Postexposure Administration of a Yersinia pestis Live Vaccine for Potentiation of Second-Line Antibiotic Treatment Against Pneumonic Plague.

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing contagious disease. In the plague mouse model, a single immunization with the EV76 live attenuated Y. pestis strain rapidly induced the expression of hemopexin and haptoglobin in the lung and serum, both of which are important in iron sequestration. Immunization against a concomitant lethal Y. pestis respiratory challenge was correlated with temporary inhibition of disease progression. Combining EV76-immunization and second-line antibiotic treatment, which are individually insufficient, led to a synergistic protective effect that represents a proof of concept for efficient combinational therapy in cases of infection with antibiotic-resistant strains.

Circumventing Y. pestis Virulence by Early Recruitment of Neutrophils to the Lungs during Pneumonic Plague.

Pneumonic plague is a fatal disease caused by Yersinia pestis that is associated with a delayed immune response in the lungs. Because neutrophils are the first immune cells recruited to sites of infection, we investigated the mechanisms responsible for their delayed homing to the lung. During the first 24 hr after pulmonary infection with a fully virulent Y. pestis strain, no significant changes were observed in the lungs in the levels of neutrophils infiltrate, expression of adhesion molecules, or the expression of the major neutrophil chemoattractants keratinocyte cell-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2) and granulocyte colony stimulating factor (G-CSF). In contrast, early induction of chemokines, rapid neutrophil infiltration and a reduced bacterial burden were observed in the lungs of mice infected with an avirulent Y. pestis strain. In vitro infection of lung-derived cell-lines with a YopJ mutant revealed the involvement of YopJ in the inhibition of chemoattractants expression. However, the recruitment of neutrophils to the lungs of mice infected with the mutant was still delayed and associated with rapid bacterial propagation and mortality. Interestingly, whereas KC, MIP-2 and G-CSF mRNA levels in the lungs were up-regulated early after infection with the mutant, their protein levels remained constant, suggesting that Y. pestis may employ additional mechanisms to suppress early chemoattractants induction in the lung. It therefore seems that prevention of the early influx of neutrophils to the lungs is of major importance for Y. pestis virulence. Indeed, pulmonary instillation of KC and MIP-2 to G-CSF-treated mice infected with Y. pestis led to rapid homing of neutrophils to the lung followed by a reduction in bacterial counts at 24 hr post-infection and improved survival rates. These observations shed new light on the virulence mechanisms of Y. pestis during pneumonic plague, and have implications for the development of novel therapies against this pathogen.

Adjunctive Corticosteroid Treatment Against Yersinia pestis Improves Bacterial Clearance, Immunopathology, and Survival in the Mouse Model of Bubonic Plague.

BACKGROUND: Plague is initiated by Yersinia pestis, a highly virulent bacterial pathogen. In late stages of the infection, bacteria proliferate extensively in the internal organs despite the massive infiltration of neutrophils. The ineffective inflammatory response associated with tissue damage may contribute to the low efficacy of antiplague therapies during late stages of the infection. In the present study, we address the possibility of improving therapeutic efficacy by combining corticosteroid administration with antibody therapy in the mouse model of bubonic plague. METHODS: Mice were subcutaneously infected with a fully virulent Y. pestis strain and treated at progressive stages of the disease with anti-Y. pestis antibodies alone or in combination with the corticosteroid methylprednisolone. RESULTS: The addition of methylprednisolone to antibody therapy correlated with improved mouse survival, a significant decrease in the amount of neutrophils and matrix metalloproteinase 9 in the tissues, and the mitigation of tissue damage. Interestingly, the combined treatment led to a decrease in the bacterial loads in infected organs. CONCLUSIONS: Corticosteroids induce an unexpectedly effective antibacterial response apart from their antiinflammatory properties, thereby improving treatment efficacy.

Transendothelial migration of effector T cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans.

Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1ß stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.

Rapid Induction of Protective Immunity against Pneumonic Plague by Yersinia pestis Polymeric F1 and LcrV Antigens.

In a recent study, we demonstrated that vaccination with the polymeric F1 capsule antigen of the plague pathogen Yersinia pestis led to the rapid induction of a protective humoral immune response via the pivotal activation of innate-like B1b cells. Conversely, the monomeric version of F1 failed to promptly protect vaccinated animals in this model of the bubonic plague. In this study, we examined the ability of F1 to confer the rapid onset of protective immunity in the more challenging mouse model of the pneumonic plague. Vaccination with one dose of F1 adsorbed on aluminum hydroxide elicited effective protection against subsequent lethal intranasal exposure to a fully virulent Y. pestis strain within a week. Interestingly, the addition of the LcrV antigen shortened the time required for achieving such rapid protective immunity to 4-5 days after vaccination. As found previously, the polymeric structure of F1 was essential in affording the accelerated protective response observed by covaccination with LcrV. Finally, in a longevity study, a single vaccination with polymeric F1 induced a higher and more uniform humoral response than a similar vaccination with monomeric F1. However, in this setting, the dominant contribution of LcrV to long-lasting immunity against a lethal pulmonary challenge was reiterated.

Phage Therapy Potentiates Second-Line Antibiotic Treatment against Pneumonic Plague.

Plague pandemics and outbreaks have killed millions of people during the history of humankind. The disease, caused by the bacteria Yersinia pestis, is currently treated effectively with antibiotics. However, in the case of multidrug-resistant (MDR) bacteria, alternative treatments are required. Bacteriophage (phage) therapy has shown efficient antibacterial activity in various experimental animal models and in human patients infected with different MDR pathogens. Here, we evaluated the efficiency of фA1122 and PST phage therapy, alone or in combination with second-line antibiotics, using a well-established mouse model of pneumonic plague. Phage treatment significantly delayed mortality and limited bacterial proliferation in the lungs. However, the treatment did not prevent bacteremia, suggesting that phage efficiency may decrease in the circulation. Indeed, in vitro phage proliferation assays indicated that blood exerts inhibitory effects on lytic activity, which may be the major cause of treatment inefficiency. Combining phage therapy and second-line ceftriaxone treatment, which are individually insufficient, provided protection that led to the survival of all infected animals-a synergistic protective effect that represents a proof of concept for efficient combinatorial therapy in an emergency event of a plague outbreak involving MDR Y. pestis strains.

An Improvement in Diagnostic Blood Culture Conditions Allows for the Rapid Detection and Isolation of the Slow Growing Pathogen Yersinia pestis.

Plague, caused by the human pathogen Yersinia pestis, is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca+2)- and iron (Fe+2)-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca+2 and Fe+2. The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an increased sensitivity and without any detectable compromise of the accuracy of the antibiotic susceptibility testing (E-test), critical for subsequent successful therapeutic interventions.

Screening of an FDA-Approved Library for Novel Drugs against Y. pestis.

Yersinia pestis is a Gram-negative pathogen that causes plague, a devastating disease that kills millions worldwide. Although plague is efficiently treatable by recommended antibiotics, the time of antibiotic therapy initiation is critical, as high mortality rates have been observed if treatment is delayed for longer than 24 h after symptom onset. To overcome the emergence of antibiotic resistant strains, we attempted a systematic screening of Food and Drug Administration (FDA)-approved drugs to identify alternative compounds which may possess antibacterial activity against Y. pestis. Here, we describe a drug-repurposing approach, which led to the identification of two antibiotic-like activities of the anticancer drugs bleomycin sulfate and streptozocin that have the potential for designing novel antiplague therapy approaches. The inhibitory characteristics of these two drugs were further addressed as well as their efficiency in affecting the growth of Y. pestis strains resistant to doxycycline and ciprofloxacin, antibiotics recommended for plague treatment.

Novel RNA Extraction Method for Dual RNA-seq Analysis of Pathogen and Host in the Early Stages of Yersinia pestis Pulmonary Infection.

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing lethal infection. The various phases of pneumonic plague are yet to be fully understood. A well-established way to address the pathology of infectious diseases in general, and pneumonic plague in particular, is to conduct concomitant transcriptomic analysis of the bacteria and the host. The analysis of dual RNA by RNA sequencing technology is challenging, due the difficulties of extracting bacterial RNA, which is overwhelmingly outnumbered by the host RNA, especially at the critical early time points post-infection (prior to 48 h). Here, we describe a novel technique that employed the infusion of an RNA preserving reagent (RNAlater) into the lungs of the animals, through the trachea, under deep anesthesia. This method enabled the isolation of stable dual mRNA from the lungs of mice infected with Y. pestis, as early as 24 h post-infection. The RNA was used for transcriptomic analysis, which provided a comprehensive gene expression profile of both the host and the pathogen.

Fc-Independent Protection from SARS-CoV-2 Infection by Recombinant Human Monoclonal Antibodies.

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.

Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice.

Neutralizing antibodies represent a valuable therapeutic approach to countermeasure the current COVID-19 pandemic. Emergence of SARS-CoV-2 variants emphasizes the notion that antibody treatments need to rely on highly neutralizing monoclonal antibodies (mAbs), targeting several distinct epitopes for circumventing therapy escape mutants. Previously, we reported efficient human therapeutic mAbs recognizing epitopes on the spike receptor-binding domain (RBD) of SARS-CoV-2. Here we report the isolation, characterization, and recombinant production of 12 neutralizing human mAbs, targeting three distinct epitopes on the spike N-terminal domain of the virus. Neutralization mechanism of these antibodies involves receptors other than the canonical hACE2 on target cells, relying both on amino acid and N-glycan epitope recognition, suggesting alternative viral cellular portals. Two selected mAbs demonstrated full protection of K18-hACE2 transgenic mice when administered at low doses and late post-exposure, demonstrating the high potential of the mAbs for therapy of SARS-CoV-2 infection.

Post-exposure protection of SARS-CoV-2 lethal infected K18-hACE2 transgenic mice by neutralizing human monoclonal antibody.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterize and further evaluate the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection, while administration of the MD65 antibody as late as 3 days after exposure rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data demonstrate the therapeutic value of human monoclonal antibodies as a life-saving treatment for severe COVID-19 infection.

Development of Improved Devices for Handling and Restraining Experimental Laboratory Mice.

Introduction: Most animal handling procedures are associated with injuries among veterinary staff and laboratory animal researchers. However, much of the currently available animal handling equipment is inadequate, limiting access to the treated animal or making workflow cumbersome. Moreover, restraining animals to perform procedures, such as blood collection or injection, elicits stress in both the animal and the worker. Herein, we present 4 home-built restraint and blood collection devices in extensive use in our institute. Methods: Animal laboratory workers and experienced veterinarians regularly using the devices (n = 14) were asked to complete a survey ranking the contribution of the devices to worker safety and procedural efficiency. Results: The overwhelming majority of responders (≥75%) associated all 4 devices with substantial improvements in worker safety and procedural efficiency. There were no reports of impaired workflow or safety when using the devices. Discussion: Infection and exposure control may be implemented on various levels, including use of safer procedures, such as injection and blood collection devices. The presented intuitive handling and restraint devices allow the animal worker/researcher to perform various procedures safely and efficiently while eliciting less animal and worker stress. The devices can be easily adjusted to accommodate animal size and disease status. Conclusion: The current devices will serve as prototypes for design of devices for larger laboratory animals.

Influenza virus infection augments susceptibility to respiratory Yersinia pestis exposure and impacts the efficacy of antiplague antibiotic treatments.

Various respiratory viral infections in general and seasonal influenza in particular may increase the susceptibility to bacterial infections. Plague caused by Yersinia pestis endangers large populations during outbreaks or bioterrorism attacks. Recommended antibiotic countermeasures include well-established protocols based on animal studies and corroborated by effective treatment of human cases. Until now, prior exposure to viral respiratory infections was not taken into consideration when selecting the appropriate treatment for plague. Here, we show that as late as 25 days after exposure to influenza virus, convalescent mice still exhibited an increased susceptibility to sublethal doses of Y. pestis, presented with aberrant cytokine expression, and impaired neutrophil infiltration in the lungs. Increased levels of M2 alveolar macrophages and type II epithelial cells, as well as induction in metalloproteases expression and collagen and laminin degradation, suggested that the previous viral infection was under resolution, correlating with enhanced susceptibility to plague. Surprisingly, postexposure prophylaxis treatment with the recommended drugs revealed that ciprofloxacin was superior to doxycycline in mice recovering from influenza infection. These results suggest that after an influenza infection, the consequences, such as impaired immunity and lung tissue remodeling and damage, should be considered when treating subsequent Y. pestis exposure.

A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes.

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

Disruption of the NlpD lipoprotein of the plague pathogen Yersinia pestis affects iron acquisition and the activity of the twin-arginine translocation system.

We have previously shown that the cell morphogenesis NlpD lipoprotein is essential for virulence of the plague bacteria, Yersinia pestis. To elucidate the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis of the wild-type Y. pestis strain and an nlpD mutant under conditions mimicking early stages of infection. The analysis suggested that NlpD is involved in three phenomena: (i) Envelope stability/integrity evidenced by compensatory up-regulation of the Cpx and Psp membrane stress-response systems in the mutant; (ii) iron acquisition, supported by modulation of iron metabolism genes and by limited growth in iron-deprived medium; (iii) activity of the twin-arginine (Tat) system, which translocates folded proteins across the cytoplasmic membrane. Virulence studies of Y. pestis strains mutated in individual Tat components clearly indicated that the Tat system is central in Y. pestis pathogenicity and substantiated the assumption that NlpD essentiality in iron utilization involves the activity of the Tat system. This study reveals a new role for NlpD in Tat system activity and iron assimilation suggesting a modality by which this lipoprotein is involved in Y. pestis pathogenesis.

Early Diagnosis of Pathogen Infection by Cell-Based Activation Immunoassay.

Diagnostic identification of pathogens is usually accomplished by isolation of the pathogen or its substances, and should correlate with the time and site of infection. Alternatively, immunoassays such as enzyme-linked immunosorbent assay (ELISA) tests for quantification of serum antibodies are expedient and are usually employed for retrospective diagnostic of a particular infective agent. Here, the potential of cell-based immunoassays for early pathogen detection was evaluated by quantification of specific, antigen-activated, low-frequency IFNγ-secreting cells in mouse spleens following infection with various pathogens. Using enzyme-linked immunospot (ELISPOT) assays, specific responses were observed within 3-6 days following infection with F. tularensis, B. anthracis, Y. pestis, or Influenza virus. Blood samples collected from F. tularensis-infected mice revealed the presence of IFNγ-producing activated cells within one week post infection. When non-human primates were infected with B. anthracis, cellular response was observed in peripheral blood samples as early as five days post infection, 3-5 days earlier than serum antibodies. Finally, the expression pattern of genes in splenocytes of F. tularensis-infected mice was inspected by a transcriptomic approach, enabling the identification of potential host targets for the future development of genetic-based cellular immunoassays. Altogether, the data demonstrate the potential of cell-based immunoassays for early pathogen detection.

Targeting of the Yersinia pestis F1 capsular antigen by innate-like B1b cells mediates a rapid protective response against bubonic plague.

The generation of adaptive immunity by vaccination is usually a prolonged process that requires multiple dosing over several months. Hence, vaccines are administered for disease prevention a relatively long time prior to possible infection as opposed to post-exposure prophylaxis, which typically requires rapid intervention such as antibiotic therapy. The emergence of pathogens resistant to common antibiotic treatments has prompted the search for alternative therapeutic strategies. We previously demonstrated that vaccination of mice with the F1 capsular antigen of Yersinia pestis elicits specific and effective yet, unexpectedly, rapid anti-plague immunity. Here, we show by applying genetic and immunological approaches that the F1 antigen is targeted by peritoneal innate-like B1b cells that generate a prompt T-independent (TI) anti-F1 humoral response. The rapid F1-mediated defense response was diminished in Xid (Btkm) mice in which B1 cell numbers and activity are limited. Binding of fluorophore-labeled F1 to peritoneal B1b cells was detected as soon as 6 h post vaccination, emphasizing the high speed of this process. By assessing the ability to achieve rapid immunity with monomerized F1, we show that the natural polymeric structure of F1 is essential for (i) rapid association with peritoneal B1b cells, (ii) early induction of anti-F1 titers and (iii) rapid TI immunity in the mouse model of bubonic plague. These observations shed new light on the potential of novel as well as well-known protective antigens in generating rapid immunity and could be implemented in the rational design of future vaccines.

Inhalational Gentamicin Treatment Is Effective Against Pneumonic Plague in a Mouse Model.

Pneumonic plague is an infectious disease characterized by rapid and fulminant development of acute pneumonia and septicemia that results in death within days of exposure. The causative agent of pneumonic plague, Yersinia pestis (Y. pestis), is a Tier-1 bio-threat agent. Parenteral antibiotic treatment is effective when given within a narrow therapeutic window after symptom onset. However, the non-specific "flu-like" symptoms often lead to delayed diagnosis and therapy. In this study, we evaluated inhalational gentamicin therapy in an infected mouse model as a means to improve antibiotic treatment efficacy. Inhalation is an attractive route for treating lung infections. The advantages include directly dosing the main infection site, the relative accessibility for administration and the lack of extensive enzymatic drug degradation machinery. In this study, we show that inhalational gentamicin treatment administered 24 h post-infection, prior to the appearance of symptoms, protected against lethal intranasal challenge with the fully virulent Y. pestis Kimberley53 strain (Kim53). Similarly, a high survival rate was demonstrated in mice treated by inhalation with another aminoglycoside, tobramycin, for which an FDA-approved inhaled formulation is clinically available for cystic fibrosis patients. Inhalational treatment with gentamicin 48 h post-infection (to symptomatic mice) was also successful against a Y. pestis challenge dose of 10 i.n.LD50. Whole-body imaging using IVIS technology demonstrated that adding inhalational gentamicin to parenteral therapy accelerated the clearance of Y. pestis from the lungs of infected animals. This may reduce disease severity and the risk of secondary infections. In conclusion, our data suggest that inhalational therapy with aerosolized gentamicin may be an effective prophylactic treatment against pneumonic plague. We also demonstrate the benefit of combining this treatment with a conventional parenteral treatment against this rapidly progressing infectious disease. We suggest the inhalational administration route as a clinically relevant treatment modality against pneumonic plague and other respiratory bacterial pathogens.

Disparity between Yersinia pestis and Yersinia enterocolitica O:8 in YopJ/YopP-dependent functions.

YopP in Y. enterocolitica and YopJ in Y. pseudotuberculosis, have been shown to exert a variety of adverse effects on cell signaling leading to suppression of cytokine expression and induction of programmed cell death. A comparative in vitro study with Y. pestis and Y. enterocolitica O:8 virulent strains shows some critical disparity in YopJ/YopP-related effects on immune cells. Involvement of yopJ in virulence was evaluated in mouse model of bubonic plague.