lcrH, a gene necessary for virulence of Yersinia pestis and for the normal response of Y. pestis to ATP and calcium.

We are investigating the functions of the three proteins encoded by the V operon (lcrGVH) of the low-calcium response virulence plasmid pCD1 of Yersinia pestis KIM5. The purpose of this study was to define the role of the 18-kilodalton protein encoded by lcrH, the third gene of the V operon. Using marker exchange mutagenesis, we constructed a Y. pestis mutant that failed to express the LcrH protein. This LcrH- mutant was "ATP blind" in that it failed to show altered growth and V-antigen expression at 37 degrees C when 18 mM ATP was present. It also showed only a partial response to 2.5 mM Ca2+. The parental Y. pestis strain showed full growth yield at 37 degrees C and depressed expression of V antigen and of yop (yersinial pCD1-encoded outer membrane protein) genes in response to ATP or Ca2+. In contrast, the LcrH- mutant failed to grow at 37 degrees C in the presence of ATP and showed only limited growth when Ca2+ was present. V-antigen expression in the mutant was not depressed by ATP and only partially depressed by Ca2+. These findings show that LcrH is necessary for the normal response of Y. pestis to ATP and that LcrH contributes to Ca2+ responsiveness. The mutant also showed abnormal yopJ expression, indicating that LcrH also is necessary for normal yop regulation. The LcrH- mutant was avirulent in mice, probably because of its compromised growth at 37 degrees C. These findings indicate that the responses of Y. pestis to ATP and Ca2+ are distinct and that lcrH encodes a protein that is an important mediator of Ca2+ and ATP regulation of pCD1-encoded virulence determinant(s) in Y. pestis.     

YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice.

In Yersinia pestis KIM there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only YopM and YopN are found in easily detectable amounts in the culture medium. In our previous work, we characterized the yopM gene. In the present study, we constructed a YopM- mutant to elucidate the role of YopM in the virulence of Y. pestis. A lacZYA sequence was inserted 126 base pairs downstream from the start codon of the yopM gene in pCD1. The YopM- mutant had the same growth properties as the parent, Y. pestis KIM5-3001. The inserted lacZ gene was regulated by the promoter of the yopM gene. Accordingly, it was expressed strongly at 37 degrees C in the absence of Ca2+ and was decreased in expression when Ca2+ was present. Northern blot (RNA blot) analysis revealed that the yopM gene was in a monocistronic operon, suggesting that the yopM insertion mutation was unlikely to have polar effects on other genes. The YopM- mutant had strongly decreased virulence in mice, with a 50% lethal dose of 3.4 x 10(5) CFU. Virulence was restored by the cloned yopM-containing 5.5-kilobase HindIII F fragment of pCD1. However, supplying a cloned 1.57-kilobase fragment containing little more than the yopM structural gene caused the yopM mutant to significantly overexpress YopM and failed to restore virulence. The infection kinetics of the YopM- mutant revealed growth in both spleens and livers from days 2 to 4 after infection, followed by a precipitous clearance of the bacteria. YopM-containing supernatant proteins of Y. pestis inhibited thrombin- or ristocetin-induced platelet aggregation, whereas there was no inhibition by supernatant proteins from the YopM- Y. pestis mutant. Accordingly, YopM may prevent platelet-mediated events and serve as an important strategy for the yersiniae in the initial stages of a plague infection.     

Transfer of the virulence plasmid of Yersinia pestis to Yersinia pseudotuberculosis.

Transposon Tn5 insertion derivatives of the virulence plasmid pYV019 of Yersinia pestis were transferred by P1 transduction into a plasmid-free strain of Y. pseudotuberculosis. One of these plasmid derivatives conferred virulence upon the Y. pseudotuberculosis strain. This strain had the ability to express temperature-inducible plasmid-coded outer membrane proteins and was also found to be Ca2+ dependent.     

Calcium-regulated type III secretion of Yop proteins by an Escherichia coli hha mutant carrying a Yersinia pestis pCD1 virulence plasmid

A series of four large deletions that removed a total of ca. 36 kb of DNA from the ca. 70-kb Yersinia pestis pCD1 virulence plasmid were constructed using lambda Red-mediated recombination. Escherichia coli hha deletion mutants carrying the virulence plasmid with the deletions expressed a functional calcium-regulated type III secretion system. The E. coli hha/pCD1 system should facilitate molecular studies of the type III secretion process.     

Polymorphisms in the lcrV gene of Yersinia enterocolitica and their effect on plague protective immunity

Current efforts to develop plague vaccines focus on LcrV, a polypeptide that resides at the tip of type III secretion needles. LcrV-specific antibodies block Yersinia pestis type III injection of Yop effectors into host immune cells, thereby enabling phagocytes to kill the invading pathogen. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by Yersinia enterocolitica strains and suggested that lcrV polymorphisms may provide for escape from LcrV-mediated plague immunity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIM D27 LcrV (LcrV(D27)) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrV(W22703)) or O:8 strain WA-314 (LcrV(WA-314)) but are otherwise unable to block type III injection by Y. enterocolitica strains. Replacing the lcrV gene on the pCD1 virulence plasmid of Y. pestis KIM D27 with either lcrV(W22703) or lcrV(WA-314) does not affect the ability of plague bacteria to secrete proteins via the type III pathway, to inject Yops into macrophages, or to cause lethal plague infections in mice. LcrV(D27)-specific antibodies blocked type III injection by Y. pestis expressing lcrV(W22703) or lcrV(WA-314) and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrV(D27) are unable to block the type III injection of Y. enterocolitica strains, expression of lcrV(W22703) or lcrV(WA-314) in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model.     

Complete DNA sequence of Yersinia enterocolitica serotype 0:8 low-calcium-response plasmid reveals a new virulence plasmid-associated replicon

The complete nucleotide sequence and organization of the Yersinia enterocolitica serotype 0:8 low-calcium-response (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for ylpA. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in Yersinia pestis plasmid pCD1 or in Y. enterocolitica serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, ISYen1, was a new IS belonging to the IS256 family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by Y. enterocolitica serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the spy/sopABC loci of pCD1 and pYVe227. Sequence analysis suggests that Yersinia LCR plasmids are from a common ancestor but that Y. enterocolitica serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of Y. enterocolitica serotype 0:8 plasmids from those of Y. enterocolitica serotype 0:9 and Y. pestis LCR plasmids.     

Expression of the plague plasminogen activator in Yersinia pseudotuberculosis and Escherichia coli

Enteropathogenic yersiniae (Yersinia pseudotuberculosis and Yersinia enterocolitica) typically cause chronic disease as opposed to the closely related Yersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage by Y. pestis of a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared approximately 70-kb low-calcium-response, or Lcr, plasmids (pCD in Y. pestis and pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (alpha-Pla) and slightly smaller (beta-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only alpha-Pla accumulates in Escherichia coli LE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble alpha and beta forms possessing biological activity. This process also converted cell-bound alpha-Pla to beta-Pla and smaller forms in Y. pestis KIM/pPCP1 and Y. pseudotuberculosis PB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops in Y. pseudotuberculosis PB1/+/pPCP1 occurred more slowly than that in Y. pestis but was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP by Y. pseudotuberculosis did not significantly influence virulence in mice.     

Expression of antigens encoded by the virulence plasmid of Yersinia enterocolitica under different growth conditions.

The expression of polypeptides of the virulence plasmid of Yersinia enterocolitica serotype O:3 was studied with the immunoblotting technique and specifically absorbed antisera to Y. enterocolitica O:3. At least 16 polypeptides were apparently specified by the virulence plasmid when plasmid-bearing bacterial were grown at 37 degrees C or intraperitoneally in semipermeable capsules. The different growth media used (also with added Ca2+) had quantitatively or qualitatively only a little influence on the expression of these polypeptides, whereas the growth temperature had a stronger influence. The best expression was achieved at 37 degrees C, at 22 degrees C the expression was weak, and at 4 degrees C the plasmid genes were inactive. Two chromosomally encoded polypeptides were expressed only at 37 degrees C, whereas the expression of eight polypeptides expressed at 22 degrees C was repressed at 37 degrees C. The intraperitoneal growth in capsules was used to detect the virulence plasmid-associated polypeptides of Yersinia pestis. Four plasmid-associated polypeptides were detected in Y. pestis with antiserum to Y. enterocolitica virulence plasmid antigens, and three were detected with antiserum to Y. pestis EV76. These results suggested that the virulence plasmid of Y. pestis was activated in the interstitial environment in vivo, where Ca2+ concentration was high, and also that the virulence plasmids of Y. enterocolitica and Y. pestis have three to four immunologically related polypeptides in common.     

Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins.

The virulence plasmids pYV019, pYV8081, and pIB1 from Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis, respectively, were characterized by restriction endonuclease analysis. The three plasmids exhibited a region of common DNA previously shown to encode determinants which confer Ca2+ dependence. The plasmids from Y. pestis and Y. pseudotuberculosis were similar throughout their genomes. In contrast, a region of the plasmid from Y. enterocolitica which contained an origin of replication differed from the other two plasmids as determined by DNA homology and replication properties. Plasmid-associated outer membrane proteins from all three species of Yersinia were characterized by polyacrylamide gel electrophoresis. There were no differences in the outer membrane protein profiles between plasmid-containing and homogenic strains lacking the plasmid after growth at 28 degrees C. After growth at 37 degrees C, both Y. enterocolitica and Y. pseudotuberculosis showed at least four major plasmid-associated outer membrane proteins. Y. pestis did not show any discernible changes after growth at 37 degrees C. It was shown by using E. coli minicell analysis that the plasmid DNA from all three species of Yersinia contained the coding capacity for production of the novel outer membrane proteins.     

A low-Ca2+ response operon encodes the V antigen of Yersinia pestis.

Yersinia pestis has a virulence regulon called the low-Ca2+ response that is mediated by the plasmid pCD and manifested as regulation of growth and of expression of several virulence-associated properties by Ca2+ and temperature. We used Mu dI(Ap lac) to obtain a mutation in pCD1 of Y. pestis KIM that rendered the bacteria unable to express one of these properties, the V antigen. This mutant also had lost the Ca2+ requirement for growth at 37 degrees C and was avirulent in mice. Two-dimensional protein gel electrophoresis showed that the Mu dI(Ap lac) insertion had eliminated 13,000- and 18,000-molecular-weight proteins in addition to the V antigen. We mapped the Mu dI(Ap lac) insertion within pCD1, cloned the HindIII fragment spanning the insertion location, prepared two subclones of this fragment, and identified the proteins these clones expressed in Escherichia coli minicells. The data indicated that the V gene lies within an operon containing three genes; lcrG (encoding the 13,000-molecular-weight protein), lcrV (encoding the 38,000-molecular-weight V antigen), and lcrH (encoding the 18,000-molecular-weight protein). Therefore, the V operon contains the structural gene for V antigen, at least one virulence gene, and at least one Ca2+-dependence gene.     

Influence of Na(+), dicarboxylic amino acids, and pH in modulating the low-calcium response of Yersinia pestis

The virulence of yersiniae is promoted in part by shared approximately 70-kb plasmids (pCD in Yersinia pestis and pYV in enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica) that mediate a low-calcium response. This phenotype is characterized at 37 degrees C by either bacteriostasis in Ca(2+)-deficient medium with expression of pCD/pYV-encoded virulence effectors (Yops and LcrV) or vegetative growth and repression of Yops and LcrV with > or =2.5 mM Ca(2+) (Lcr(+)). Regulation of Yops and LcrV is well defined but little is known about bacteriostasis other than that Na(+) plus l-glutamate promotes prompt restriction of Y. pestis. As shown here, l-aspartate substituted for l-glutamate in this context but only Na(+) exacerbated the nutritional requirement for Ca(2+). Bacteriostasis of Y. pestis (but not enteropathogenic yersiniae) was abrupt in Ca(2+)-deficient medium at neutral to slightly alkaline pH (7.0 to 8.0), although increasing the pH to 8.5 or 9.0, especially with added Na(+) (but not l-glutamate), facilitated full-scale growth. Added l-glutamate (but not Na(+)) favored Ca(2+)-independent growth at acidic pH (5.0 to 6.5). Yops and LcrV were produced in Ca(2+)-deficient media at pH 6.5 to 9.0 regardless of the presence of added Na(+) or l-glutamate, although their expression at alkaline pH was minimal. Resting Ca(2+)-starved Lcr(+) cells of Y. pestis supplied with l-glutamate first excreted and then destroyed l-aspartate. These findings indicate that expression of Yops and LcrV is necessary but not sufficient for bacteriostasis of Ca(2+)-starved yersiniae and suggest that abrupt restriction of Y. pestis requires Na(+) and the known absence of aspartate ammonia-lyase in this species.     

Role of a New Intimin/Invasin-Like Protein in Yersinia pestis Virulence

A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1(+)) Pgm(-) (pigmentation negative)/, KIM6(pCD1(-)) Pgm(+), and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δilp had a 55-fold increase in the 50% lethal dose ([LD(50)] 1.64 × 10(4) CFU) compared to the parental wild-type strain LD(50) (2.98 × 10(2) CFU). These findings identified Ilp as a novel virulence factor of Y. pestis.     

Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae

Three bacterial species within the genus Yersinia are causative agents of human disease. Yersinia pestis is transmitted by fleas or in aerosols, infects regional lymph nodes or lungs, and causes the highly lethal disease known as plague. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteric pathogens most commonly associated with self-limiting infections of the mesenteric lymph nodes. Although Y. pestis and the enteropathogenic Yersinia species utilize different modes of transmission and cause different diseases, they rely on a common set of "core" virulence determinants to successfully infect a mammalian host. These virulence factors are encoded on the bacterial chromosome and on an approximately 70-kb plasmid. Once established in lymphoid tissue, all three Yersinia species replicate as aggregates of extracellular bacteria within necrotic lesions or abscesses. At this stage of the infectious process, the bacteria resist phagocytosis by neutrophils, which are able to destroy the bacteria if they are internalized. A type III secretion system encoded on the 70-kb plasmid functions to export multiple proteins (the Yops and LcrV) that are delivered to the extracellular milieu, the plasma membrane, or the cytosol of a host target cell. The Yops and LcrV act in concert to inhibit phagocytosis and downregulate inflammation. Although it is clear that the bulk of bacterial multiplication occurs in an extracellular phase, there is also evidence that all three pathogenic Yersinia survive and multiply in macrophages. Survival and replication of Yersinia in macrophages may occur throughout the infection, but is likely to be of greatest importance at early stages of colonization. That macrophages can serve as permissive sites for bacterial replication in vivo is supported by in vitro experiments, which demonstrate that Y. pestis, Y. peudotuberculosis, and Y. enterocolitica share the ability to survive and multiply in macrophage phagosomes. There is also evidence that the bacteria can subvert the functions of macrophages from within, by inhibiting phagosome acidification (Y. pseudotuberculosis) and the production of nitric oxide (Y. pestis and Y. pseudotuberculosis). Although considerable attention has been focused on how Yersinia subverts the functions of phagocytes from the outside, the study of how these bacteria subvert macrophage functions from the inside will lead to a better overall understanding of Yersinia pathogenesis.     

The pH 6 antigen is an antiphagocytic factor produced by Yersinia pestis independent of Yersinia outer proteins and capsule antigen

The pH 6 antigen (pH 6 Ag; PsaA) of Yersinia pestis has been shown to be a virulence factor. In this study, we set out to investigate the possible function of Y. pestis PsaA in a host cell line, RAW264.7 mouse macrophages, in order to better understand the role it might play in virulence. Y. pestis KIM5 derivatives with and without the pCD1 plasmid and their psaA isogenic counterparts and Escherichia coli HB101 and DEta5alpha carrying a psaA clone or a vector control were used for macrophage infections. Macrophage-related bacteria and gentamicin-resistant intracellular bacteria generated from plate counting and direct microscopic examinations were used to evaluate these RAW264.7 macrophage infections. Y. pestis psaA isogenic strains did not show any significant difference in their abilities to associate with or bind to mouse macrophage cells. However, expression of psaA appeared to significantly reduce phagocytosis of both Y. pestis and E. coli by mouse macrophages (P < 0.05). Furthermore, we found that complementation of psaA mutant Y. pestis strains could completely restore the ability of the bacteria to resist phagocytosis. Fluorescence microscopy following differential labeling of intracellular and extracellular Y. pestis revealed that significantly lower numbers of psaA-expressing bacteria were located inside the macrophages. Enhanced phagocytosis resistance was specific for bacteria expressing psaA and did not influence the ability of the macrophages to engulf other bacteria. Our data demonstrate that Y. pestis pH 6 Ag does not enhance adhesion to mouse macrophages but rather promotes resistance to phagocytosis.     

Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis

Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37 degrees C. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is unique to Y. pestis. F1 is a surface polymer composed of a protein subunit, Caf1, with a molecular mass of 15.5 kDa. The secretion and assembly of F1 require the caf1M and caf1A genes, which are homologous to the chaperone and usher protein families required for biogenesis of pili. F1 has been implicated to be involved in the ability of Y. pestis to prevent uptake by macrophages. In this study we addressed the role of F1 antigen in inhibition of phagocytosis by the macrophage-like cell line J774. The Y. pestis strain EV76 was found to be highly resistant to uptake by J774 cells. An in-frame deletion of the caf1M gene of the Y. pestis strain EV76 was constructed and found to be unable to express F1 polymer on the bacterial surface. This strain had a somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, which is cured for the virulence plasmid common to the pathogenic Yersinia species, was, as expected, much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid and caf1M was even further hampered in the ability to prevent uptake and, in this case, essentially all bacteria (95%) were phagocytosed. Thus, F1 and the virulence plasmid-encoded type III system act in concert to make Y. pestis highly resistant to uptake by phagocytes. In contrast to the type III effector proteins YopE and YopH, F1 did not have any influence on the general phagocytic ability of J774 cells. Expression of F1 also reduced the number of bacteria that interacted with the macrophages. This suggests that F1 prevents uptake by interfering at the level of receptor interaction in the phagocytosis process.     

Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins.

The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid.     

Integration of the plasmid encoding the synthesis of capsular antigen and murine toxin into Yersinia pestis chromosome

Yersinia pestis, the causative agent of plague, usually carries three plasmids. The largest of them, a 60 megadalton (MDa) replicon designated pFra determines the synthesis of capsular antigen (fraction I) and murine toxin. Both products are involved in the expression of virulence. Previously, several cases of integration into the bacterial chromosome of the calcium dependence plasmid common to pathogenic Yersinia species have been described. In this study, using the Southern hybridization method, we have shown that the plasmid pFra of Y. pestis also integrates into the host chromosome in some clones of strain populations. The integration could be observed for both the intact plasmid and its mutant derivatives unable to express murine toxin or mediating a dramatically reduced level of capsular antigen synthesis.     

The role of the phoPQ operon in the pathogenesis of the fully virulent CO92 strain of Yersinia pestis and the IP32953 strain of Yersinia pseudotuberculosis

At the genomic level, Yersinia pestis and Yersinia pseudotuberculosis are nearly identical but cause very different diseases. Y. pestis is the etiologic agent of plague; whereas Y. pseudotuberculosis causes a gastrointestinal infection primarily after the consumption of contaminated food. In many gram-negative pathogenic bacteria, PhoP is part of a two-component global regulatory system in which PhoQ serves as the sensor kinase, and PhoP is the response regulator. PhoP is known to activate a number of genes in many bacteria related to virulence. To determine the role of the PhoPQ proteins in Yersinia infections, primarily using aerosol challenge models, the phoP gene was deleted from the chromosome of the CO92 strain of Y. pestis and the IP32953 strain of Y. pseudotuberculosis, leading to a polar mutation of the phoPQ operon. We demonstrated that loss of phoPQ from both strains leads to a defect in intracellular growth and/or survival within macrophages. These in vitro data would suggest that the phoPQ mutants would be attenuated in vivo. However, the LD(50) for the Y. pestis mutant did not differ from the calculated LD(50) for the wild-type CO92 strain for either the bubonic or pneumonic murine models of infection. In contrast, mice challenged by aerosol with the Y. pseudotuberculosis mutant had a LD(50) value 40× higher than the wild-type strain. These results demonstrate that phoPQ are necessary for full virulence by aerosol infection with the IP32953 strain of Y. pseudotuberculosis. However, the PhoPQ proteins do not play a significant role in infection with a fully virulent strain of Y. pestis.