Yersinia pestis caf1 variants and the limits of plague vaccine protection

Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.     

Virulence Role of V Antigen of Yersinia pestis at the Bacterial Surface

Yersinia pestis, the etiologic agent of plague, secretes a set of environmentally regulated, plasmid pCD1-encoded virulence proteins termed Yops and V antigen (LcrV) by a type III secretion mechanism (Ysc). LcrV is a multifunctional protein that has been shown to act at the level of secretion control by binding the Ysc inner-gate protein LcrG and to modulate the host immune response by altering cytokine production. LcrV also is essential for the unidirectional targeting of Yops to the cytosol of infected eukaryotic cells. In this study, we constructed an in-frame deletion within lcrG (DeltalcrG3) to further analyze the requirement of LcrV in Yop targeting. We confirmed the essentiality of LcrV and found that LcrG may have a facilitative role, perhaps by promoting efficient secretion of LcrV. We also constructed mutants of lcrV expressing LcrV truncated at the N or C terminus. Both the N and C termini of LcrV were required for the secretion of LcrV into the medium and targeting of Yops. LcrV was detected in punctate zones on the surface of fixed Y. pestis by laser-scanning confocal microscopy, and this localization required a functional Ysc. However, the truncated LcrV proteins were not found on the bacterial surface. Finally, we tested the ability of LcrV-specific Fab antibody fragments or full-length antibody to interfere with Yop targeting and found no interference, even though this antibody protects mice against plague. These results indicate that LcrV may function in Yop targeting at the extracellular surface of yersiniae and that the protective efficacy of LcrV-specific antibodies can be manifested without blocking Yop targeting.     

Plague in Guinea pigs and its prevention by subunit vaccines

Human pneumonic plague is a devastating and transmissible disease for which a Food and Drug Administration-approved vaccine is not available. Suitable animal models may be adopted as a surrogate for human plague to fulfill regulatory requirements for vaccine efficacy testing. To develop an alternative to pneumonic plague in nonhuman primates, we explored guinea pigs as a model system. On intranasal instillation of a fully virulent strain, Yersinia pestis CO92, guinea pigs developed lethal lung infections with hemorrhagic necrosis, massive bacterial replication in the respiratory system, and blood-borne dissemination to other organ systems. Expression of the Y. pestis F1 capsule was not required for the development of pulmonary infection; however, the capsule seemed to be important for the establishment of bubonic plague. The mean lethal dose (MLD) for pneumonic plague in guinea pigs was estimated to be 1000 colony-forming units. Immunization of guinea pigs with the recombinant forms of LcrV, a protein that resides at the tip of Yersinia type III secretion needles, or F1 capsule generated robust humoral immune responses. Whereas LcrV immunization resulted in partial protection against pneumonic plague challenge with 250 MLD Y. pestis CO92, immunization with recombinant F1 did not. rV10, a vaccine variant lacking LcrV residues 271-300, elicited protection against pneumonic plague, which seemed to be based on conformational antibodies directed against LcrV.     

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.     

Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 x 10(7) CFU of the Deltalpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Deltalpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD(50)). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Deltalpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Deltalpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Deltalpp mutant than in those infected with WT CO92. Additionally, the Deltalpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Deltalpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.     

Vaccination with live Yersinia pestis primes CD4 and CD8 T cells that synergistically protect against lethal pulmonary Y. pestis infection

Vaccination with live attenuated Yersinia pestis confers protection against pneumonic plague but is not considered safe for general use. Subunit plague vaccines containing the Y. pestis F1 and LcrV proteins prime robust antibody responses but may not provide sufficient protection. To aid the development of a safe and effective plague vaccine, we are investigating roles for T cells during defense against Y. pestis infection. Here we demonstrate that vaccination of mice with live Y. pestis primes specific CD4 and CD8 T cells that, upon purification and direct transfer to na?ve mice, synergistically protect against lethal intranasal Y. pestis challenge. While not preventing extrapulmonary dissemination, the coadministered T cells promote bacterial clearance and reduce bacteremia. These observations strongly suggest that development of pneumonic plague vaccines should strive to prime both CD4 and CD8 T cells. Finally, we demonstrate that vaccination with live Y. pestis primes CD4 and CD8 T cells that respond to Y. pestis strains lacking the capacity to express F1, LcrV, and all pCD1/pPCP-encoded proteins, suggesting that protective T cells likely recognize antigens distinct from those previously defined as targets for humoral immunity.     

Passive immunity to yersiniae mediated by anti-recombinant V antigen and protein A-V antigen fusion peptide.

LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, has been implicated as a regulator of the low-calcium response, virulence factor, and protective antigen. In this study, lcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yielding major peptides of 36, 35, 34, and 32 to 29 kDa. Rabbit gamma globulin raised against this mixture of cleavage products provided significant protection against 10 minimum lethal doses of Y. pestis (P < 0.01) and Y. pseudotuberculosis (P < 0.02). To both stabilize V antigen and facilitate its purification, plasmid pPAV13 was constructed so as to encode a fusion of lcrV and the structural gene for protein A (i.e., all but the first 67 N-terminal amino acids of V antigen plus the signal sequence and immunoglobulin G-binding domains but not the cell wall-associated region of protein A). The resulting fusion peptide, termed PAV, could be purified to homogeneity in one step by immunoglobulin G affinity chromatography and was stable thereafter. Rabbit polyclonal gamma globulin directed against PAV provided excellent passive immunity against 10 minimum lethal doses of Y. pestis (P < 0.005) and Y. pseudotuberculosis (P < 0.005) but was ineffective against Y. enterocolitica. Protection failed after absorption with excess PAV, cloned whole V antigen, or a large (31.5-kDa) truncated derivative of the latter but was retained (P < 0.005) upon similar absorption with a smaller (19.3-kDa) truncated variant, indicating that at least one protective epitope resides internally between amino acids 168 and 275.     

Evaluation of the role of LcrV-Toll-like receptor 2-mediated immunomodulation in the virulence of Yersinia pestis

Pathogenic members of the Yersinia genus require the translocator protein LcrV for proper function of the type III secretion apparatus, which is crucial for virulence. LcrV has also been reported to play an independent immunosuppressive role via the induction of interleukin-10 (IL-10) through stimulation of Toll-like receptor 2 (TLR2). To investigate the LcrV-TLR2 interaction in vitro, His-tagged recombinant LcrV (rLcrV) from Yersinia pestis was cloned and expressed in Escherichia coli and purified through Ni-nitrilotriacetic acid column chromatography. High concentrations (5 microg/ml) of rLcrV stimulated TLR2 in vitro. Fractionation of rLcrV preparations via gel filtration revealed that only a minor component consisting of high-molecular-weight multimers or aggregates has TLR2 stimulating activity. Dimer and tetramer forms of rLcrV, which constitute the bulk of the material, do not have this activity. To investigate the potential role of LcrV/TLR2 in plague pathogenesis, we infected wild-type and TLR2(-/-) mice with virulent Y. pestis. No discernible difference between the two mouse strains in severity of disease or kinetics of survival after subcutaneous challenge was observed. IL-6, tumor necrosis factor, and IL-10 levels from spleen homogenates; bacterial load; and the extent of inflammation observed in organs from mice infected intravenously were also indistinguishable in both mouse strains. Taken together, our data indicate that the most abundant molecular species of Y. pestis LcrV do not efficiently activate TLR2-signaling and that TLR2-mediated immunomodulation is unlikely to play a significant role in plague.     

The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis

Yersinia pestis, the agent of plague, has arisen from a less virulent pathogen, Yersinia pseudotuberculosis, by a rapid evolutionary process. Although Y. pestis displays a large number of virulence phenotypes, it is not yet clear which of these phenotypes descended from Y. pseudotuberculosis and which were acquired independently. Y. pestis is known to replicate in macrophages, but there is no consensus in the literature on whether Y. pseudotuberculosis shares this property. We investigated whether the ability to replicate in macrophages is common to Y. pestis and Y. pseudotuberculosis or is a unique phenotype of Y. pestis. We also examined whether a chromosomal type III secretion system (TTSS) found in Y. pestis is present in Y. pseudotuberculosis and whether this system is important for replication of Yersinia in macrophages. A number of Y. pestis and Y. pseudotuberculosis strains of different biovars and serogroups, respectively, were tested for the ability to replicate in primary murine macrophages. Two Y. pestis strains (EV766 and KIM10(+)) and three Y. pseudotuberculosis strains (IP2790c, IP2515c, and IP2666c) were able to replicate in macrophages with similar efficiencies. Only one of six strains tested, the Y. pseudotuberculosis YPIII(p(-)) strain, was defective for intracellular replication. Thus, the ability to replicate in macrophages is conserved in Y. pestis and Y. pseudotuberculosis. Our results also indicate that a homologous TTSS is present on the chromosomes of Y. pestis and Y. pseudotuberculosis and that this secretion system is not required for replication of these bacteria in macrophages.     

Antibody against V antigen prevents Yop-dependent growth of Yersinia pestis

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously by Y. pestis KIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary for Yersinia growth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10-/- mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs of Y. pestis lacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.     

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.     

Immune response to plasmid- and chromosome-encoded Yersinia antigens.

The immune response of humans and mice to temperature-specific, plasmid- or chromosome-encoded proteins of yersinia pestis and Yersinia enterocolitica was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Extracts from Y. pestis and Y. enterocolitica strains with and without the virulence plasmids pYV019 and pYV8081, respectively, were resolved by denaturing electrophoresis, and the major antigens were visualized with sera from convalescing plague patients, individuals immunized with plague vaccine, and mice and rabbits immunized with avirulent live yersiniae. The Y. pestis grown in vitro in this study did not express detectable amounts of plasmid-encoded antigens. The sera from plague patients recognized Y. pestis and Y. enterocolitica antigens ranging from 15 to 72 kilodaltons (kDa), whereas sera from immunized subjects recognized four antigenic components in Y. pestis ranging from 17 to 64 kDa and five antigens in Y. enterocolitica ranging from 16 to 68 kDa. Sera from mice reacted with 7 antigens in Y. pestis and 12 antigens in Y. enterocolitica ranging from 14 to 68 kDa, and sera from rabbits reacted with 7 and 10 antigens in Y. pestis and Y. enterocolitica, respectively. All of the plague patient sera, as well as the sera from immunized mice and rabbits, reacted with a 22-kDa Y. enterocolitica plasmid-associated polypeptide, and five of the patient sera also recognized a Y. enterocolitica plasmid-associated 31-kDa protein. The results indicate a common immune response to at least these two plasmid-specified Yersinia outer membrane proteins. Y. pestis apparently expresses these components only in vivo, and in vitro, Y. enterocolitica expresses a greater number of plasmid-associated antigens than does Y. pestis.     

Novel virulence-associated type II secretion system unique to high-pathogenicity Yersinia enterocolitica

Yersinia enterocolitica strains comprise an important group of bacterial enteropathogens that cause a broad range of gastrointestinal syndromes. Three groups are distinguishable within this bacterial species, namely, the nonpathogenic group (biotype 1A strains), the low-pathogenicity, non-mouse-lethal group (biotypes 2 to 5), and the high-pathogenicity, mouse-lethal group (biotype 1B). To date, the presence of the high-pathogenicity island (HPI), a chromosomal locus that encodes the yersiniabactin system (involved in iron uptake), defines essentially the difference between low-pathogenicity and high-pathogenicity Y. enterocolitica strains, with the low-pathogenicity strains lacking the HPI. Using the powerful tool of representational difference analysis between the nonpathogenic 1A strain, NF-O, and its high-pathogenicity 1B counterpart, WA-314, we have identified a novel type II secretion gene cluster (yts1C-S) occurring exclusively in the high-pathogenicity group. The encoded secreton, designated Yts1 (for Yersinia type II secretion 1) was shown to be important for virulence in mice. A close examination of the almost completed genome sequence of another high-pathogenicity representative, Y. enterocolitica 8081, revealed a second putative type II secretion cluster uniformly distributed among all Y. enterocolitica isolates. This putative species-specific cluster (designated yts2) differed significantly from yts1, while resembling more closely the putative type II cluster present on the genome of Y. pestis. The Yts1 secreton thus appears to have been additionally acquired by the high-pathogenicity assemblage for a virulence-associated function.     

Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ

Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1beta (IL-1beta) in na?ve macrophages infected with Yersinia enterocolitica. Na?ve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1beta was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1beta following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1beta were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.     

Virulence markers of LCR plasmid in Indian isolates of Yersinia pestis

Presence of 10 important yop genes in Yersinia pestis isolates (18 in number) of Indian origin from 1994 plague outbreak regions of Maharashtra (6 Rattus rattus & Tetera indica rodents) and Gujarat (11 from human patients, 1 from R. rattus) and from plague endemic regions of the Deccan plateau (8 from T. indica) was located by PCR and specific enzyme immunoassay. PCRs were standardized for six effector yops (YopE, YopH, YopJ, YopM, YopO and YopT), three translocator yops (YopB, YopD and YopK) and a regulator LcrV gene. Amplification of all the 10 yop genes was observed in isolates recovered from pneumonic patients and in 5 of 7 rodents from outbreak regions. Among these, amplification of the yopD gene was absent in all eight isolates, and that of yopM in all except one (10R). One of the isolates from rodents of the Deccan plateau (24H) was consistently negative for all the yops. Cloning and expression of truncated yopM (780 bp), yopB (700 bp) and lcrV (796 bp) genes in pQE vectors with SG13009 host cells yielded recombinant proteins for generation of monoclonal antibodies for further use in enzyme immunoassay. Ten stable reactive clones for YopB, nine for YopM and six for LcrV were obtained, all of them exhibiting specific reactions only to Y. pestis. Testing of 26 Y. pestis isolates by monoclonal antibody dot-ELISA and Western blotting provided results identical to PCR, suggesting that the isolates that failed to show PCR amplification also had no expression of their respective proteins. The Y. pestis isolates of outbreak regions had their virulence factors intact in the LCR plasmid. Yersinia pestis isolates recovered from rodents of the Deccan plateau were relatively heterogeneous. It appears that a long residency of Y. pestis of nearly 100 years in the enzootic plague foci has resulted in shedding of virulence genes in the LCR plasmid region in a fairly large proportion of the organisms, possibly due to natural recombination.     

Yersinia enterocolitica O:9 as a potential live oral carrier for protective antigens

Yersinia enterocolitica has the capacity to invade the intestinal tissue and to resist the primary host resistance. The former is chromosome coded while the second largely depends on the presence of a 70 kb plasmid called pYV. This plasmid directs the conditional synthesis of high amounts of proteins (YOPs) that are secreted and inserted in the outer membrane. In order to evaluate Y. enterocolitica W22703 as a potential live carrier for immunization, three strains expressing beta-galactosidase (GZ), were tested for their ability to induce an antibody response to this antigen in mice. The first strain contained plasmid pGC1256, a mutated pYV plasmid containing lacZ transcribed from a yop gene promoter. This strain produced high amounts of GZ instead of a YOP protein and was shown to be hypovirulent. The other strains tested were W22703 pYV+ and pYV- containing a derepressed lac operon carried on an independent plasmid. Immunoblot analysis of sera of mice having received by oral inoculation, W22703(pGC1256) or the pYV+ GZ producing strain revealed the presence of antibodies to GZ. The response to GZ after inoculation of W22703(pGC1256) was shown by ELISA to be only slightly inferior to that obtained by subcutaneous injection of GZ. No response was obtained after oral inoculation of the pYV-GZ producing strain. This showed that the presence of pYV was necessary to obtain an antibody response in this system.     

LcrV plague vaccine with altered immunomodulatory properties

Yersinia pestis, the causative agent of plague, secretes LcrV (low-calcium-response V or V antigen) during infection. LcrV triggers the release of interleukin 10 (IL-10) by host immune cells and suppresses proinflammatory cytokines such as tumor necrosis factor alpha and gamma interferon as well as innate defense mechanisms required to combat the pathogenesis of plague. Although immunization of animals with LcrV elicits protective immunity, the associated suppression of host defense mechanisms may preclude the use of LcrV as a human vaccine. Here we show that short deletions within LcrV can reduce its immune modulatory properties. An LcrV variant lacking amino acid residues 271 to 300 (rV10) elicited immune responses that protected mice against a lethal challenge with Y. pestis. Compared to full-length LcrV, rV10 displayed a reduced ability to release IL-10 from mouse and human macrophages. Furthermore, the lipopolysaccharide-stimulated release of proinflammatory cytokines by human or mouse macrophages was inhibited by full-length LcrV but not by the rV10 variant. Thus, it appears that LcrV variants with reduced immune modulatory properties could be used as a human vaccine to generate protective immunity against plague.     

LcrV capture enzyme-linked immunosorbent assay for detection of Yersinia pestis from human samples

In the United States, there is currently a major gap in the diagnostic capabilities with regard to plague. To address this, we developed an antigen capture assay using an essential virulence factor secreted by Yersinia spp., LcrV, as the target antigen. We generated anti-LcrV monoclonal antibodies (MAbs) and screened them for the ability to bind bacterially secreted native Yersinia pestis LcrV. Anti-LcrV MAb 19.31 was used as a capture antibody, and biotinylated MAb 40.1 was used for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay is 0.1 ng/ml. The assay detected LcrV from human sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV. This assay could be used as a tool to help confirm the diagnosis of plague in patients presenting with pneumonia.     

The plague virulence protein YopM targets the innate immune response by causing a global depletion of NK cells

Yersinia pestis, the etiologic agent of plague, delivers six Yersinia outer proteins (Yops) into host cells upon direct bacterial contact. One of these, YopM, is necessary for virulence in a mouse model of septicemic plague, but its pathogenic function is unknown. We report here the immune processes affected by YopM during infection. To test whether the innate or adaptive immune system is targeted by YopM, C57BL/6 (B6) and B6 SCID mice were infected with either the conditionally virulent Y. pestis KIM5 or a yopM deletion mutant and evaluated for bacterial growth in spleen and liver. Both B6 and SCID mice succumbed to infection with Y. pestis KIM5, whereas both mouse strains survived infection by the YopM(-) mutant. These data showed that YopM counteracts innate defenses present in SCID mice. The YopM(-) strain grew more slowly than the parent Y. pestis during the first 4 days of infection in both mouse strains, indicating an early pathogenic role for YopM. In B6 mice, populations of cells of the immune system were not differentially affected by the two Y. pestis strains, with one major exception: the parent Y. pestis KIM5 but not the YopM(-) mutant caused a significant global decrease in NK cell numbers (blood, spleen, and liver), beginning early in infection. NK cells and macrophages isolated early (day 2) from livers and spleens of mice infected with either Y. pestis strain contained comparable levels of cytokine mRNA: interleukin (IL)-1 beta, IL-12, IL-15, IL-18, and tumor necrosis factor alpha in macrophages and gamma interferon in NK cells. However, by day 4 postinfection, cells from mice infected with the parent Y. pestis expressed lower levels of these messages, while those from mice infected with the mutant retained strong expression. Significantly, mRNA for the IL-15 receptor alpha chain was not expressed in NK cells from Y. pestis KIM5-infected mice as early as day 2 postinfection. These findings suggest that YopM interferes with innate immunity by causing depletion of NK cells, possibly by affecting the expression of IL-15 receptor alpha and IL-15.     

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.