destruction of Yops, we pulsed restricted Lcr+, Pst+ yersiniae for 1 min with 35S-methionine in a similar manner but extended the period of chase with unlabeled methionine for up to 6 h. As shown in autoradiograms, previously defined (36) major stable peptides of 70,000 Da (p70), 56,000 Da (p56), 38,000 Da (p38 or V antigen), and 20,000 Da (p20) remained intact throughout this prolonged chase in cultures of both Lcr+, Pst+ (Fig. 1) and Lcr+, Pst‘ yersiniae (Fig. 2). Only Yops of Lcr+, Pst“ mutants exhibited equivalent stability (Fig. 2). As anticipated, pulsed radioactive Yops of 36 Eiguze_1. Stained gel (A) and corresponding autoradiogram (B) of trichloroacetic acid-precipitated material from cultures of Lcr+, Pst+ cells of Xerginia pestis strain KIM after 6 h of cultivation at 37°C in Ca2+- deficient medium (at which time vegetative growth had ceased). Bacteria were pulsed for 1 min with [35S]- methionine and then chased with excess unlabeled methionine for 0 (lane 1), 5 min (lane 2), 15 min (lane 3), 30 min (lane 4), l h (lane 5), 2 h (lane 6), 3 h (lane 7), 4 h (lane 8), 5 h (lane 9), and 6 h (lane 10): molecular weight markers (kDa) are shown in lane M. Peptides were separated by 12.5% SDS-PAGE. Open arrowheads represent stable peptides known to be produced during the LCR and closed arrowheads indicate Yops: stable degradation products (p33 and p24) are shown by arrows. 37 m-‘j—fii—uv—m— —-.. C—p33 2 . _ .. .1 sis «Began. '1 .4 =—Q§§5fi_m=fiua 2. saris-salsa . . awn-uga—Efifl. .4. . a. 3 ESSEHEBEHHM =__M§a—!um=m§~ figs—935355»? ... I. ”Else—Sleafim . a use- «535“. s ~ ..Q 0 ~ \ J 2 0. O 5 W % “a N 2 ‘. \. w a . ‘ a l . . ru- ‘I-Iu- -¢Ir.-:oanlap —..-‘——.-‘— YopF)» — _ ...- QQQQ-Q- {970 fig” e m m I e L _. W: Z, 1. a a: _. : a: a 2 a: a u: a: a ..s :- fi _ “a. . . YopD,N>- ‘d ' . uni-“and- “:9-” '4. «.3... Mini. "... as“ g'Nflqa...'ap-:Eo"‘ \_ . c——— Figure 2. Same as Figure 1 except that an isogenic Lcr+ Pst' isolate was used. 39 Lcr+, Pst+ organisms underwent rapid degradation (Fig. 1). Nevertheless, reduced but detectable levels of peptides with molecular weights corresponding to Yap H (47,000 Da), Yap B (44,000 Da), Yap C (42,000 Da) PAC or p36 (36,000 Da) (20, 35, 37) and Yap D (34,500 Da) occurred in all lanes of the corresponding stained gel of Lcr+, Pst+ yersiniae (Fig. 1). This finding suggests that diminished but nevertheless significant levels of at least these four Yops are maintained by Lcr+, Pst+ organisms in the steady state. We have shown previously that a unique 24,000 Da peptide (p24) is generated during degradation of Yops in Lcr+, Pst+ yersiniae (38). As shown in Fig. 1, this hydrolytic product remained intact throughout the extended chase used in the present study. This observation, plus occurrence of significant concentrations of the peptide in all lanes of the stained gel (Fig. 1), demonstrates that p24 is a major stable intermediate. A radioactive peptide of 33,000 Da (p33) accumulated in Lcr+, Pst+ (Fig. 1) but not Lcr+, Pst‘ yersiniae (Fig. 2) after chase for 2 h with unlabeled methionine. Like p24, p36 and the four Yops already noted, this structure was present in all lanes of the corresponding stained gel (Fig. 1) indicating that, in the steady state, it exists as a major processed peptide. In view of prior work showing slow conversion of PAC to a smaller processed form (41), we 40 assume that p33 is identical to the smaller (33,000 Da) of two outer membrane peptides previously equated with this activity (37,42,44). The larger such peptide (p36 of 36,000 Da) could thus serve as the precursor of p33. A radioactive structure of this size was initially present (Fig. 1) which underwent degradation at a rate sufficient to account for generation of p33. lentietheezenreem The kinetics of p24 synthesis suggested that this structure arose as a degradation product of Yap E (38). To establish this relationship, we shortened the pulse of Lcr+, Pst+ organisms from 1 min to 15 s and also decreased the period of chase. This modification illustrated a transient increase in p24 accompanied by reduction in concentration of Yap E [Fig. 3(A)]. To prove that Yap E per se was the precursor of p24, the same determination was performed with a mutant of the Lcr+, Pst+ parent possessing a Mud11(Apr lac) insertion in yng (45). As shown in Fig. 3(B), neither Yap E nor p24 were evident thus demonstrating a precursor- product relationship. However, this isolate expressed at least two stable peptides (102,000 and 42,000 Da) not produced by the parent. To ascertain whether these structures represented either some unprocessed form of Yop E or, more likely Mudl1(Aprlac) dependent functions, we performed a similar determination with a yap; mutant 41 do; c. 5593 Se 2:925 5.3222 .9595 3 :32.» En motion. Eooceaop -cofia. 3.: .33 :u as. Annmarie; “.320 .3 “.8355 En ewe use mao> .woaiimow $02 .3 US$33 e53 maniac“. .8— ace: 2 — use .3 can: SF. on 8 ecu: SE 3 .Ah use: EE m .3 can: EE 9 .3 can: SE N .3 ace: 55 p .8 ace: m an ..N can: m mp .3 use: 0 .o. o:_:oE.oE 3.32:: 9.083 2:3 bongo :2: use ochELoEm: 5.3 m mp .2 comic 225 5.280 .333 on; 5306 3:239. 95. 5...; E 52an Eons—op. . «so E 0.5 E 5.32:3 .o e c .23 .23. 52.... 3an 2.5me .0 £3 5. ..m... 439: ..o._ can .35 .7... Ammo: .54 43 ..md ...u._ .o 35:30 Ea: 3:25: 3.53.8565: 2.85028: .o Ee.uo_m~_o.:< .n .uE O_mm~.wmvn~_ o_mo~mnvmm_ o_mmhmnvm~_ i. _c ll A’II'I'IIQOO‘Pa 111 Ill-Ill!!! o._m O.nv 04110.4?“ . -n-- ---lfln ”Hugh? New I 0.- .. . l n.~m 42 Emoswnvnm. o.mmhwmvn~_ gambwmvnN. .osllll "I'JHI .. l 0.6 l ‘ O‘i"0‘"“1 . . " ..O""""NQO> Ill -... 1.. lo;' .. _ 1“!" -.‘Olll I ..-.......3.......-m Iluxauunnmm ..lllclll'tllillcl. ll II‘I‘-"IO‘ I 8.1"! b.0”--’.la . I N3 l l ”' 3"... n “.mm ' U _ in < 0.00 43 possessing an analogous Mud11(Aprlac) insertion in the Lcr plasmid. Identical extra peptides were expressed in this isolate [Fig. 3(C)] indicating that they are fusion- dependent functions rather than specific Yop precursors. The kinetics of Yop E degradation with conversion to p24 in this mutant were similar to those of the parent. To further clarify these relationships, this determination was repeated with the same strains after cure of the Pst plasmid. As shown in Fig. 4, both Yop E and Yop J remained stable in isolates capable of their production, p24 was never detected, and the extra fusion-dependent peptides were similarly expressed in the ygpfi and ygpg mutants. Irsnzdsssndsnf pbsnssxpis suppression sf sxirulssss is 1225 mutants Mice were injected either intravenously or intraperitoneally with Lcr+, Pst+ yersiniae or their ygpfi and ygpg mutants. As expected, these non-pigmented isolates were all avirulent via the intraperitoneal route (L050 > 107) (23,24,46) but the parent (46) and 299; (45) strains were fully lethal by intravenous injection (Table 1). The corresponding yng mutant was essentially avirulent by this route thus verifying the results of Straley and Bowmer (45). This finding also demonstrated that the necessity to synthesize the extra fusion-dependent peptides described 44 Table l Interperitoneal and intravenous LD50 of Yop- deficient and non-pesticinogenic strains of zersinia pestis strain KIM in normal and iron-treated micea Injected iron No injected iron Organism injected Bacteria injected Bacteria injected Bacteria injected Bacteria injected i.p. i.v. i.p. i.v. Lcr +» Pst+ 2.4x101 43x101 > 107 1.3x102 Lcr+ @313), Pst+ 1.4x103 2.3xm5 > 107 35x106 Lcr" M), Pst+ 2.5x101 6.0x101 > 107 65x101 Lcr+ Pst“ zmo3 2.0x106 > 107 7.8x105 La+ 19215), Pst‘ 1.7x106 3.7x106 > 107 9.8x106 Lcr+ (Leg), Pst' 5.4x101 2.2x104 > 107 3.1x103 Lcr'Pst + > 107 > 107 > 107 > 107 aIron was injected as FeClz in arachis oil (40 ug/ mouse). (37) 45 above in ygpfi mutants does not inhibit expression of disease because the same peptides were produced by the virulent ygpg isolate. However, virulence of the ygpfi mutant was essentially restored by intraperitoneal (LD50 = 1.4 x 103) but not by intravenous injection (LD50 = 2.3 x 105) in mice treated with exogenous iron (Table 1). This pattern is distinct from that of the parent and yogi mutant where lethality could be enhanced by either use of the intravenous route (with or without injected iron) or by intraperitoneal injection in mice receiving injected iron. The Lcr‘, Pst+ mutant (cured of the Lcr plasmid) was unable to kill either normal or iron-treated mice at sub-endotoxic doses (intravenous and intraperitoneal L050 >107). Accordingly, exogenous iron can phenotypically suppress the ygpfi mutation to avirulence whereas this treatment is unable to restore virulence of mutants lacking the Lcr plasmid. As anticipated, the Lcr+, Pst’ isolate was attenuated under all tested conditions (Table 1) although virulence was significantly enhanced via intraperitoneal infection in mice receiving exogenous iron (L050 = 2.1 x 103) (7). This route of injection did not significantly suppress avirulence of the Pst' yQpE mutant (LD50 = 1.7 x 105), an observation that would be expected if processing of Yop E to p24 is required for lethality. Curiously, the control Pst' yopg mutant was 46 somewhat more virulent under most conditions of assay than was its Lcr+, Pst" parent. Exszsssisn 91 £3321: Esatisss during gas 19! sslsisn £§§EQD§§ Since the consequences of mutation to ygpfi but not avirulence resulting from cure of the Lcr plasmid, was phenotypically suppressed by injection of iron, we assumed that Lcr+-dependent functions other than Yops fulfill essential roles in causing disease. To identify such candidates without interference by Yops and to define more fully regulatory events associated with onset of the LCR, we prepared a series of parallel cultures of Lcr+, Pst+ yersiniae in Ca2+-deficient medium. The latter were aerated at 26'C until logarithmic growth was assured and then shifted to 37'C. At different times thereafter, each culture was pulsed for 1 min with 35S-methionine and then chased for 1 h with unlabeled methionine to assure complete degradation of Yops. Growth in these cultures (plus that of a control which was maintained at 26'C) and the times that each culture was pulsed is shown in Fig. 5(A). The autoradiogram of the corresponding samples prepared after incubation with excess unlabeled methionine for 1 h [Fig 5(8)] demonstrated that no significant difference occurred if the cultures were chased at 26'C or 37'C and that bulk protein, contributing to 47 Eigure 5, (A) Optical density of parallel cultures of Lcr+, Pst+ cells of Xersinia pestis strain KIM versus time (h) showing points (arrows) where samples were pulsed for 1 minute with [3531-methionine prior to chase for 1 h with unlabeled methionine and preparation for autoradiography. Cultures (lacking Ca2+) were shifted from 26’C to 37°C at an optical density of 0.25. Growth was then monitored during onset and expression of the LCR (O) and in a control culture maintained at 26'C (0). Protein synthesized during corresponding pulses were separated by 12.5% SDS-PAGE (B): 0 chased at 26’C(lane 1), 0 chased at 37‘C(1ane 2), 1 h(lane 3), 2 h(lane 4), 4 h(lane 5), 6 h (lane 6), 8 h(lane 7), 10 h(lane 8), and 12 h(lane 9); all chases after 0 h were performed at 37°C. Open arrowheads indicate Lcr plasmid- mediated peptides and closed arrowheads represent chromosomal (p70, p56) or Pst plasmid-mediated structures. Molecular weights are shown in kDa. 48 fl. : . _ :3 :2: 450- fig: : z. : z. : . 5:: : :4. j. ass-Ens” E . a: BEES: Inn _ _ 5. 2 0 2 6 I. 9 .0 3 I! hi I 0/ 0].! k I 0/0/ K .r... e p p . . .s . . . . .f w 0 J N I. 0 £2... 32:6 2|.5- l6 ‘ €920 ——‘-*---- _ a: a: Time (h) 4 re 5. Pi 49 vegetative growth, continued to be synthesized during the two anticipated (53) residual post-shift doublings. However, two evident heat-shock peptides of 20,500 and 40,500 Da were induced within 1 h after shift. Although appearance of these structures is the first known detectable event associated with onset of the LCR, their synthesis was not maintained after cell-division had ceased. At this time (6 h after shift to 37'C) and thereafter, the organisms continued to synthesize detectable levels of only p70, p56, p38, p36 and p20. As already noted, p38 is V, p36 is a Pst plasmid- mediated precursor of p33 (either or both of which serve as the PAC) (41,42) and p20 is a major, but unknown, Lcr plasmid-mediated function (38,unpublished results). Both p70 and p56 were detected as major temperature-dependent peptides 30 min after shift from 26°C to 37‘C in both Lcr+ and Lcr’ organisms [Fig. 6(A)]. This observation suggests that these structures are chromosomally encoded but, like Lcr plasmid-mediated functions, are regulated by a mechanism that abrogates repression during the LCR. The identity of p56 has not been determined but p70 reacted in immunoblots with monospecific polyclonal rabbit antiserum (6) directed against antigen E [Fig. 6(B)], a structure probably identical (25) to the classical temperature-dependent antigen 5 of Crumpton and DaVies (17). Antigen 5 was also 50 92.5- __ .- — ._ l- L "_ 47231.0 4.U"N to- . ljii .. E5 “121.5 ”um: . "E "U 40bJ~I " UH“ -o‘o- no“, 12345678 Eigg;§_z. Silver-stained gel of whole cells of x; pseudotuberculosis PB1 (lanes 1-4) and X; pestis KIM (lanes 5-8) grown at 37'C in Ca2+—deficient media. Peptides were separated by 12.5% SDS-PAGE. Each lane contains isogenic strains lacking one or both plasmids as follows: Lcr+, Pst+ (lanes 1 and 5), Lcr+, Pst‘ (lanes 2 and 6), Lcr', Pst+ (lanes 3 and 7), Lcr', Pst“ (lanes 4 and 8). Bla= beta- lactamase and p36 is a Pst specific peptide. 77 specific for those strains carrying the Lcr plasmid (Figure 2, Lanes 1, 2, 5, and 6) and this is V antigen. In the Pst+ x; pestis strains (Figure 2. lanes 5 and 7) there is a band at about 36 kilodaltons that represents the plasminogen activator/coagulase factor encoded on this plasmid. This band cannot be seen in the Pst+ x. pseggotuberculosis strains as seen in Figure 2, lanes 1 and 3. However, there is a band at about 29 kilodaltons found in these lanes and this protein is the processed form of the beta-lactamase gene product. Only some of the Yops could be detected on the silver- stained gels so a Western blot was done on these same samples using polyclonal antisera raised against Yops of x; pestis. Again the Yops are most distinct in the Lcr+, Pst' strains as shown in Figure 3 lanes 2 and 6. In the Lcr+, Pst' x; pestis strain only Yops of 45 and 34 kilodaltons are distinct but there are a few of the less prominent Yops visible which are clustered around 45 kilodaltons in size. In the Lcr+, Pst‘ 1; pseudotubgrculosig only bands of 45, 43 and 34 kilodaltons are visible which probably represent Yops H, B, and D respectively. The surprising result is the appearance of a Yop at 40 kdal in the Lcr+, Pst+ x. psguggtubgrgglggig strain. There are no detectable Yops in the Lcr+, Pst+ I; pestis control and there is no Yop of that size reported in the literature (57). Since this Yop is 78 Eiggrg_3. Immunoblot with antisera raised against Yops of x. pestis KIM. The blot contains whole cell extracts of x; pseugotgbgzcglgsig PBl (lanes 1-4) and I. pestis KIM (lanes 5-8) grown at 37'C in Ca2+-deficient media. Peptides were separated by 12.5% SDS-PAGE. Each lane contains isogenic strains lacking one or both plasmids as follows: Lcr+, Pst+ (lanes 1 and 5), Lcr+, Pst' (lanes 2 and 6), Lcr' , Pst+ (lanes 3 and 7), Lcr‘, Pst' (lanes 4 and 8). 79 only in the Lcr+, Pst+ I; pgggggtgbgrgglgsig strain it probably represents a stable degradation product of one of the higher molecular weight Yops. MW+J§L+W Yop production could not be detected in any great quantity in the Lcr+, Pst+ x; pgeudgtgbgrgglgsig strain. Therefore, to show that Yops are produced and subsequently hydrolyzed in this strain is was necessary to use a radiolabeled amino acid to follow protein production. The cells were grown at 37'C in Ca+ deficient media to induce the production of Yops. They were then pulsed with [358]- methionine for 15 seconds and then chased with an excess of unlabeled methionine for one hour. Aliquots were taken at timed intervals, and then these protein samples were separated by SDS-PAGE and autoradiographed. From the autoradiogram in Figure 4, Yops are being produced with molecular weights of 76, 46, 44, 42, 34, and 25 Kdals. These correspond to Yops F, H, B, C, D, and E respectively. All of these Yops undergo hydrolysis and are not detectable after the one hour chase. A control experiment was performed with Lcr+, Pst+ 1; pestis (Figure 5) to compare the kinetics of degradation between the two species. A comparison of Figures 4 and 5 reveals an almost identical degradation pattern between the two organisms. The only 80 £igurg_1. Stained gel (left) and corresponding autoradiogram (right) of trichloroacetic-acid precipitated material from cultures of x; pseudotuberculosis PB1 grown at 37'C in Ca2+-deficient medium. Bacteria were pulsed for 15 s with [3SSJ-methionine and then chased with excess unlabeled methionine for 0 (lane 1), 15 5 (lane 2), 30 5 (lane 3), 1 min (lane 4), 2 min (lane 5), 4 min (lane 6), 8 min (lane 7), 15 min (lane 8), 30 min (lane 9), and 1 h (lane 10). Control lanes (pulsed for 15 s and then precipitated) contained the following isogenic derivatives of X; pseudgtuberculosis PBl Lcr+, Pst’ (lane 11), Lcr', Pst+ (lane 12), Lcr', Pst' (lane 13). Lane M contains molecular weight markers (kDa). Peptides were separated by 12.5% SDS-PAGE. Closed arrowheads indicate Yops and open arrowheads indicate Pst plasmid specific products. Arrows indicate stable degradation products (p24 and p40). 81 "_———"-—~HH"" ..au-~.-..... . . ”tihtisSSJR'... . rynv-vrofi—O-ov-Gu I. ~. E a ——————— '23: Eggegen 35 Bl.> flfiWHH——¢~_ YopF> “--H-‘- - --—¢~- . in: __HF‘ I M12345678910111213M 12345373910111213 2mm. 82 -' :..f ...‘E 92.5 '5 x .: n “,pr 66.2 ""‘F‘ 0.9-» on. ‘ ‘ 1'17!!! ant-7"“ H "‘77:?" """ —r"‘" 45° - Emma—- :. WE=§=-::EEE__’_'_‘5"' :3: ff‘"- :9”: Yopn>g,_.._...._ a; 31.0~ i: ‘22-; 2'3: E 2: "is 'figfignmggzfl E v°p4::_____._._- "’ Sgfifiééfifiéfifi -. .---.. . r. 14.4 "" " 5””39'5: g. . .,,._._:_.;___,,iau' I Illfigflfiuu u u” ..- M‘2345678910m2‘3'“ 12345678910111213 EM. Essentially the same as Figure 4 except that a Lcr+, Pst+ L m strain KIM was used in the pulse- chase. Control lanes (11-13) contained isogenic derivatives oprssugmmms. 83 major difference is the presence of two distinct bands in X; pseudgtnhgrgnlgsis. The smaller band is the 29 kdal form of the beta-lactamase (Bla) and 33 kdal band that may represent the larger form of the beta-lactamase or the smaller processed form of the PAC (38,52). A comparison of individual Yops shows that there is not much difference in how the PAC degrades the Yops. Yop E is probably the most important Yop and, like 1; pestis, this Yop is degraded into a 24 kdal product. However, this hydrolysis is slower in X; psggggtgbgrgglggig in which it takes about 15 minutes for the labeled Yop E to be degraded, while it only takes about 1 minute in X; pestis. The degradation of Yop D is also slower in 1; pseudgtgbgrgglgsis (Figure 4) with detectable quantities throughout the one hour chase while it is lost after 8 minutes in X; pestis (Figure 5). The hydrolysis of Yops H, B, C and F is nearly identical between the two organisms. The stable degradation product in the Lcr+, Pst+ 1; pgggggtgpgrgglgsis seen in the immunoblot (Figure 3) was not detectable in the autoradiogram. However, this band can be seen on the stained gel (Figure 4). Also, on the same gel 3 distinct degradation products from the Yop A protein that is unique to I; psggggtgbgzgnlggis. This protein is not well labeled in the 15 second pulse, but these degradation products can be seen in the autoradiogram (Figure 4) from 1 84 minute into the chase until one hour. Yops G and I are also unique to X‘,p§gndgtubgrgulg§i§ but they cannot be detected in either the stained gel or the autoradiogram. DIEQQEfiIQH The construction of a pesticinogenic 1L pgggggtgbgrgglggig strain allowed us to compare the production and degradation of Yops between species. The products associated with the pesticin plasmid are produced in I; pgggggtgbgrgglgsis and there is no interference by the Tnl transposon that was used as a marker for the plasmid because all of the products of the plasmid are produced at levels similar to that of I; pestis (Table 1). This plasmid was stably maintained even without selection with ampicillin. However, this is to be expected since this I; psgnggtnbgrgglgsis strain is sensitive to pesticin. Hence, production of the bactericin would eliminate any organism that lost the plasmid. The most interesting aspect of this comparison between the pesticinogenic strain and 1; pestis is the similarity in Yop production and processing. The Yops form obvious structures in the protein profile of Pst“ strains but they seem to disappear when the pesticin plasmid is present. The only visible differences between the pesticin plasmid containing species are 2 protein bands (Figure 2). The Pst+ I; psendgtubgrgnlgsis has a band at 29 kdal which represents 85 the Beta-Lactamase gene product from the transposon. The Pst+ .1; pestis has a band at about 36 kdal which is one form of the PAC (38,52). There is no similar band in the Pst+ 1; psggdggnbgrgnlggig strains but it has the same amount of fibrinolytic activity per gram of cells as 1; pestis. Immunoblotting is a more sensitive method for detection of protein production. However, the antisera only identified a few of the Yop proteins. One of these was a novel stable degradation product of 40 kdal only found in the Lcr+, Pst+ x; pgggggtgbgrgglggig strain. This product probably represents either Yop H or Yop B and probably represents a difference in primary structure in that particular Yop between the two species. This Yop has a structure that is only partially degraded by the PAC while its counterpart in x; pestis is completely degraded. The presence of this single Yop did not actually show that Yops in general are produced in this strain. Therefore, it was necessary to use a radioactively labeled amino acid to trace protein production. A possible problem with this approach is the fact that the enteropathogenic strains continue to replicate during the LCR while 1; pestis does not replicate (18). Therefore, the bulk cellular proteins were labeled in 1; pgggggtgbgrgglggig but not in x; pestis. It was not know ahead of time whether or not Yops could be detected in x; psggggtubgrgglgsis. To help 86 alleviate this problem, isogenic strains lacking one or both plasmids were also labeled with the radioactive amino acid to help identify the origins of plasmid-specific proteins. Most of the Yops were easily distinguished in X; psggdgtgbgrgnlggig, with the exception of the Yops that are made in small amounts such as Yops J, G, and I (57). The short labeling time (15 s) also does not allow a lot of time for incorporation of the label into the bulk proteins, thus, cutting down on the background but not allowing these Yops to be well labeled. The kinetics of degradation of Yops between these two species is very similar (Figures 4 and 5). This result is not surprising because the two plasmids found in these species are highly related (42). Sequence analysis of the Yop E gene between I; pgggggtgbgzgglggis and 1; pestis EV76 show that only 2 out of 219 amino acids are different (24), while immunological similarity of Yop E between these species is also quite high (19). This similarity can be seen in the degradation pattern between these species (Figures 4 and 5). This Yop is quickly degraded in a 24 kdal protein in I; pestis (38,47) (Figure 5) and this also occurs in 1‘ pseudotgbergglgsig (Figure 4). Therefore, this important protein may still function in the same manner as the Lcr+, Pst' strains (38). The other visible Yops also behave in a similar manner as the counterparts in 1; pestis including another important Yop, Yop H. This information 87 gives indirect evidence that these Yops are highly related between these two species and probably function in a similar manner. Yops A, G and I are unique to x&,p§§9ggtgbgrgglg§i§ P31 (57) and it was of interest to see how they were affected by the fibrinolytic activity of the PAC. Yops G and I are minor Yops (57) and their production could not be detected due to the short pulse time as well as the effect of being masked by labeling of the bulk protein. Yop A, an adhesion protein, shows a distinctive degradation pattern. This protein has a normal molecular weight of between 200-240 kdal when run on SDS-PAGE (51). This protein can be dissociated into 45-52.5 kdal subunits if the sample is boiled for a prolonged time (15 min) or by addition of 8M urea in the gel (48,50,59). The size of these subunits varies between different serotypes of 1; pseudotgberculosis (50). This Yop can be clearly seen on the stained gel in the radiolabeling experiment (Figure 4) as a large band and also as a series of large degradation products. These large degradation products differ in size by about 40-50 kdal which may represent fragments removed from all of the subunits or the loss of an entire subunit. How this processing effects its role in pathogenesis is not known. However, other studies have indicated that loss of this protein and another adhesion protein needed for invasion of non-phagocytic cells (ivn+) (22,29) actually increases the 88 virulence of this organism in mice (44). The ivn gene is chromosomally encoded but does not seem to be expressed in 1‘ pestis (44) or it can not function properly in X; pestis due to production of the species specific capsular antigen (1). X; pestis has an similar coding region for Yop A but it is not produced due to a 1 bp deletion (44). If production of Yop A is restored in 1‘ pestis, its virulence is decreased (44). Therefore, this protein, along with ivn protein, seems to be important in virulence of Xersinia and whether its causes a chronic or an acute disease. It is interesting to speculate on how the addition of the pesticin plasmid affects the virulence of X; s c ’s. In this study we used a purine auxotroph, thus this strain is avirulent by any route of injection (12). Studies are under way to see how the pesticin plasmid affects the virulence of x; pgguggtubgrgulgsig in a wild type strain. The pesticin plasmid is thought to be responsible for the invasiveness of 1‘ pestis and the spread of the organism from peripheral sites (18). However, 1; pestis does not produce any adhesion proteins that are found in I; psggggtubgrgglggig (44). These proteins may be needed for the enteropathogenic type of disease produced by x; psgggotubgrculgsis and 1; gntgrggglitiga. These same proteins seem to limit the virulence of the organisms since the loss of these proteins increases the virulence of the organisms (44). Therefore, 89 it seems that the pesticin plasmid may only contribute a small amount to the virulence of I; psggggtgbgrgglgsig. However, the Lcr-mediated virulence factors appeared to be highly conserved and most likely play the same role in both organisms. 90 REFERENCES Baker, E. E., H. Sammer, L. E. Faster, E. Meyer, and K. F. Meyer. 1952. Studies on immunization against plague. I. The isolation and characterization of the soluble antigen of W pesti . J. Immunol. 68:131-145. Balligand, G., Y. Laroche, and G. Cornelis. 1985. Genetic analysis of virulence from a serogroup 9 legginig gntgrggglitig; strain: role of outer membrane protein P1 in resistance to human serum and autoagglutination. Infect. Immun. 48:782-786. Beesley, E. D., R. R. Brubaker, W. A. Janssen, and M. J. Surgalla. 1967. Pesticins. III. Expression of coagulase and mechanism of fibrinolysis. J. Bacterial. 94:19-26. Ben-Gurian, R., and I. Hertman. 1958. Bacteriocin- like material produced by Eagtggrglla pg§t1_. J. Gen. Microbial. 19:289-297. Bercovier, H., H. H. Mallaret, J. M. Alonso, J. Brault, G. R. Fanning, A. G. Steigerwalt, and D. J. Brenner. 1980. Intra- and interspecies relatedness of Xersinia M by DNA hybridization and its relationship to Xsrsinia W. Curr. Microbiol- 4:225- 229. Balin, I., and H. Walf-Watz. 1984. Molecular cloning of the temperature-inducible outer membrane protein 1 of Xersinig pseuggtgbergulasis. Infect. Immun. 43:72- 78. Balin, I., D. A. Portnoy, and H. Walf-Watz. 1985. Expression of the temperature-inducible outer membrane proteins of yersiniae. Infect. Immun. 48:234-240. Balin, I., and H. Walf-Watz. 1988. The virulence plasmid-encoded Yap2b protein of , Xersinia pseudatuberculasis is a virulence determinant regulated by calcium and temperature at transcriptional level. Mal. Microbial. 2:237-245. Brenner, D. J., A. G. Steingerwalt, D. P. Falcaa, R. E. Weaver, and G. R. Fanning. 1976. Characterization of Minis snisreselitiga and 22:31:11; seudo era by deaxyribanucleic acid hybridization and by biochemical reactions. Int. J. Syst. Bacterial. 26:180-194. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 91 Brubaker, R. R., and M. J. Surgalla. 1964. The effect of Ca++ and Mg++ an lysis, growth, and production of virulence antigens by Pasteurglla pestis. CL. Infect. Dis. 114813-25. Brubaker, R. R., E. D. Beesley, and M. J. Surgalla. 1965. Ezgtggrgllg pestis: role of pesticin I and iron in experimental plague. Science 149:422-424. Brubaker, R. R. 1970. Interconversion of purine mononucleotides in Eagtggrgllg pestis. Infect. Immun. 1:446-454. Brubaker, R. R. 1983. The Vwa+ virulence factor of yersiniae: molecular basis of the attendant nutritional requirement for Ca2+. Rev. Infect. Dis. 5 (Suppl. 4):s748-s753. Brubaker, R. R. 1984. Molecular biology of the dread black death. ASM News 50:240-245. Brubaker, R. R., A. K. Sample, D.-z. Yu, R. J. Zahorchak, P. C. Hu, and J. M. Fowler. 1987. Proteolysis of V antigen from x_e_r_s_in_i_a pest; . Microbial Path. 2:49-62. Bukholm, G., G. Kapperud, and M. Skurnik. 1990. Genetic evidence that the yoga, gene-encoded ‘Xgrsinia outer membrane protein Yapl mediates inhibition of the anti- invasive effect of interferon. Infect. Immun. 58:2245- 2251. Burrows, T. W., and G. A. Bacon. 1956. The basis of virulence in Eastggrg11§_ pestis; an antigen determining virulence. Brit. J. Exp. Pathol. 37:481- 493. Carter, P. B., R. J. Zahorchak, and R. R. Brubaker. 1980. Plague virulence antigens from EXersinia gnggrgggligiga. Infect. Immun. 28:638-640. Chalvignac, M.-A., E. Carniel, C. Tram, A. Joseph- Francois, and H. H. Mollaret. 1988. In vitra expression of a 22-kilodalton legginig pestis polypeptide immunologically related to the 25- kilodalton plasmid-encoded protein of the three pathogenic mini; species. Infect. Immun. 5632576- 2580. Ferber, D. M., and R. R. Brubaker. 1979. Made of action of pesticin: N-acetylglucosaminidase activity. J. Bacterial. 139:495-501. “amt“ “I- 17*. A . ‘_ h." .e-wnrfi 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 92 Ferber, D. M., and R. R. Brubaker. 1981. Plasmids in W pestis. Infect. Immun. 31:839-841. Finlay, B. B., and S. Falkow. 1989. Common themes in microbial pathogenicity. Microbiol. Rev. 53:210-230. Forsberg A., and H. Wolf-Watz. 1988. The virulence protein Yops of (legginig, psggdgtgbezcglosis is regulated at transcriptional level by a repressor and an activator controlled by temperature and calcium. M01 . Microbial . 2 : 121-133 . Forsberg, A., and H. Wolf-Watz. 1990. Genetic analysis of the yogi; region of 1225.211; spp . : identification of a novel conserved locus, yerA, regulating 19,95 expression. J. Bacterial. 172:1547- 1555. ‘ Gaguen, J. D., J. Yother, and S. C. Straley. 1984. Genetic analysis of the low calcium response in Yersinia pestis Mu d1 (Ap lac) insertion mutants. J. Bacterial. 160:842-848. Guan, R., and J.E. Dixon. 1990. Protein Tyrosine Phosphatase activity of an essential virulence determinant in Xgrsinia. Science 249:553-556. Higuchi, R., L. L. Kupferberg, and J. L. Smith. 1959. Studies on the nutrition and physiology of Pasteurglla pestis: III. Effects of calcium ions on the growth of virulent and avirulent strains of W pestis. J. Bacterial. 77:317-321. Hu, P. C., and R. R. Brubaker. 1974. Characterization of pesticin: separation of antibacterial activities . J. Biol. Chem. 24934749-4753. Isberg, R. R. 1989. ‘ Mammalian cell adhesion of enteropathogenic ,1gr_inig, species. Mol. Microbiol. 381449- -1453. , Jackson, S., and T. W. Burrows. 1956. The pigmentation of Pagtgurgllg pestis on a defined medium containing haemin. Brit. J. Exp. Pathol. 37:570-576. Kapperud, G., E. Namark, and H.-J. Skarpeid. 1985. Temperature- inducible surface fibrillae associated with the virulence plasmid of Legging W193 and xsrsinia Wis Infect. Immun- 45:561-566. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 93 Kapperud, G., E. Namark, M. Skurnik, and T. Nesbakken. 1987. Plasmid-mediated surface fibrillae of mini; pseudgtubgrcglosig and Ygzsinig gntgrocalitigg: relationship to the outer membrane protein YOPl and possible importance for pathogenesis. Infect. Immun. 55:2247-2254. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lawton, W. D., R. L. Erdman, and M. J. Surgalla. 1963. Biosynthesis and purification of V and W antigen in Pasteurgllg 2§§§i§- J. Immunol. 91:179-184. Lawry, O. H., N. J. Rosebraugh, A. L. Farr, and R. J. Randall. 1951. Protein. measurement. with. the falin phenol reagent. J. Biol. Chem. 193:265-275. Maniatis, T., E. F. Fritsch, and J. Sambroak. 1982. Molecular cloning. Cold Spring Harbor Lab. Cold Spring Harbor, NY. McDonaugh, K.A., and S. Falkow. 1989. A Xersinia pestis-specific fragment encodes temperature dependent coagulase and fibrinolysin-associated phenotypes. Mol. Microbiol. 3:767-775. Mehigh, R. J., A. K. Sample, and R. R. Brubaker. 1989. Expression of the law-calcium response in (lgr§131_ pestis. Microbial Path. 6:203-217. Moore, R. L., and R. R. Brubaker. 1975. Hybridization of deoxyribonucleotide sequences of legginia entgrggglitigg and other selected members of Entergbagtgriaggag. Int. J. Syst. Bacterial. 25:336-339. Marrisey J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310. Portnoy, D. A., S. L. Moseley, and S. Falkow. 1981. Characterization of plasmids and plasmid-associated determinants of 151513;; W pathogenesis. Infect. Immun. 31:775-782. Portnoy, D. A., H. Wolf-Watz, I. Bolin, A. B. Beeder, and S. Falkow. 1984. Characterization of common virulence plasmids in Xerginig species and their role in the expression of outer membrane proteins. Infect. Immun. 43:108-114. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 94 Protsenko, O. A., P. I. Anisimav, 0. T. Mosarov, N. P. Donnov, Y. .A. Papav, and. A. 2M. Kokushkin. 1983. Detection and characterization of Xersinia pestis plasmids determining pesticin I, fraction I antigen and mouse toxin synthesis. Genetika 19:1081-1090. Rosqvist, R., M. Skurnik, and H. Wolf-Watz. 1988. Increased virulence of mg psguggtgbergglosis by two independent mutations. Nature 334:522-525. Rosqvist, R., A. Forsberg, M. Rimpilainen, T. Bergman, and H. Wolf-Watz. 1990. The cytotoxic protein YopE of Xersinig obstructs the primary host defence. Mal. Microl. 4:657-667. Sample, A. K., J. M. Fowler, and R. R. Brubaker. 1987. Modulation of the low calcium rsponse in legginia pestis by plasmid-plasmid interaction. Microbial Path. 2:443-453. Sample A. K., and R. R. Brubaker. 1987. Pasttranslational regulation. of Lcr’ plasmid-mediated peptides in pesticinogenic Xerginig pestis. Microbial Path. 3:239-248. Skurnik, M., I. Balin, H. Heikkinen, S. Piha, and H. Wolf-Watz. 1984. Virulence plasmid-associated autoagglutination in 1gz§inia spp. J. Bacteriol. 158:1033. Skurnik, M. 1985. Expression of antigens encoded by the virulence plasmid of Xgrsinig enterogglitica under different growth conditions. Infect. Immun. 47:183-190. Skurnik, M., and K. Poikonen. 1986. Monoclonal antibody to the autoagglutination protein P1 of Xgrsinia. In W W in we o lo m. Lark, D. L. (ed). New York: Academic Press, pp. 355-357. Skurnik, M., and H. Walf-Watz. 1989. Analysis of the ygpa gene encoding the Yopl virulence determinants of 1gzsigi§ spp. Mol. Microbiol. 3:517-529. Sodeinde O. A., and J. D. Gaguen. 1988. Genetic analysis of the 9.5- kilobase virulence plasmid of legginia pestis. Infect. Immun. 56:2743-2748. Sodeinde, O. A., A. K. Sample, R. R. Brubaker, and J. D. Gaguen. 1988. Plasminogen activator/coagulase gene of legging pestis is responsible for degradation of plasmid-encoded outer membrane proteins. Infect. Immun. 56:2749-2752. Iii-‘1 “‘"m‘f‘ ""5 3:1! 54. 55. 56. 57. 58. 59. 95 Straley, S. C., and R. R. Brubaker. 1981. Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment. Proc. Natl. Acad. Sci. (Wash.) 78:1224- 1228. Straley, s. C., and R. R. Brubaker. 1982. Localization in Xgrginig pg_§i§ of peptides associated with virulence. Infect. Immun. 36:129-135. Straley, S. C., and W. S. Bowmer. 1986. Virulence genes regulated at the transcriptional level by Ca2+ in pestis, include structural genes for outer membrane proteins. Infect. Immun. 51:445-454. Straley, S. C. 1988. The plasmid-encoded outer- membrane proteins of W pestis. Rev. Infect. Dis. 10:5323- 5326. Zahorchak, R. J., and R. R. Brubaker. 1982. Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in zerginig pestis. Infect. Immun. 38:953- 959. Zaleska, M. , K. Lounatmaa, N. Nurminen, E. Wahlstram, and P . H . Makela . 1985 . A novel virulence-associated cell surface structure composed of 47-kd protein subunits in Ersinia snisrssslitiss EMBO J- 4:1013- 1018. .-hM4 . _ _ 2‘... CHAPTER 4 Major stable peptides of legginig pestis produced during the low calcium response by Richard J. Mehigh and Robert R. Brubaker (manuscript to be submitted to Infection and Immunity) 96 97 ABSTRACT The ~70-kb Lcr plasmid of yersiniae contains 2 functionally distinct sets of genes known to mediate the low calcium response (Lcr+). Those of the first set encode activities that promote restriction of cell division at 37°C in Ca2+-deficient medium. Products of the second set are selectively synthesized within this environment and serve as virulence factors (soluble V antigen and outer membrane peptides termed Yops). Ability of restricted Lcr+ cells of Yersigig pestis (incapable of synthesizing vegetative protein) to produce additional virulence functions was defined in this study. Bacteria were pulsed with [355]- methionine and then chased to assure ~10 kb pesticin plasmid-encoded plasminogen activator (PAC)-catalyzed degradation of Yops. The 14 remaining radioactive peptides were cytoplasmic and could be separated by conventional chromatography. They consisted of V antigen plus other Lcr- mediated peptides of about 20 and 15 kd, pesticin plasmid- encoded pesticin and a 35 kd degradation product of PAC, and ~110 kb cryptic plasmid-mediated fraction 1 (capsular antigen) plus 3 peptides comprising the murine exotoxin. Evident chromosome-encoded activities were the 15 kd antigen 4 (pH 6 antigen) and temperature-inducible peptides of about 70 and 56 kd tentatively identified as a unique catalase (antigen 5) and Gro EL, respectively. The p56 peptide has 98 also been equated with W antigen as a p56/V antigen complex. This is the first report of production of other virulence factors during the Lcr. INTRODUCTION lensinig pestis, the causative agent of bubonic plaque, and enteropathogenic yersinig pgguggtgpgrgglggis are facultative intracellular parasites capable of growth within Ca2+-deficient host cell cytoplasm and in Ca2+-enriched extracellular fluids (8,40). Virulence of these species is dependent upon carriage of a common ~70 kb Lcr plasmid that mediates a unique temperature-dependent low calcium response (Lcr+). This phenomenon is characterized by shutoff of vegetative growth under restrictive conditions (cultivation at 37'C in Ca2+-deficient media) accompanied by selective synthesis of Lcr plasmid-encoded virulence functions (6,17,33,52). The latter include a series of yersiniae outer membrane peptides (33,43) termed Yops (5) and V antigen (10), a soluble peptide of 38 kd (9,43). Yops are synthesized but undergo immediate post-translational degradation in 11 pestis (28,37,38) catalyzed by plasminogen/prothrombin activator (PAC) activity (42) encoded on a species specific ~10 kb pesticin (Pst) plasmid (16). In contrast, Yops exhibit net accumulation in the enteropathogenic yersiniae (33,43) or nonpesticinogenic XL 99 pestis (36). V antigen is stable and accumulates in all 3 yersiniae (7,38). Onset of the low calcium response reflects an ordered nutritional stepdown as judged by typical reduction of adenylate energy charge (51) and shutoff of stable but not mRNA synthesis (12). Mutation to Lcr' caused by cure of the plasmid results in loss of both the nutritional requirement for Ca 2+ and the ability to express Yops and V antigen (7,34). Although a number of Lcr-plasmid-encoded genes regulate the low calcium response (17,46,49,50), the sequences whereby they or their products interact together, or with environmental sensors are not yet fully resolved. This process is evidently complex in that loss of only one of these regulatory functions often mimics cure of the Lcr plasmid in that significant growth occurs under restrictive conditions with greatly reduced synthesis of Yops and V antigen (17,46). The assumption that Lcr+-specific virulence functions are selectively induced following uptake by host cells (45) was directly substantiated (45). These functions, therefore, may contribute to survival within intracellular niches. However, the ability of yersiniae to synthesize distinct virulence factors encoded by other plasmids or the chromosome during residence within these or other Ca2+- deficient environments is unknown. To resolve this question, we cultivated Lcr+ cell of I; pestis under 100 restrictive conditions and then, after growth had ceased, determined their ability to synthesize virulence factors other than V antigen and Yops. Results demonstrated that possibly all established virulence determinants of the species were produced during restriction even though these organisms are known to be incapable of synthesizing bulk vegetative protein (28). These factors included capsular or fraction 1 antigen (1), antigen 4 or "pH 6" antigen (3,25), plague murine exotoxin (29), PAC (2,41), and 2 small Lcr plasmid-mediated peptides in addition to V antigen. Other major peptides synthesized by restricted yersiniae were tentatively identified as a novel catalase (antigen 5,28) and the protein export function Gro EL (22,25). The Gro EL factor has also been correlated with W antigen believed to be a Gro EL/V antigen complex. MATERIALS AND METHODS 3119:2112 A methionine-independent meiotrophic mutant (15) of nonpigmented (21) 1; pestis KIM, known to lack outer membrane peptides associated with assimilation of iron (39,44), was used in all experiments. This isolate possesses the 3 plasmids typical of wild type (16,35) and is virulent by intravenous (47) but not periphera1(47) routes of injection. 101 911W Bacteria, stored in liquid buffered glycerol at -20'C, were transferred to slopes of Tryptose blood agar (Difco Laboratories, Detroit, Michigan), incubated for 2 days at 26'C, and then transferred to flasks containing the chemically defined medium of Higuchi gt 31 (19) as modified by Zahorchak and Brubaker (52), except that L-methionine was omitted (the concentration of Mg2+ was 20 mM and no Ca2+ was added). After two pregrowths that were at 26°C as previously defined (38), a subculture of the same medium was inoculated at an optical density (620 nm) of 0.1 (200 ml/ 2 L Erlenmeyer flask). This flask was aerated at 26°C until the optical density was 0.25 and then shifted to 37'C where, after further aeration for 6 hours, vegetative growth completely ceased (28,38,51). The culture was then pulsed by addition of carrier-free [3581-methionine (new England Nuclear, Boston Mass.) to a final concentration of 10 uCi/ml. After 1 minute an excess of unlabeled L-methianine (final cancentration=1.6 mM) was added to the culture to chase the radioactive label from the unstable Yops. After 1 hour of chase, the cells were centrifuged at 10,000 X g and washed twice in ice-cold phosphate buffer. The cells were resuspended to an optical density at 620 nm of ~20 and then sonicated for 2 minutes to disrupt the cells. The cell debris was removed by centrifugation at 10,000 X g and the supernatant was 102 filtered through a 0.22 micron membrane to remove any remaining cells. This radioactively labeled cytoplasmic extract was mixed with about 4 times as much unlabeled cytoplasmic extract grown and prepared in the exact same manner as described above. Aliquots of this cytoplasmic mixture were separated on the columns and used in the lane gels and immunoblots. Radiolabeling of Lcr+ and Lcr' cells found in Figure 1 were prepared in a similar manner. Smaller quantities of cells (10 ml) were pregrown and then shifted to restrictive conditions as above. The cultures were then pulsed for one minute in 10 uCi/ml [3581-methionine and then chased for 1 hour with an excess unlabeled L-methionine. Aliquots of the cultures were removed at 0 and 1 hour into the chase and precipitated with an equal volume of cold 10% trichloroacetic acid. These samples were then prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Aniissra_and_1mmunsblsts- Manospecific polyclonal antisera to the p56 and p70 peptides was prepared by injecting partially purified peptides into rabbits with the RIBI conjugate (RIBI Immunochem research Inc. Hamilton, MT) according to the manufactures instructions. To make these antisera monospecific, anti-p70 sera was absorbed (9) with 103 lyophilized, disrupted, Lcr+ 11 gntgrggglitiga which does not produce p70 while anti-p56 was clarified against a lyophilized cytoplasmic protein mixture that did not contain p56. Immunoblots were performed as previously described (9). W An Lcr+ 11 pestis cytoplasmic fraction was separated on a DEAE cellulose column. Fractions were assayed for the presence of W antigen by the double diffusion method of Ouchterloney (32) using monospecific anti-W obtained from Dr. William Lawton (23). The fractions that contained W antigen were combined and precipitated and then separated on a Sepharose S-200 sizing column. These fractions were again checked for W antigen and positive fractions were pooled. This is crude W antigen and a cytoplasmic sample were separated by two-dimensional gel electrophoresis as described by O'Farrell (31). The amount of protein loaded on each gel was determined by the method of Loweryet al (26). A Lcr’, Pst‘ strain of X; pestis KIM was grown at 37°C in large fermenter vessels, containing a complex NZ amine medium (9), to an optical density at 620 nm of ~8. The 104 cells were harvested, washed twice in ice-cold phosphate buffer, and resuspended in 50 mM Tris-HCl pH 8.0. The cells were then disrupted by passing ~80 g of cell paste through an ice-cold French pressure cell twice. The cell debris was removed by centrifugation at 17,000 X g for 20 minutes. This crude extract was loaded on a 225 ml (2.5 x 45 cm) DEAE cellulose (Whatman Biosystems. Maidstone, England) column equilibrated with 50 mM Tris-HCl pH 8.0 buffer. The p70 protein eluted in the wash as a brown pigmented protein. The first 32 ml containing p70 were combined and brought to 1 M [NH4]ZSO4. This sample was then loaded on a 50 ml (1.5 x 30 cm) Phenyl-sepharose CL-4B (Pharmacia. Uppsala, Sweden) column equilibrated with 50 mM Tris-HCl pH 7.0 + 1M [NH4JZSO4. After the sample was loaded, it was washed with 50 ml of the equilibration buffer. The p70 peptide was eluted with a step gradient using 50 mM Tris-HCl pH 7.0 buffer, and the pigmented fractions were subsequently combined. This protein solution was precipitated by addition of solid [NH4JZSO4 to 80% saturation. The precipitated protein was collected by centrifugation at 27,000 X g for 30 minutes. The pellet was resuspended in 1.5 ml of 50 mM CHES buffer pH 10 . This sample was concentrated to ~0.5 ml in a centricon-lo tube (Amicon. Danvers, MA). This extract was loaded on a 150 ml (1.5 x 170 cm) Sephacryl S-300 (Pharmacia. Uppsala, Sweden) sizing column equilibrated in 50 mM CHES pH 10.0 + 0.25 M 105 [NH4]2804. About 150 ml was collected before 4 ml fractions were taken. The fractions with positive optical density at 280 nm were separated by SDS-PAGE and those fractions which were seen to contain pure p70 were combined. Detection of catalase activity was performed as described by the Worthington Biochemical Corporation (Freehold, NJ). RESULTS a ' s ' t s 's To examine protein production during the low calcium response, growing cells were shifted to restrictive conditions (37'C, no Ca2+) and pulsed for 1 minute with [3581-methionine. These cells were chased for 1 hour with an excess of unlabeled methionine. Aliquots taken at the beginning of the chase and 1 hour later show that peptides of 70, 56, 38, 20, and 15 kdal remain stable throughout the chase in the Lcr+ cells (Figure 1). The other peptides in the Lcr+ cells that are labeled at the beginning of the chase but are subsequently lost 1 hour later represent the unstable Yops which are degraded (28,38) (Figure 1). An isogenic strain of 11 pestis lacking the Lcr plasmid was labeled in a similar manner. Both the p70 and p56 peptides can be seen in this strain, while p38, p20, and p15 are missing. The p38 peptide is V antigen and it is encoded on the Lcr plasmid (16). The p20 and p15 peptides are unknown and may be encoded on the Lcr plasmid or require the 49.-1| 106 Egan-925 .:;:-662 -45.0 '1‘-..“ 2:. :il'Hl { t-t ll Elfin"? ”in?!“ --.—31.0 -21.5 920-“ - .. ”5”?“ -14.4 .0. ...- .. - - 0I1h Lcr’ 0I1h Lcr' Eiggzg_1. Autoradiogram of protein profiles of Lcr+ and Lcr‘ cells of 21 pestis KIM grown at 37'C in Ca2+-deficient medium. Cells were pulsed for one minute with [358]- methionine and then chased with an excess of unlabeled methionine. Aliquots of the culture were precipitated with trichloroacetic acid at the beginning of the chase and one hour later. Peptides were separated by 12.5% SDS-PAGE. Small arrows represent unstable Yops, open arrowheads represent Lcr-mediated stable peptides and closed arrowheads represent chromosomally encoded stable products. Molecular weight markers are in kilodaltons. .- -9.mAI-I—m —~__._ .m‘fin 107 Lcr plasmid for expression. The p70 and p56 peptides are found in large amounts in the Lcr' strain and represent chromosomally encoded peptides (38). C e l o s Most of the stable peptides were found to be located in the cytoplasm (38, unpublished results), so to further characterize and identify these peptides, we labeled Lcr+ cells with [3581-methionine and extracted the cytoplasm. Aliquots of the labeled cytoplasm were fractionated on a Sepharose A-1.5 sizing column,a DEAE cellulose column, and a calcium hydroxyapatite column. The protein profile and radioactivity were determined for each fraction from each of the columns and plotted as shown in Figure 2. The protein profiles are typical for a Lcr+ X; pestis cytoplasmic extract (unpublished results). In each of the columns there are distinct peaks of radioactivity which indicates the presence of one or more stable peptides. To correlate these radioactive peaks with specific stable peptides, aliquots of the column fractions were precipitated with 10% trichloroacetic acid and then separated on a denaturing gels. To identify the exact location of the labeled peptides the gels were dried and 108 £1gurg_z. Fractionation of radioactively labeled 1‘ pestis KIM cytoplasm extracts of (A) Sepharose A-1.5, (B) DEAE cellulose, and (C) calcium hydrayapatite. Eluted macromolecules were monitored by absorbance at 280 nm (0) and cpm/ml (O). Buffers used were 0.05 M Tris-HCl pH 8.0 (A), 0.05 M Tris-HCl pH 8.0 with introduction at arrow of gradient (dashed line) of NaCl (0 to 0.5 M) in same buffer (B), and 0.5 M Tris-HCl pH 8.0 with introduction at arrow of gradient (dashed line) of phosphate (0 to 0.5 M). Fraction sizes were 6.0 ml (A) and 4.0 ml (B and C). 109 3.0- DENSITY OPTICAL Fracflon kcpm/ml 110 exposed to film. The results of this analysis are shown in Figures 3-5. An unexpected result of this analysis was the appearance of other radioactively labeled peptides in the autoradiograms of the gels. These new peptides were labeled F1, Toxin, p35, and Pst (Figures 3-5). MW It was of interest at this stage to see if any of these stable products could be correlated with any known virulence factor. To accomplish this task, a series of immunoblots were performed of the radioactive cytoplasm using antisera to pH 6 antigen (antigen 4), V antigen, the bacteriocin pesticin, the plague murine exotoxin, and to monospecific polyclonal sera prepared against the p70 and p56 peptides. In addition to these antisera, a comparison was made to purified capsular antigen (fraction 1) and pH 6 antigen. These immunoblots were compared to a stained gel of the labeled cytoplasm and its corresponding autoradiogram shown in Figure 6. The antisera to pH 6 antigen, kindly provided by Dr. Susan Stratley, recognized a 15 kdal peptide. This antisera also reacted with all of the column fractions that contained the radioactive p15 peptide (unpublished data), thus indicating that the major p15 peptide is the pH 6 antigen. 111 -fl.5 “00.2 ”45.0 02.5- 00.2- 45.0“ ' -ul' " 31.0 14.4- ' “A -.2.5 *2 -21.5 404 1 as - a a no a Ftactian £1ggrg_1. Coamassie blue stained gels (top) and corresponding autoradiograms (bottom) of trichloroacetic acid precipitated proteins of the column fractions of the Sepharose A-1.5 sizing column. Molecular weight markers are in kilodaltons. Labeled proteins are discussed in text. 112 an: HJ- . “ _’Pn '3‘ "a". '. | ‘fi. 45.0 V 5", up ' 4" ; an 1 «u I u u a a a n a a n Fraction Figure 5. Coomassie blue stained gels (top) and corresponding autoradiograms (bottom) of trichloroacetic acid precipitated proteins of the column fractions of the DEAE cellulose column fractionation of the labeled cytoplasmic proteins of g; pestis. Molecular weight markers are in kilodaltons. Labeled proteins are discussed in text. 113 us- | ~ on u:- . 5 -u.a '" ~45» -ato ~21; an au- 42.5 “3' ,nno- .: mm- ’" ~ 45. «a- ,___ o t?- ” " pu- _ -n.o n5- .aor- 41.5 '17-- It‘- p'l' ' —-- ..u,‘ no a 00 u u to F r a c I la n ziggrg_§. Coomassie blue stained gels (top) and corresponding autoradiograms (bottom) of trichloroacetic acid precipitated proteins of the column fractions of the calcium hydroxyapatite column fractionation of the labeled cytoplasmic proteins of X; pestis. Molecular weight markers are in kilodaltons. Labeled proteins are discussed in text. 114 Purified fraction 1, the capsular antigen, has a molecular weight of about 18 kdal and has the same apparent molecular mass as the radioactive peptide labeled F1 in each of the columns. This peptide is found in nearly all of the fractions in the sizing column fractionation (Figure 3) which would indicate a diverse native molecular weight that would be expected of this capsular antigen (4). The anti-V and anti-pesticin antibodies recognized peptides of 38 and 45 kdal, respectively. These correspond to the p38 and Pst radioactive peptide bands. These results were verified by immunoblots of the column fractions which contain the respective peptides using antisera to them (unpublished data). The murine exotoxin (toxin) is a distinct 3 subunit polypeptide in which the largest band is about 58 kdal (unpublished data, Figure 3). In Western blots prepared against whole cytoplasm only the largest subunit is identified (Figure 6). This toxin was clearly visible in the stained gels of the Sepharose A-1.5 column and the DEAE cellulose column (Figures 3 and 4). However, it was not labeled well with the radioactive amino acid which indicates that this peptide is only expressed at low levels during the low calcium response. 115 .-. - —.-——_———.~»- .. - .- ——-—...—.- .. .-————.- —. W'F'"-' .. . _ ———_—— .— - —q-—- .— ; I ' 5 . I. "“ ‘ ‘ 1 : ' , » . .. - - . .~ I ' ' - w. - r -. , ‘ T“. 1. fl ”’ - ' ' ' I . r: .i ' ' ~ 3 ' . I _ 5 . . ' ~ .75 . £1gurg__. Stained gel (lane 1) and corresponding autoradiogram (lane 2) of radioactively labeled 1; pestis KIM cytoplasm as described in materials and methods. Immunoblots prepared against this cytoplasm used monospecific polyclonal rabbit antisera raised against: p70 (lane 3), p56 (lane 4), murine exotoxin (lane 5), pesticin (lane 6), and V antigen (lane 7). Antisera to pH 6 antigen (lane 9) was kindly provided by Dr. Susan Straley. Lane 8 contains purified fraction 1 (capsular) antigen. Molecular weight markers are in kilodaltons. Eigg;g_1. Immunoblots of whole cell extracts of S typhimurium LT2 (lanes 1 and 4), E; 9911 K-12 (lanes 2 and 5), E; 9911 B (lanes 3 and 6), and 1; pestis KIM (lane 7) developed with monospecific polyclonal rabbit antisera to the p56 peptide. Cells were grown at 26'C (lanes 1-3) or 37°C (lanes 4-7) in medium that lacked Ca2+. Peptides were separated on 12.5% SDS-PAGE and molecular weight markers are in kilodaltons. 117 The p20 and p35 peptides could not be correlated with any known virulence factor. However, the p35 peptide may represent a processed form of the plasminogen activator/coagulase (PAC) encoded on the pesticin plasmid (28,41). mass The p56 product was found in all strains of yersiniae in our laboratory and did not appear to be correlated with any of the plasmids (unpublished data). Monospecific polyclonal antisera to the p56 peptide crass-reacted with a 58 kdal peptide in S; typhimurigm and E; ggli (Figure 7). This is the same molecular weight as the Gro EL peptide in E; 991; (18). The p56 peptide purifies in the same manner as Gro EL (unpublished results) and is probably serves the equivalent function in yersiniae. The p56 peptide also migrated to the same point in two-dimensional gels as partially purified W antigen (Figure 8). The p56 peptide is also the major cytoplasmic protein (Figure 8). W The p70 peptide was purified as described in the materials and methods. SDS-PAGE analysis of the proteins during this purification can be seen in Figure 9. This 91 -'—-’ - '1. .. . . J --__ 118 £1gu;g_§. Stained two-dimensional gels of 21 pestis KIM cytoplasmic peptides of cells grown at 37'C (A) and of purified W antigen (C). Immunoblot of x; pestis KIM cytoplasmic peptides was developed with monospecific polyclonal antisera to the p56 peptide. The isoelectric focusing of the peptides is in the horizontal direction with the acidic peptides to the right side. The second dimension was a 6-15% exponential gradient SDS-PAGE. Arrowhead indicates location of p56 peptide. 119 brown pigmented peptide had a sorét band at 405 nm associated with a heme-containing enzyme and also stained with a hemoprotein specific stain (unpublished results). This protein exhibited moderate catalase activity, but no peroxidase activity. Purification of this catalase resulted in only about 5% of the total catalase activity as shown in Table 1. A majority of the catalase activity comes from a second catalase distinct from the p70 peptide (Figure 10). These are distinct enzymes since the p70 peptide is eluted in the wash of a DEAE column (Figure 4) while the second activity is very acidic because it is eluted at the very end of the DEAE column (unpublished results). Overall, 1; pestis has an enormous catalase activity as compared to E; 2211 (13.14)- 120 11.1mm 3:1 92.5 66.2 a- 45.0- l I 31.0 21.5 I It '1 Z .1 N 0 I} £1gurg_2. Silver stained 12.5% gel of the purification of the p70 peptide (as described in the materials and methods) showing crude extract (lane 1), after the DEAE cellulose (lane 2), after the phenyl-sepharose CL-4B (lane 3), and after the Sephacryl S-300 (lane 4). The molecular weight markers can be seen in lane M and they are in kilodaltons. 121 Table 1. Purification of the p70 protein Step Volume [protein] total specific total units yield Purification protein activity (m1) (mg/ml) (mg) (II/mg) (%) Crude 26 63.8 1658.8 468 776,318 100 1.0 extract DEAE 32 2.6 83.2 658 54,746 7.0 1.4 Phenyl- 63 0.88 55.4 704 44,352 5.7 1.5 Sepharose S-300 15.6 0.94 14.7 2459 36,147 4.7 5.3 122 Cytoplasmic Catalase Activity Protein (mg/ ml) Catalasc Activity (U/ml) 00 600 150 100 50 1 1 l 111 run-H4441. o .. .-..::e::::s:::!e:s:.. .m! 0 o 20 4o 60 , 80 100 Fraction Number —b Protein _‘— Catalase Activity Eiggrg_1g. A Lcr’, Pst' x; pestis strain KIM cytoplasm was separated on a Sepharose A-1.5 column in 0.05 M Tris-HCl and fractions were assayed for protein by the method of Lowery (26) and for catalase activity. As verified by stained gels and immunoblots (Figure 3,unpublished data), the peak of the p70 peptide elutes at fraction 24 with small amounts found in fractions 70-75. No p70 can be detected in fraction 35-40 where the peak of catalase activity is found. 123 DISCUSSION Much of the research done on the low calcium response has focused on the Yops. While these structures are stable in I; psggggtgbgrgglggis and x; gntezgcolitica they undergo degradation in x; pestis (38,42). This degradation in 1; pestis has allowed us a unique opportunity to examine how the LCR effects other virulence factors. The restriction of growth in X; pestis also allows us to distinguish proteins um.-.‘ .t_ ..1. _ “h“ that may be important in virulence without the interference m of protein production needed for vegetative growth. Labeling protein production during restriction is a sensitive method for determining what proteins are produced during the LCR, but lane gels may obscure minor but important proteins. In Figure 1, 5 major stable peptides could be visualized. The p38, p20 and p15 peptides are specific for the Lcr plasmid and cannot be detected upon loss of this plasmid. The p70 and p56 peptides are chromosomally encoded (28, unpublished results) and they are able to bypass the restriction normally found on chromosomal peptides during the low calcium response. In addition to p70 and p56, the pH 6 antigen is another chromosomally encoded product (25) that circumvents the restriction during the LCR. This protein is a homopolymer with a base subunit size of 15 kdal (25). This can be verified by its presence in multiple fractions in the sizing column (Figure 3). This peptide is normally found on the 124 exterior of the cell but is a soluble peptide and can be found in the cytoplasmic fraction when the cells are disrupted by sonication. This peptide was designated a virulence factor because 1; pestis strains that produce it kill mice faster than those that don't and the purified protein was found to be cytotoxic to cultured macrophages (25). This peptide was named pH 6 antigen because it was only made in the greatest amounts at pH values below 6.7, and at host body temperatures (37°C) (3). These conditions would be expected in a macrophage phagolysosome. This same niche would also induce the LCR, therefore, this result is not that surprising. Two other peptides produced during the LCR are the murine exotoxin and the capsular antigen termed fraction 1. Both of these peptides are believed to be encoded on the 110 kb cryptic plasmid (16,35). Their production during the LCR is very low at best, but it is detectable above backround. Fraction 1 production is greater than the production of toxin. The toxin protein reported here is much different that reported in the literature (29) and work is continuing to further characterize this protein. Fraction 1 is a glycoprotein homopolymer with a subunit size of 18 kdal with native complexes ranging up to 300 kdal (4). This size diversity can be seen in the sizing column fractionation where this peptide is found in almost all of the fractions (Figure 3). Purification of this peptide also resulted in 125 the copurification of the pH 6 antigen. These two proteins may interact in some manner or possibly act in a synergistic way. Another protein that is produced during the LCR is the bacteriocin pesticin. This peptide is encoded on a third plasmid named after it, the pesticin plasmid (16). This plasmid also encodes the PAC which has both fibrinolytic and coagulase activities (27,41). This protein is already known 1% to be encoded during the LCR because it is responsible for ll degradation of the Yops (28,38,41). The protein has a 37 and 35 kdal forms (41) and the latter form probably represents the p35 peptide seen in the autoradiograms (Figures 3-5). This is normally an outer membrane peptide (41,44) but small quantities of this protein remain in solution upon disruption of cells by sonication. The p20 peptide is a minor peptide and could not be purified to any great degree. It is Lcr specific and it may represent a regulatory peptide, but at this time its identity and function remain unknown. The p56 peptide is the major cytoplasmic peptides made during the LCR (Figure 8). This peptide represents the yersiniae equivalent to the Gro EL peptide in E1 2911 or the common antigen (20). This protein has been identified in many pathogens the Chlamydia species Gro EL is cross reactive to N... We. 8... 11121523511 B... W and u;_tgbgr§glg§1§ (30). The Gro EL protein is a large 126 acidic peptide that is believed to "chaperone" proteins for export (22,24). This "chaperone" protein holds newly produced proteins in an "open” conformation so that they can be exported or be assembled into large complexes. It seems reasonable to assume that this peptide is necessary during the LCR because there is a large number of proteins being exported at this time. Most of these proteins are Yops, but they only seem to need the L993 gene for export (36). Therefore, Gro EL is may not needed for export of Yops. An important virulence factor of yersiniae is the Lcr- mediated V antigen which has been shown to prevent granuloma formation during infection (48). This protein has a subunit molecular weight of 38 kdal (9) and can be seen in the figures as the p38 peptide. This peptide is mainly found in the cytoplasm but significant quantities, of the intact protein, can be found in the culture supernatant (9). Since the Gro EL peptide (p56) has been shown to be involved in the export of some proteins, we believe that the p56 peptide is responsible for the export of V antigen. At the same time V antigen was identified as a virulence factor, there was also another peptide thought to be important in virulence termed W antigen (11). This antigen was found to be chromosomally encoded and, until recently, seems to have been forgotten. This protein has been purified in using the original anti-W antisera (23). A crude purification revealed a peptide of 56 kdal. Two- 127 dimensional gel electrophoresis of this crude W antigen preparation and a cytoplasmic extract revealed that W antigen is the same as the p56 peptide (Gro EL). Therefore, we propose that W antigen is actually a V antigen/p56 complex in which the V antigen is in the process of being exported. V antigen could not be detected in this complex due to its instability (9) during the purification of the W antigen. Purified V antigen is not stable in the purified form unless special measures are undertaken (9). However, anti-W antigen sera cross reacted with V antigen although W antigen was found to be much larger (90 kdal to 140 kdal) and have a more acidic isoelectric point (23). The second chromosomally encoded peptide, p70, was found to be a catalase. Since this organism can survive intracellularly, it not unreasonable to assume that this type of enzyme would be necessary to help protect the organism from oxygen radicals. However, this peptide only makes up between 7-20% of the total catalase activity of the cytoplasm (Figure 10). Therefore, its role remains unclear inside the cell. The purified form of the enzyme has a specific activity level comparable to that of the hydroperoxidases of E9 9911 (13,14). However, this is the first reported catalase with an isoelectric point that is highly basic and it is very different than that of any other reported catalase. 128 The restriction of cells during the Lcr in 11 999919 results in the shut down of protein production except for Lcr-plasmid encoded protein which are induced. In this report, we have shown that non-Lcr virulence factors also continue to be produced under these energy limiting conditions (51). More work will have to be done to determine if there is some type of coordinate regulate of these virulence factors. We have also identified 2 chromosomally important peptides that may be required for the Lcr-mediated virulence factors to be effective during infection by 19 pest19. 10. 129 REFERENCES Baker, E. E., H. Sommer, L. E. Foster, E. Meyer, and K. F. Meyer. 1952. Studies on immunization against plague. I. The isolation and characterization of the soluble antigen of 29999999119 999919. J. Immunol. 68:131-145. Beesley, E. D., R. R. Brubaker, W. A. Janssen, and M. J. Surgalla. 1967. Pesticins. III. Expression of coagulase and mechanism of fibrinolysis. J. Bacteriol. 94:19-26. Ben-Efraim, S., M. Aronson, and L. Bichowsky-Slomnicki. 1961. New antigenic componet of 29999999119, 999919 formed under specific conditions of pH and temperature. J. Bacteriol. 81:704-714. Bennett , L . G . and T . G . Tornabene . 19 7 4 . Characterization of the antigenic subunits of the envelope protein of 19991919, 99991_, J. Bacteriol. 117:48-55. Bolin, I., D. A. Portnoy, and H. Wolf-Watz. 1985. Expression of the temperature-inducible outer membrane proteins of yersiniae. Infect. Immun. 48:234-240. Brubaker, R. R., and M. J. Surgalla. 1964. The effect of Ca++ and Mg++ on lysis, growth, and production of virulence antigens by 29599399119 999919. J. Infect. Dis. 114:13-25. Brubaker, R. R. 1983. The Vwa+ virulence factor of yersiniae: molecular basis of the attendant nutritional requirement for Ca2+ . Rev . Infect . Dis . 5 (Suppl. 4):S748-S758. Brubaker, R. R. 1985. Mechanisms of bacterial virulence. Ann. Rev. Microbiol. 39:21-50. Brubaker, R. R., A. K. Sample, D.-z. Yu, R. J. Zahorchak, P. C. Hu, and J. M. Fowler. 1987. Proteolysis of V antigen from 19991919 999919. Microbial Path. 2:49-62. Burrows, T. W., and G. A. Bacon. 1956. The basis of virulence in 29999999119_ 999919: an antigen determining virulence. Brit. J. Exp. Pathol. 37:481- 493. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20 21. 22. 130 Burrows, T. W., and G. A. Bacon. 1960. V and W antigens in strains of Eastsurslla nssssstnnsrsnlssis- Br. J. Exp. Pathol. 41:38-44. Charnetzky, W. T., and R. R. Brubaker. 1982. RNA synthesis in 19991919 999919 during growth restriction in calcium deficient medium. J. Bacteriol. 149:1089- 1095. Claiborne, A., and I. Fridovich. 1979. Purification of the o-dianisidine peroxidase from 29999919919 9911 B. J. Biol. Chem. 254:4245-5252. Claiborne, A., D. P. Malinowski, and I. Fridavich. 1979. Purification and characterization of hydroperoxdase II of 29999919919 9911 B. J. Biol. Chem. 254:11664-11668. Englsberg, E., and L. Ingraham. 1957. Meiotrophic mutants of 299999999119 999919 and their use in elucidation of nurtritional requirements. Proc. Natl. Acad. Sci. USA. 43:369-372. Ferber, D. M., and R. R. Brubaker. 1981. Plasmids in 19991919 99991_. Infect. Immun. 31:839-841. Gaguen, J. D.,~J. Yother, and S. C. Straley. 1984. Genetic analysis of the low calcium response in Yersinia pestis Mu d1 (Ap lac) insertion mutants. J. Bacteriol. 160:842-848. Hendrix, R. W. 1979. Purification and properties of 9995, a host protein involved in bacteriophage assembly. J. Mol. Biol. 129:375-392. Higuchi, K., L. L. Kupferberg, and J. L. Smith. 1959. Studies on the nutrition and physiology of 29999999119 999919: III. Effects of calcium ions on the growth of virulent and avirulent strains of 29999999119 99s9is. J. Bacteriol. 77:317-321. Hoiby, N. 1975. Cross reactions between 2se9d9909as and thirty-six other bacterial species. Scand. J. Immunol. 4(Suppl. 2):187-l92. Jackson, S., and T. W. Burrows. 1956. The pigmentation of 29999999119 pestis on a defined medium containing haemin. Brit. J. Exp. Pathol. 37:570-576. Kusukawa, N., T. Yura, C. Ueguchi, Y. Akiyama, and K. Ito. 1989. Effects of mutations in heat-shock genes 9:955 and grsEL on protein export in Esshsrisnis ssli EMBO J. a: 3517- 3521. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 131 Lawton, W. D., R. L. Erdman, and M. J. Surgalla. 1963. Biosynthesis and purification of V and W antigen in 29999999119 999919. J. Immunol. 91:179-184. Lecker, S., R. Lill, T. Ziegelhoffer, C. Georgopoulos, P.J. Bassford, Jr., C. A. Kumamoto, and W. Wickner. 1989. Three pure chaperone proteins of {29999919919 9911-SecB, trigger factor and GroEL-form soluble complexes with precursor proteins 19 9199_. EMBO J. 8:2703-2709. Lindler, L. E., M. S. Klempner, and S. C. Straley. 1990. 19991919 999919 pH 6 antigen: genetic, biochemical, and virulence characterization of a protein involved in the pathogenesis of bubonic plague. Infect. Immun. 58:2569-2577. Lawry, O. H., N. J. Rosebraugh, A. L. Farr, and R. J. Randall. 1951. Protein :measurement. with the folin phenol reagent. J. Biol. Chem. 193:265-275. McDonaugh, K.A., and S. Falkow. 1989. A 19991919 999919-specific fragment encodes temperature dependent coagulase and fibrinolysin-associated phenotypes. Mol. Microbiol. 3:767-775. Mehigh, R. J., A. K. Sample, and R. R. Brubaker. 1989. Expression of the low-calcium response in 19991919 999919. Microbial Path. 6:203-217. Montie, T. C., and D. B. Montie. 1971. Protein toxins of M9119 999919. Subunit composition and acid binding, Biochemistry 10:2094-2100. Morrison, R.P., R.J. Beland, K. Lyng, and H.D. Caldwell. 1989. Chlamydial disease pathogenesis. J. Exp. Med. 170:1271-1283. O'Farrell, P. H. 1975. High resolution two- dimensional electrophoresis of proteins.‘ J. Biol. Chem. 250:4007-4021. Ouchterlony, O. 1949. Antigen-antibody reactions in gel Acta. Pathol. Microbiol. Scand. 26:507 Portnoy, D. A., S. L. Moseley, and S. Falkow. 1981. Characterization of plasmids and plasmid-associated determinants of 19991919 Moo—0119199 pathogenesis. Infect. Immun. 31:775-782. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 132 Portnoy, D. A., and R. J. Martinez. 1985. Role of a plasmid in the pathogenicity of 19991919 species. Curr. Top. Microbiol. Immunol. 118329-51. Protsenko, O. A., P. I. Anisimav, O. T. Mosarov, N. P. Donnov, Y. A. Papav, and..A. M. Kokushkin. 1983. Detection and characterization of 19991919 999919 plasmids determining pesticin I, fraction I antigen and mouse toxin synthesis. Genetika 19:1081-1090. Rosqvist, R., A. Forsberg, M. Rimpiléinen, T. Bergman, and H. WOlf-Watz. 1990. The cytotoxic protein YopE of ' obstructs the primary host defence. Mal. Microl. 4:657-667. Sample, A. K., J. M. Fowler, and R. R. Brubaker. 1987. Modulation of the low calcium rsponse in 19991919 999919 by plasmid-plasmid interaction. Microbial Path. 23443-453. Sample A. K., and R. R. Brubaker. 1987. Pasttranslational regulation. of Lcr jplasmid-mediated peptides in pesticinogenic 19991919 9est1s. Microbial Path. 33239-248. Sikkema, D. J., and R. R. Brubaker. 1989. Outer- membrane peptides of 19991919, 999919_ mediating siderophore-independent assimilation of iron. Biol. Metals 2:174-184. Simonet, M., S. Richard, and P. Berche. 1990. Electron microscopic evidence for 19 yivo extracellular localization of mm W harboring the pYV plasmid. Infect. Immun. 58:841-845. Sodeinde O. A., and J. D. Gaguen. 1988. Genetic analysis of the 9.5- kilobase virulence plasmid of 19991919 99991_. Infect. Immun. 56:2743-2748. Sodeinde, O. A., A. K. Sample, R. R. Brubaker, and J. D. Gaguen. 1988. Plasminogen activator/coagulase gene of 19991919 995918 is responsible for degradation of plasmid-encoded outer membrane proteins. Infect. Immun. 56:2749-2752. Straley, S. C., and R. R. Brubaker. 1981. Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment. Proc. Natl. Acad. Sci. (Wash.) 78:1224- 1228. 44. 45. 46. 47. 48. 49. 50. 51. 52. 133 Straley, S. C., and R. R. Brubaker. 1982. Localization in 19991919 999919 of peptides associated with virulence. Infect. Immun. 36:129-135. Straley, S. C., and P. A. Harmon. 1984. 19991919 999919 grows within phagolysosomes in mouse peritoneal macrophages. Infect. Immun. 45:655-659. Straley, S. C., and W. S. Bowmer. 1986. Virulence genes regulated at the transcriptional level by Ca2+ in 999919 include structural genes for outer membrane proteins. Infect. Immun. 51:445-454. Une, T., and R. R. Brubaker. 1984. In vivo comparison of avirulent Vwa- and Pg'm- or Pstr phenotypes of yersiniae. Infect. Immun. 43:895-900. Une, T., R. Nakajima, and R. R. Brubaker. 1986. Roles of V antigen in promoting virulence in Yersi919. Contrib. Microbiol. Immunol. 9:179-185. Viitanen, A. -M., P. Toivanen, and M. Skurnik. 1990. The 1993 gene is part of an operon in the 199 region 19991919 99999999119199 0:3. J. Bacteriol. 172: 3152- 3162. Yother, J., L. W. Chamness, and J. D. Gaguen. 1986. Temperature-controlled plasmid regulon associated with low calcium response in 199s1ni9 99991_. J. Bacteriol. 165:443-447. Zahorchak, R. J., W. T. Charnetzky, R. V. Little, and R. R. Brubaker. 1979. Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in 19991919 999919. J. Bacteriol. 1393792-799. Zahorchak, R. J., and R. R. Brubaker. 1982. Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in 19991919 999919. Infect. Immun. 38:953- 959. 134 SUMMARY AND CONCLUSIONS The low calcium response (Lcr) is a very complex virulence factor in yersiniae. The Lcr induces several distinct peptides that are necessary for virulence. Among these is a group of outer membrane peptides termed Yops. These Yops undergo a pesticin plasmid-mediated degradation in 11 999919, that does not occur in the enteropathogenic strains. In this thesis, I have shown that despite this degradation there are sufficient levels of important Yops maintained. This Yap degradation can also occur in 11 999999999999919919 upon transformation of the pesticin plasmid. The resulting degradation is very similar to 11 999919 indicating that these two species have a similar virulence factor but yet cause two distinct types of disease. This report has also identified the production of numerous other peptides during the LCR in 19 999919 when most vegetative protein production has ceased. Among these were several other virulence factors not associated with the LCR and some new products. Two of these virulence factors (fraction 1 and murine exotoxin) were encoded on the large cryptic plasmid while the plasminogen activator/coagulase and the bacteriocin pesticin were encoded on the pesticin plasmid. The pH 6 antigen was also produced and it is a chromosomal peptide. During the onset of the LCR, 2 heat- shock proteins were produced. Another heat-shock protein, 135 Gro EL, was identified as a major cytoplasmic peptide that may be necessary for the export of a known virulence factor (V antigen). A second chromosomally encoded product was identified as a novel catalase whose role is as yet unclear. Therefore, the LCR allows expression of other proteins that are important besides the Lcr-encoded products.