Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD₅₀) equivalent to 6,800 LD₅₀s of WT CO92. The mutant-infected animals developed balanced T_H1- and T_H2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD₅₀s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant''s sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.     

Deletion of Braun Lipoprotein and Plasminogen-Activating Protease-Encoding Genes Attenuates Yersinia pestis in Mouse Models of Bubonic and Pneumonic Plague

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4⁺ and CD8⁺ T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.     

High-Throughput,Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD₅₀) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD₅₀ when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD₅₀. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD₅₀ in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD₅₀ in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD₅₀. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD₅₀ were 90% protected upon subsequent challenge with 12 LD₅₀ of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s).     

Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1⁻ strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1⁻ mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1⁻ CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.     

Analysis of Autoinducer-2 Quorum Sensing in Yersinia pestis

The autoinducer-2 (AI-2) quorum-sensing system has been linked to diverse phenotypes and regulatory changes in pathogenic bacteria. In the present study, we performed a molecular and biochemical characterization of the AI-2 system in Yersinia pestis, the causative agent of plague. In strain CO92, the AI-2 signal is produced in a luxS-dependent manner, reaching maximal levels of 2.5 μM in the late logarithmic growth phase, and both wild-type and pigmentation (pgm) mutant strains made equivalent levels of AI-2. Strain CO92 possesses a chromosomal lsr locus encoding factors involved in the binding and import of AI-2, and confirming this assignment, an lsr deletion mutant increased extracellular pools of AI-2. To assess the functional role of AI-2 sensing in Y. pestis, microarray studies were conducted by comparing Δpgm strain R88 to a Δpgm ΔluxS mutant or a quorum-sensing-null Δpgm ΔypeIR ΔyspIR ΔluxS mutant at 37°C. Our data suggest that AI-2 quorum sensing is associated with metabolic activities and oxidative stress genes that may help Y. pestis survive at the host temperature. This was confirmed by observing that the luxS mutant was more sensitive to killing by hydrogen peroxide, suggesting a potential requirement for AI-2 in evasion of oxidative damage. We also show that a large number of membrane protein genes are controlled by LuxS, suggesting a role for quorum sensing in membrane modeling. Altogether, this study provides the first global analysis of AI-2 signaling in Y. pestis and identifies potential roles for the system in controlling genes important to disease.     

Characterization of an F1 deletion mutant of Yersinia pestis CO92, pathogenic role of F1 antigen in bubonic and pneumonic plague, and evaluation of sensitivity and specificity of F1 antigen capture-based dipsticks

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37 °C, although the mutant's growth dropped slightly during the late phase at 37 °C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 10(5) to 5 × 10(5) CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 10(8) CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.     

A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague

The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD₅₀. Intranasal infection of mice with 15 LD₅₀ of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24–72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of antimicrobial countermeasures in real time.     

yadBC of Yersinia pestis, a new virulence determinant for bubonic plague

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.     

Intranasal administration of an inactivated Yersinia pestis vaccine with interleukin-12 generates protective immunity against pneumonic plague

Inhalation of Yersinia pestis causes pneumonic plague, which rapidly progresses to death. A previously licensed killed whole-cell vaccine is presently unavailable due to its reactogenicity and inconclusive evidence of efficacy. The present study now shows that vaccination intranasally (i.n.) with inactivated Y. pestis CO92 (iYp) adjuvanted with interleukin-12 (IL-12) followed by an i.n. challenge with a lethal dose of Y. pestis CO92 prevented bacterial colonization and protected 100% of mice from pneumonic plague. Survival of the vaccinated mice correlated with levels of systemic and lung antibodies, reduced pulmonary pathology and proinflammatory cytokines, and the presence of lung lymphoid cell aggregates. Protection against pneumonic plague was partially dependent upon Fc receptors and could be transferred to naïve mice with immune mouse serum. On the other hand, protection was not dependent upon complement, and following vaccination, depletion of CD4 and/or CD8 T cells before challenge did not affect survival. In summary, the results demonstrate the safety, immunogenicity, and protective efficacy of i.n. administered iYp plus IL-12 in a mouse model of pneumonic plague.     

Evaluation of a Yersinia pestis mutant impaired in a thermoregulated type VI-like secretion system in flea,macrophage and murine models

Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499–YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 °C in comparison to 37 °C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499–YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 °C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499–YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 °C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.     

LcrV Delivered via Type III Secretion System of Live Attenuated Yersinia pseudotuberculosis Enhances Immunogenicity against Pneumonic Plague

Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC P_BAD crp) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δasd-206 ΔmsbB868::P_msbB msbB_(EC) ΔP_crp21::TT araC P_BAD crp] for use with a balanced-lethal Asd-positive (Asd⁺) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopE_Nt138-LcrV) was synthesized in χ10057 harboring an Asd⁺ plasmid (pYA5199, yopE_Nt138-lcrV) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ∼240 median lethal doses (LD₅₀) (2.4 × 10⁴ CFU) of Y. pestis KIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.     

Cynomolgus macaque model for pneumonic plague

A recombinant vaccine (rF1V) is currently being developed for protection against pneumonic plague. An essential component in evaluating efficacy of the rF1V vaccine is the development of a well-understood animal model that shows similarity to human disease. The objective of this study was to determine the inhaled median lethal dose (LD₅₀), evaluate the pathophysiology of disease and identify appropriate study endpoints in a cynomolgus macaque (CM) model of pneumonic plague. Eighteen CMs were challenged by head-only aerosol exposure with seven dosages of Yersinia pestis CO92. An LD₅₀ of 24 colony forming units was estimated using Probit analysis. Disease pathology was evaluated by blood culture, clinical pathology, histopathology and telemetry. CMs that died became febrile following challenge and died 34–92 h after onset of fever. Bacteremia, increased respiration and heart rate, decreased blood pressure and loss of diurnal rhythm were also observed in conjunction with onset of fever. Histopathological examinations revealed significant findings in the lungs (intra-alveolar neutrophils and fibrinous pleuritis) consistent with pneumonic plague. These data indicate that the disease pathology observed in CMs following aerosol exposure to Y. pestis CO92 is similar to that of pneumonic plague in humans. Thus, the CM is an appropriate model to evaluate efficacy of a recombinant F1V vaccine candidate.     

Deletion of Braun lipoprotein gene (lpp) attenuates Yersinia pestis KIM/D27 strain: Role of Lpp in modulating host immune response,NF-κB activation and cell death

The pathogenic species of yersiniae potently blocks immune responses in host cells by using the type III secretion apparatus and its effector proteins. In this study, we characterized potential mechanisms associated with the Braun lipoprotein (Lpp) that contributed to a further attenuation of a pigmentation locus-minus Yersinia pestis KIM/D27 mutant strain and its ability to generate immune responses in mice. The lpp gene encodes one of the major outer membrane lipoproteins that is involved in inflammatory responses and septic shock. We found that sera and splenocytes from Δlpp mutant-immunized mice, when transferred to naïve animals, provided protection to the latter against challenge with a lethal dose of the Y. pestis parental strain. Further, the Δlpp mutant promoted ex vivo a significantly higher interleukin (IL)-2 and interferon-gamma production from T cells of immunized mice, when compared to those from animals infected with the sub-lethal dose of the parental Y. pestis KIM/D27 strain. Likewise, murine primary macrophages infected with the mutant, when compared to those infected with the parental strain in vitro, produced significantly higher IL-12 levels. Importantly, increased nuclear factor-kappa B activation and decreased apoptosis were noted in splenocytes and primary macrophages of mice challenged with the Δlpp mutant, when compared to those in animals infected with the parental Y. pestis KIM/D27 strain. Finally, significantly higher levels of antibodies specific for the parental Y. pestis antigens were developed in mice first immunized with the Δlpp mutant and then challenged with the parental strain, compared to the antibody levels in animals that were immunized and then infected with the parental KIM/D27 strain. To our knowledge, this is the first report of a mechanistic basis for attenuation and immunological responses associated with deletion of the lpp gene from the Y. pestis KIM/D27 strain.     

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.     

Gr1+ Cells Control Growth of YopM-Negative Yersinia pestis during Systemic Plague

YopM, a protein toxin of Yersinia pestis, is necessary for virulence in a mouse model of systemic plague. We previously reported YopM-dependent natural killer (NK) cell depletion from blood and spleen samples of infected mice. However, in this study we found that infection with Y. pestis KIM5 (YopM⁺) caused depletion of NK cells in the spleen, but not in the liver, and antibody-mediated ablation of NK cells had no effect on bacterial growth. There was no YopM-associated effect on the percentage of dendritic cells (DCs) or polymorphonuclear leukocytes (PMNs) in the early stage of infection; however, there was a YopM-associated effect on PMN integrity and on the influx of monocytes into the spleen. Ablation of Gr1⁺ cells caused loss of the growth defect of YopM⁻ Y. pestis in both the liver and spleen. In contrast, ablation of macrophages/DCs inhibited growth of both parent and mutant bacteria, accompanied by significantly fewer lesion sites in the liver. These results point toward PMNs and inflammatory monocytes as major cell types that control growth of YopM⁻ Y. pestis. Infection with fully virulent Y. pestis CO92 and a YopM⁻ derivative by intradermal and intranasal routes showed that the absence of YopM significantly increased the 50% lethal dose only in the intradermal model, suggesting a role for YopM in bubonic plague, in which acute inflammation occurs soon after infection.Yersinia pestis, the infective agent of bubonic and pneumonic plague, has caused widespread loss of human life during recurrent pandemics. Y. pestis still infects rodent populations in large geographic zones where Y. pestis is endemic, and there are cases of human plague reported annually (15, 19, 56). Y. pestis and the closely related food-borne pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica share a 70-kb plasmid carrying genes that encode a major set of proteins involved in pathogenic properties that compromise the host immune system (60). These include a type 3 secretion system (T3SS) that at mammalian body temperature delivers a set of six Yersinia outer protein (Yop) effector proteins into host cells once the bacteria contact host target cells. Enzymatic and cell biological mechanisms of five of the Yops, YopH, YopE, YopT, YpkA/YopO, and YopJ, have been elucidated. YopJ interferes with signal transduction through acetyltransferase activity but is not required for virulence in either a mouse model of systemic plague (57) or mouse and rat models of bubonic plague (28, 65). In tissue culture infection models, YopH, YopE, YopT, and YopO have been shown to antagonize focal complex formation and activity of Rho family GTPases and synergistically inhibit phagocytosis by mammalian cells. YopH and YopE have been shown to be crucial for lethality in a mouse model of systemic plague (intravenous [i.v.] infection), and a ΔyopH strain is attenuated for both bubonic and pneumonic plague (9). In addition, Y. pestis virulence proteins, such as the surface fibrils F1 and PsaA, have antiphagocytic effects and also have been found to contribute to virulence in systemic plague (7, 31). Accordingly, Y. pestis is believed to exist predominantly in an extracellular location in vivo, although initially the bacteria might invade resting tissue macrophages (Mφs) and dendritic cells (DCs), based on assays of mouse spleens in the systemic phase of bubonic plague (33). The intracellular versus extracellular locations of Y. pestis during the peripheral phases of plague on skin or in the lung have not yet been studied.It is believed that tissue Mφs, DCs, and polymorphonuclear leukocytes (PMNs) are early target cells for Yop delivery in vivo, because these cells are present before or soon after infection begins and function to initiate the innate defenses that are undermined by Yops. Consistent with this hypothesis, Y. pestis has been found in association with alveolar Mφs early during lung infection of mice (6) and likewise in association with Mφs, DCs, and PMNs in the spleens of mice infected i.v., and YopM can be injected into these cells (34). However, it is becoming clear that spleens and lungs present distinctly different inflammatory environments when infected by Y. pestis, with PMNs migrating rapidly into spleens infected by the i.v. route but not appearing in lungs until 36 h after intranasal (i.n.) infection (27, 58). Accordingly, some virulence properties required for lethality of systemic plague are not required in pneumonic plague. Examples are the capsular fibril F1 and the antiphagocytic adhesin PsaA (7, 11, 12). There is no information yet on the target cells or relative importance of Yops other than YopH in pneumonic or bubonic plague.The sixth effector Yop, YopM, is essential for virulence in the mouse model of systemic plague: in C57BL/6 mice, a YopM⁻ strain of Y. pestis KIM5 is reduced in lethality by at least 4 orders of magnitude (29). However, the function of YopM has not been defined. YopM is a 46.2-kDa acidic protein made up almost entirely of 15 repeats of a 19-residue leucine-rich repeat motif (30). The YopM monomer is horseshoe shaped and has the potential to form tetramers in which the monomers stack together to form a hollow cylinder; however, the form that YopM assumes within the mammalian cell is not known (16). After delivery to the host cell cytoplasm, YopM localizes to the nucleus in a process that is facilitated by vesicular trafficking (53). YopM was reported to form a complex with the serine/threonine kinases PRK2 (protein kinase C-related kinase 2) and RSK1 (90-kDa ribosomal S6 kinase) in HEK293 cells infected with Y. pseudotuberculosis (36), leading to activation of both kinases. However, the biological significance of this complex is not known. There is no visible effect of delivery of YopM into cultured cells, and microarray analysis of Mφ-like cell lines infected with Y. enterocolitica having or lacking YopM also has not yielded any clue to YopM''s mechanism of action (21, 50).Because these and other in vitro approaches to defining the pathogenic mechanism of YopM have not been fruitful, we have begun to characterize YopM''s effects in vivo. Previously we found that YopM was still required for lethality in i.v. infected SCID mice, showing that YopM''s virulence mechanism does not require B or T cells and indicating that early in systemic plague, YopM''s main function is to counteract a component of innate immunity (25). A striking YopM-specific effect during systemic plague in wild-type C57BL/6 mice was the depletion of natural killer (NK) cells from the spleen and a reduction of NK cell numbers in blood, suggesting that YopM might cause a loss of the NK cell compartment during systemic plague. Correlated with this effect, there was a YopM-associated loss of mRNA for gamma interferon (IFN-γ) by NK cells in infected spleens and diminished expression of mRNAs in splenic Mφs for cytokines that are required for viability and activation of NK cells (interleukin 15 [IL-15], IL-18, and IL-12). These findings supported the hypothesis that YopM may function to inhibit IFN-mediated activation of Mφs through the depletion of NK cells (25).In this study, we tested the hypothesis that NK cells are critical for controlling Y. pestis pathogenesis early in plague and that NK cells are the primary target of YopM. We characterized infection dynamics and leukocyte populations in both the liver and spleen during systemic plague in mice ablated for Mφs/DCs or for Gr1⁺ cells. The data point to PMNs and inflammatory monocytes as critical cells affected by YopM and to Mφs/DCs as an important early reservoir for bacterial growth. Consistent with a role in undermining acute inflammation, YopM was found to be important for the lethality of bubonic plague, but not pneumonic plague.     

Oral vaccination against bubonic plague using a live avirulent Yersinia pseudotuberculosis strain

We evaluated the possibility of using Yersinia pseudotuberculosis as a live vaccine against plague because it shares high genetic identity with Y. pestis while being much less virulent, genetically much more stable, and deliverable orally. A total of 41 Y. pseudotuberculosis strains were screened by PCR for the absence of the high pathogenicity island, the superantigens YPM, and the type IV pilus and the presence of the pYV virulence plasmid. One strain (IP32680) fulfilled these criteria. This strain was avirulent in mice upon intragastric or subcutaneous inoculation and persisted for 2 months in the mouse intestine without clinical signs of disease. IP32680 reached the mesenteric lymph nodes, spleen, and liver without causing major histological lesions and was cleared after 13 days. The antibodies produced in vaccinated animals recognized both Y. pseudotuberculosis and Y. pestis antigens efficiently. After a subcutaneous challenge with Y. pestis CO92, bacteria were found in low amounts in the organs and rarely in the blood of vaccinated animals. One oral IP32680 inoculation protected 75% of the mice, and two inoculations induced much higher antibody titers and protected 88% of the mice. Our results thus validate the concept that an attenuated Y. pseudotuberculosis strain can be an efficient, inexpensive, safe, and easy-to-produce live vaccine for oral immunization against bubonic plague.     

Immunological responses against Salmonella enterica serovar Typhimurium Braun lipoprotein and lipid A mutant strains in Swiss-Webster mice: potential use as live-attenuated vaccines

We generated and characterized Salmonella enterica serovar Typhimurium mutants that were deleted for the genes encoding Braun lipoprotein (lpp) alone or in conjunction with the msbB gene, which codes for an enzyme required for the acylation of the lipid A moiety of lipopolysaccharide. Two copies of the lpp gene, designated as lppA and lppB, exist on the chromosome of S. Typhimurium. These mutants were highly attenuated in a mouse infection model and induced minimal histopathological changes in mouse organs compared to those seen in infection with wild-type (WT) S. Typhimurium. The lppB/msbB and the lppAB/msbB mutants were maximally attenuated, and hence further examined in this study for their ability to induce humoral and cellular immune responses. Importantly, infection of out-bred Swiss-Webster mice with the mutant S. Typhimurium generated superior T helper cell type 2 (Th2) responses compared to WT S. Typhimurium, as determined by measuring IgG subclasses and cytokines. WT S. Typhimurium induced higher levels of IgG2a in sera of infected mice, while the lppB/msbB and lppAB/msbB mutants mounted higher levels of IgG1 as determined by an enzyme-linked immunosorbent assay. Mice immunized with lppB/msbB and lppAB/msbB mutants rapidly cleared WT S. Typhimurium upon subsequent rechallenge, and naïve mice passively immunized with sera from animals infected with S. Typhimurium mutants were protected against subsequent challenge with WT S. Typhimurium. Splenic T cells produced higher levels of interferon-gamma following ex vivo exposure to WT S. Typhimurium, while splenic T cells infected with the above-mentioned two mutants evoked higher levels of interleukin-6. Further, mice infected with lppB/msbB and lppAB/msbB mutants showed much higher levels of splenic T cell activation as measured by CD44⁺ expression on CD4⁺ T cells by flow cytometry and by incorporation of ³H-thymidine compared to mice that were infected with WT S. Typhimurium. We expect the lppB/msbB and lppAB/msbB mutants to be excellent live-attenuated vaccine candidates, because they induced minimal inflammatory responses and evoked stronger and specific antibody and cellular immune responses.     

Yersinia pestis CO92 delta yopH is a potent live, attenuated plague vaccine

An in-frame deletion of the yopH gene in Yersinia pestis CO92 attenuates virulence in both bubonic and pneumonic plague models. When it is used as a live, attenuated vaccine, CO92 delta yopH provides a high degree of protection from parental and respiratory challenge with Y. pestis CO92.     

Three Yersinia pestis Adhesins Facilitate Yop Delivery to Eukaryotic Cells and Contribute to Plague Virulence

To establish a successful infection, Yersinia pestis requires the delivery of cytotoxic Yops to host cells. Yops inhibit phagocytosis, block cytokine responses, and induce apoptosis of macrophages. The Y. pestis adhesin Ail facilitates Yop translocation and is required for full virulence in mice. To determine the contributions of other adhesins to Yop delivery, we deleted five known adhesins of Y. pestis. In addition to Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) could mediate Yop translocation to host cells. The contribution of each adhesin to binding and Yop delivery was dependent upon the growth conditions. When cells were pregrown at 28°C and pH 7, the order of importance for adhesins in cell binding and cytotoxicity was Ail > Pla > Psa. Y. pestis grown at 37°C and pH 7 had equal contributions from Ail and Pla but an undetectable role for Psa. At 37°C and pH 6, both Ail and Psa contributed to binding and Yop delivery, while Pla contributed minimally. Pla-mediated Yop translocation was independent of protease activity. Of the three single mutants, the Δail mutant was the most defective in mouse virulence. The expression level of ail was also the highest of the three adhesins in infected mouse tissues. Compared to an ail mutant, additional deletion of psaA (encoding Psa) led to a 130,000-fold increase in the 50% lethal dose for mice relative to that of the KIM5 parental strain. Our results indicate that in addition to Ail, Pla and Psa can serve as environmentally specific adhesins to facilitate Yop secretion, a critical virulence function of Y. pestis.The causative agent of plague is the Gram-negative bacterium Yersinia pestis (54, 77). Plague is one of the most deadly infectious diseases and has decimated civilizations repeatedly throughout history (10, 54). Plague still remains a public health concern, and due to the increasing number of cases worldwide, plague is classified as a reemerging infectious disease (68). Identification of therapies or effective vaccines would provide protection against plague as a potential bioterrorism threat.There are three clinical forms of plague in humans: bubonic, pneumonic, and septicemic (54). Bubonic plague is the most common form and usually occurs following a fleabite. In bubonic plague, the organism initially spreads to the regional lymph nodes, where it replicates primarily extracellularly, and then eventually enters the bloodstream. If untreated, bubonic plague is fatal in 40 to 60% of cases (54). Pneumonic plague is the least common form, but it progresses rapidly and is the most fatal form of the disease. Pneumonic plague may occur as a complication of bubonic or septicemic plague (secondary pneumonic plague) or by inhalation of infectious droplets spread by the cough or sneeze of a person with pneumonic plague (primary pneumonic plague). If treatment is not initiated within the first 24 h after symptoms appear, it is likely to be fatal within 48 h (18, 49). Septicemic plague can occur if Y. pestis gains direct access to the bloodstream via open wounds or fleabites (primary septicemic plague) (64) or as a result of spread from the lymphatic system to the bloodstream during advanced stages of bubonic plague (54). Y. pestis can also spread to the bloodstream and blood-filtering organs during late stages of pneumonic plague (39).In order for Y. pestis to cause disease, it must harbor the 70-kb pCD1 virulence plasmid, which encodes the Ysc type III secretion system (T3SS) and the Yop effector proteins (13, 69, 70). Yops inhibit phagocytosis by disrupting the actin cytoskeleton, diminish proinflammatory cytokine responses, and induce apoptosis of macrophages (13, 30, 50, 51, 54, 60, 62). In order for Yops to be delivered efficiently to host cells, adhesins must provide a docking function to facilitate T3S (8, 22, 59).Two adhesins shown to be important for Yop delivery in the related Yersinia species Y. enterocolitica and Y. pseudotuberculosis are YadA and invasin (Inv) (8, 47, 59). Y. pestis lacks both of these adhesins due to inactivation of inv by an IS1541 element and of yadA by a frameshift mutation (17, 52, 61, 65). Thus, we focused our studies on the Y. pestis adhesins described below.We recently identified Ail, an adhesin of Y. pestis that binds host cell fibronectin (73) and plays an important role in delivery of Yops to both phagocytic and nonphagocytic cells in vitro (22). In addition to having a defect in Yop delivery in vitro, a Y. pestis KIM5 Δail mutant has a >3,000-fold increased 50% lethal dose (LD₅₀) for mice in a septicemic plague model (22). However, the virulence defect of a KIM5 Δail mutant is not as severe as that of a KIM5 strain lacking the virulence plasmid (>10⁶-fold increase in LD₅₀) (22, 69). Thus, we set out to determine if there were other Y. pestis adhesins capable of facilitating Yop delivery.Four other adhesins of Y. pestis have been described. Plasminogen activator (Pla) is an adhesin and a protease. It can mediate binding to extracellular matrix proteins (32, 37, 43) and direct invasion of tissue culture cells (14, 36). Pla may also interact with the host cell receptor DEC-205 on macrophages and dendritic cells (80). Pla is known to be required for dissemination of a bubonic plague infection from the site of inoculation, presumably due to cleavage of fibrin clots by plasmin after Pla-mediated activation of plasminogen (27, 66). Its protease activity is also required for the development of fulminant pneumonic plague (39).pH 6 antigen (Psa) is a multifunctional surface structure that is induced at 37°C under low-pH conditions (4, 57, 75) and is controlled by the regulatory protein RovA (11). Psa can bind to β1-linked galactosyl residues in glycosphingolipids (53) and to phosphatidylcholine (26) on the surfaces of host cells. Due to its ability to bind surface receptors, Psa functions as an efficient adhesin (5, 24, 26, 41, 76). Psa-mediated adhesion also prevents phagocytosis (29). Finally, Psa can bind the Fc portion of human IgG (79) and interact with plasma low-density lipoproteins (LDLs) (44).The two remaining Y. pestis adhesins are the putative autotransporter YapC (23) and the pilus associated with the chaperone usher locus y0561 to y0563 (24). Neither locus is well expressed under laboratory conditions (23, 24; S. Felek and E. S. Krukonis, unpublished data), but when expressed in Escherichia coli, they can confer an increase in E. coli adhesion to several cultured cell lines (23, 24).Here we demonstrate that Ail, Pla, and Psa can mediate Yop delivery in vitro when expressed in a KIM5 mutant lacking all five adhesins (KIM5 Δ5), whereas overexpression of YapC or y0561 to y0563 does not allow for Yop delivery from the KIM5 Δ5 strain. Furthermore, we observed cumulative defects in virulence when each of three genes, ail, pla, and psaA, was deleted and tested in a mouse model. Thus, we have shown that all three adhesins can contribute to Yop delivery in vitro and to virulence in vivo.