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.     

Nucleotide sequence of yst, the Yersinia enterocolitica gene encoding the heat-stable enterotoxin, and prevalence of the gene among pathogenic and nonpathogenic yersiniae.

The gene encoding the heat-stable enterotoxin (yst) was cloned from the chromosome of Yersinia enterocolitica W1024 (serotype O:9), and the nucleotide sequence was determined. The yst gene encodes a 71-amino-acid polypeptide. The C-terminal 30 amino acids of the predicted protein exactly correspond to the amino acid sequence of the toxin extracted from culture supernatants (T. Takao, N. Tominaga, and Y. Shimonishi, Biochem. Biophys. Res. Commun. 125:845-851, 1984). The N-terminal 18 amino acids have the properties of a signal sequence. The central 22 residues are removed during or after the secretion process. This organization in three domains (Pre, Pro, and mature Yst) resembles that of the enterotoxin STa of Escherichia coli. The degree of conservation between the E. coli and Y. enterocolitica toxins is much lower in the Pre and the Pro domains than in the mature proteins. The mature toxin of Y. enterocolitica is much larger than that of E. coli, but the active domain appears to be highly conserved. The yst gene of Y. enterocolitica introduced in E. coli K-12 directed the secretion of an active toxin. The cloned yst gene was used as an epidemiological probe among a collection of 174 strains representative of all Yersinia species except Yersinia pestis and numerous Y. enterocolitica subgroups. In Y. enterocolitica, there was a clear-cut difference between pathogenic and nonpathogenic strains: 89 of 89 pathogenic and none of 51 nonpathogenic strains contained yst-homologous DNA, suggesting that Yst is involved in pathogenesis. Among the other Yersinia species, only four strains of Yersinia kristensenii had DNA homologous to yst.     

Temperature-modulated immunogenicity to Yersinia pestis from Yersinia enterocolitica O3.

The ability of Yersinia enterocolitica O3, grown at 25 degrees C, to promote cross-immunity to Y. pestis was lost after repeated subcultures at 37 degrees C, which selected for bacterial populations having lower in vivo survival. Subculturing Y. enterocolitica O3 from 37 to 25 degrees C restored the cross-immunogenicity although the in vivo survival remained low.     

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.     

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.     

Secretion of Yop proteins by Yersiniae.

Upon incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of the genus Yersinia cease growing and produce large amounts of a series of plasmid-encoded proteins involved in pathogenicity. These proteins, called Yops (for Yersinia outer membrane proteins), are detected in both the outer membrane fraction and the culture supernatant. We present here the nucleotide sequence of genes yop20 and yop25 from Yersinia enterocolitica O:9. Protein Yop25 is very similar to YopE, the corresponding protein from Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica O:8 (A. Forsberg and H. Wolf-Watz, J. Bacteriol. 172:1547-1555, 1990). This is the first report of a yop20 sequence of yersiniae. We present evidences that Yops are not membrane proteins. Their detection in the membrane fraction results either from copurification of large aggregates of extracellular Yops with the membrane fraction or from the adsorption of released proteins to the cell surface. In contrast with Yops, protein P1 has characteristics of a true membrane protein. The release of Yops by Y. enterocolitica occurs by a novel secretion mechanism that does not involve the cleavage of a typical signal sequence or the recognition of a carboxy-terminal domain.     

Expression of the low calcium response in Yersinia pestis

Pathogenic yersiniae undergo an established low calcium response (LCR) at 37 degrees 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 Yersinia pestis but not in enteropathogenic yersiniae lacking this plasmid. Salient Yops of Y. pestis are shown here to be either maintained in the steady state or to exist as a stable degradation product (p24 of Yop E). Processing of plague plasminogen activator (p36 to p33), responsible for hydrolysis of Yops, required 2 h. Avirulence of mutants with inserted Mu dl1 (Apr lac) in yopE was verified and shown to occur independently of introduced fusion-dependent peptides. However, avirulence of such yopE mutants but not that of isolates lacking the Lcr plasmid was phenotypically suppressed in mice injected with iron. Appearance of 20,500 and 40,500 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 Yersinia pseudotuberculosis but not Yersinia enterocolitica.     

Role of the Yersinia outer membrane protein YadA in adhesion to rabbit intestinal tissue and rabbit intestinal brush border membrane vesicles

The Yersinia virulence plasmid confers on strains of Yersinia pseudotuberculosis and Y. enterocolitica an adhesive potential superior to the one encoded by the chromosome alone. We have evaluated the role of the plasmid-encoded outer membrane protein YadA (formerly called Yopl) in adhesion. Insertional inactivation of the yadA gene (formerly called yopA), which encodes YadA, led to a reduction in the capacity of plasmid-carrying strains of Y. pseudotuberculosis 0:III and Y. enterocolitica 0:9 to adhere to intestinal tissue, brush border membranes and polystyrene surfaces. The adhesive characteristics of the mutants were comparable to those of their plasmid-cured counterparts. When the yadA gene from Y. pseudotuberculosis serotype 0:III or Y. enterocolitica serotype 0:3 or 0:8 was cloned into an Escherichia coli strain, increased ability to adhere to intestinal tissue, brush border membrane vesicles and polystyrene was transferred concomitantly. The introduction of the yadA gene from Y. pestis, which is unable to express YadA due to a one base pair deletion, did not change the adhesive characteristics of E. coli. Expression of YadA in the outer membrane may, therefore, make an important contribution to intestinal adherence of the two enteropathogenic members of the Yersinia species, Y. pseudotuberculosis and Y. enterocolitica.     

The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis

The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoP mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37 degrees C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.     

Rapid diagnostic test that uses isocitrate lyase activity for identification of Yersinia pestis.

The presence of high levels of isocitrate lyase activity in Yersinia pestis grown on blood agar base medium, as compared with low levels of this enzyme in Yersinia pseudotuberculosis and Yersinia enterocolitica, suggested that the differences in the levels of this enzyme could be used for the presumptive identification of Y. pestis. A modified, semiquantitative assay for isocitrate lyase activity is described which requires no expensive instrumentation, utilizes readily available chemicals and substrates, and requires only 20 min for completion. This test yielded positive results with all 108 isolates of Y. pestis tested and negative results with all strains of Y. pseudotuberculosis (68 isolates) and Y. enterocolitica (202 isolates) tested. Less than 2% of the approximately 1,300 non-Yersinia isolates from the family Enterobacteriaceae and none of the 93 isolates from the family Pseudomonadaceae yielded positive results. We conclude that this test provides for rapid identification of Y. pestis and should be useful in the initial screening of isolates from rodent and flea populations and in the presumptive identification of this organism from suspected cases of human plague.     

Yersinia pestis V protein epitopes recognized by CD4 T cells

Pneumonic plague, an often-fatal disease for which no vaccine is presently available, results from pulmonary infection by the bacterium Yersinia pestis. The Y. pestis V protein is a promising vaccine candidate, as V protein immunizations confer to mice significant protection against aerosolized Y. pestis. CD4 T cells play central roles during vaccine-primed immune responses, but their functional contributions to Y. pestis vaccines have yet to be evaluated and optimized. Toward that end, we report here the identification of three distinct epitopes within the Y. pestis V protein that activate CD4 T cells in C57BL/6 mice. To our knowledge, these are the first identified CD4 T-cell epitopes in any Y. pestis protein. The epitopes are restricted by the I-A(b) class II major histocompatibility complex molecule and are fully conserved between Y. pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. Immunizing mice with a V protein-containing vaccine or with short peptides containing the identified epitopes primes antigen-specific production of interleukin 2 and gamma interferon by CD4 T cells upon their restimulation in vitro. Consistent with prior studies documenting protective roles for CD4 T cells during Y. enterocolitica infection, vaccinating mice with a 16-amino-acid peptide encoding one of the epitopes suffices to protect against an otherwise lethal Y. enterocolitica challenge. The identification of these epitopes will permit quantitative assessments of V-specific CD4 T cells, thereby enabling researchers to evaluate and optimize the contribution of these cells to vaccine-primed protection against pneumonic plague.     

Rapid and specific identification of Yersinia pestis by using a nested polymerase chain reaction procedure.

We developed a 4-h nested polymerase chain reaction assay that detected a region of the plasminogen activator gene of Yersinia pestis in 100% of 43 Y. pestis strains isolated from humans, rats, and fleas yet was unreactive with the closely related species Yersinia enterocolitica and Yersinia pseudotuberculosis.     

Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies.     

Evaluation of enzyme immunoassay for the detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains.

To determine the virulence plasmid-harboring strains of Yersinia enterocolitica, we prepared antiserum against plasmid-encoded proteins of Y. enterocolitica serotype O3 and carried out an enzyme immunoassay (EIA) against temperature-inducible released proteins. This serum reacted with proteins released from not only a Y. enterocolitica serotype O3 strain but also Y. enterocolitica serotype O5:27, O8, and O9 and Y. pseudotuberculosis serotype 1b, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b, 6, 7, and 8 strains, which all harbored plasmids. Plasmid-cured Y. enterocolitica and Y. pseudotuberculosis strains did not react in the EIA, nor did nonpathogenic Y. enterocolitica strains or Y. frederiksenii, Y. intermedia, and Y. kristensenii strains. These observations demonstrated that this EIA was useful for determining whether the isolated Yersinia strains were pathogenic or not.     

Pyrazinamidase activity in Yersinia enterocolitica and related organisms.

Pyrazinamidase activity was tested in 381 Yersinia strains from various ecological and geographical origins and belonging to the following species: Y. enterocolitica (five biogroups), Y. intermedia, Y. frederiksenii, Y. kristensenii, Y. aldovae, Y. pseudotuberculosis, and Y. pestis. The pyrazinamidase test was negative (Pyz-) in all bioserogroups of Y. enterocolitica, in which is usually harbored the virulence plasmid, and was involved in human or animal diseases. Y. pseudotuberculosis and Y. pestis were also Pyz-. The more ubiquitous bioserogroups of Y. enterocolitica, without naturally occurring virulence plasmid, and related species were all Pyz+. Pyrazinamidase activity allowed the separation of the pathogenic North American Y. enterocolitica isolates from other nonpathogenic strains within biogroup 1. Similarly, environmental biogroups 3A and 3B were clearly distinguished from pathogenic biogroup 3. However, the pyrazinamidase test was not linked to the presence of the virulence plasmid itself and should not replace the pathogenicity tests to assess the actual virulence of an individual strain. This test proved to be a valuable tool to distinguish potential pathogenic from nonpathogenic strains of Y. enterocolitica in epidemiological surveillance programs.     

Interaction of Yersinia pestis with macrophages: limitations in YopJ-dependent apoptosis

The enteropathogenic Yersinia strains are known to downregulate signaling pathways in macrophages by effectors of the type III secretion system, in which YopJ/YopP plays a crucial role. The adverse effects of Yersinia pestis, the causative agent of plague, were examined by infecting J774A.1 cells, RAW264.7 cells, and primary murine macrophages with the EV76 strain and with the fully virulent Kimberley53 strain. Y. pestis exerts YopJ-dependent suppression of tumor necrosis factor alpha secretion and phosphorylation of mitogen-activated protein kinases and thus resembles enteropathogenic Yersinia. However, Y. pestis is less able to activate caspases, to suppress NF-kappaB activation, and to induce apoptosis in macrophages than the high-virulence Y. enterocolitica WA O:8 strain. These differences appear to be related to lower efficiency of YopJ effector translocation by Y. pestis. The efficiencies of effector translocation and of apoptosis induction can be enhanced either by using a high bacterial load in a synchronized infection or by overexpressing exogenous YopJ in Y. pestis. Replacing YopJ with the homologous Y. enterocolitica effector YopP can further enhance these effects. Overexpression of YopP in a yopJ-deleted Y. pestis background leads to rapid and effective translocation into target cells, providing Y. pestis with the high cytotoxic potential of Y. enterocolitica WA O:8. We suggest that the relative inferiority of Y. pestis in triggering cell death in macrophages may be advantageous for its in vivo propagation in the early stages of infection.     

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.     

Expression of iron-regulated proteins in Yersinia species and their relation to virulence.

Under iron-starvation conditions, the different Yersinia species expressed various iron-regulated proteins. Among them, two high-molecular-weight outer membrane proteins were synthesized in high-virulence-phenotype Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovars O:8 and O:Tacoma but were present neither in low-virulence phenotype Y. enterocolitica serovars O:3 and O:9 nor in avirulent Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. enterocolitica serovar O:39. Thus, the degree of virulence correlates with the presence of the two high-molecular-weight proteins in Yersinia species.     

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.