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Abstract Objective Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. Data description We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.

ABSTRACT The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

The emergence of antibiotic-resistant bacteria is a pressing public health concern, highlighting the need for alternative approaches to control bacterial infections. Promising approaches include the development of therapeutic vaccines and the utilization of innate immune activation techniques, which may prove useful in conjunction with antibiotics, as well as other antibacterial modalities. However, innate activation should be fast and self- or actively- contained to prevent detrimental consequences. TLR ligand adjuvants are effective at rapidly activating, within minutes to hours, the innate immune system by inducing cytokine production and other signaling molecules that bolster the host’s immune response. Neutrophils serve as the first line of defense against invading pathogens by capturing and destroying them through various mechanisms, such as phagocytosis, intracellular degradation, and the formation of NETs. Nutritional immunity is another host defense mechanism that limits the availability of essential metals, such as iron, from invading bacterial pathogens. Thus, iron starvation has been proposed as a potential antibacterial strategy. In this review, we focus on approaches that have the potential to enhance rapid and precise antibacterial responses, bridging the gap between the onset of infection and the elimination of bacteria, hence limiting the infection by antibiotic-resistant bacteria.

Recent advances in the field of cell therapy have proposed new solutions for tissue repair and regeneration using various cell delivery approaches. Here we studied ex vivo a novel topical delivery system of encapsulated cells in hybrid polyethylene glycol-fibrinogen (PEG-Fb) hydrogel microspheres to respiratory tract models. We investigated basic parameters of cell encapsulation, delivery and release in conditions of inflamed and damaged lungs of bacterial-infected mice. The establishment of each step in the study was essential for the proof of concept. We demonstrated co-encapsulation of alveolar macrophages and epithelial cells that were highly viable and equally distributed inside the microspheres. We found that encapsulated macrophages exposed to bacterial endotoxin lipopolysaccharide preserved high viability and secreted moderate levels of TNFα, whereas non-encapsulated cells exhibited a burst TNFα secretion and reduced viability. LPS-exposed encapsulated macrophages exhibited elongated morphology and out-migration capability from microspheres. Microsphere degradation and cell release in inflamed lung environment was studied ex vivo by the incubation of encapsulated macrophages with lung extracts derived from intranasally infected mice with Yersinia pestis, demonstrating the potential in cell targeting and release in inflamed lungs. Finally, we demonstrated microsphere delivery to a multi-component airways-on-chip platform that mimic human nasal, bronchial and alveolar airways in serially connected compartments. This study demonstrates the feasibility in using hydrogel microspheres as an effective method for topical cell delivery to the lungs in the context of pulmonary damage and the need for tissue repair.

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.

Here, the authors generate a replication-competent VSV based vaccine expressing SARS-CoV-2 spike protein and show protection in the hamster model with one dose. Analysis of the antibody response in mice shows induction of neutralizing antibodies and suggests a desirable Th1-biased response to the vaccine.

The COVID-19 pandemic initiated a worldwide race toward the development of treatments and vaccines. Small animal models included the Syrian golden hamster and the K18-hACE2 mice infected with SARS-CoV-2 to display a disease state with some aspects of human COVID-19. A group activity of animals in their home cage continuously monitored by the HCMS100 (Home cage Monitoring System 100) was used as a sensitive marker of disease, successfully detecting morbidity symptoms of SARS-CoV-2 infection in hamsters and in K18-hACE2 mice. COVID-19 convalescent hamsters rechallenged with SARS-CoV-2 exhibited minor reduction in group activity compared to naive hamsters. To evaluate the rVSV-ΔG-spike vaccination efficacy against SARS-CoV-2, we used the HCMS100 to monitor the group activity of hamsters in their home cage. A single-dose rVSV-ΔG-spike vaccination of the immunized group showed a faster recovery than the nonimmunized infected hamsters, substantiating the efficacy of rVSV-ΔG-spike vaccine. HCMS100 offers nonintrusive, hands-free monitoring of a number of home cages of hamsters or mice modeling COVID-19.

The COVID-19 pandemic caused by the SARS-CoV-2 infection induced lung inflammation characterized by cytokine storm and fulminant immune response of both resident and migrated immune cells, accelerating alveolar damage. In this work we identified members of the matrix metalloprotease (MMPs) family associated with lung extra-cellular matrix (ECM) destruction using K18-hACE2-transgenic mice (K18-hACE2) infected intranasally with SARS-CoV-2. Five days post infection, the lungs exhibited overall alveolar damage of epithelial cells and massive leukocytes infiltration. A substantial pulmonary increase in MMP8, MMP9, and MMP14 in the lungs post SARS-CoV-2 infection was associated with degradation of ECM components including collagen, laminin, and proteoglycans. The process of tissue damage and ECM degradation during SARS-CoV-2 lung infection is suggested to be associated with activity of members of the MMPs family, which in turn may be used as a therapeutic intervention.

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.

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.

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.

Pneumonic plague is a lethal infectious disease caused by Yersinia pestis, a Tier-1 biothreat agent. Antibiotic treatment can save infected patients; however, therapy should begin within 24 h of symptom onset. As some Y. pestis strains showed an antibiotic resistance phenotype, an antibiotic susceptibility test (AST) must be performed. Performing the Clinical and Laboratory Standards Institute (CLSI)-recommended standard process, which includes bacterial isolation, enumeration and microdilution testing, lasts several days. Thus, rapid AST must be developed. As previously published, the Y. pestis-specific reporter phage ϕA1122::luxAB can serve for rapid identification and AST (ID-AST). Herein, we demonstrate the ability to use ϕA1122::luxAB to determine minimal inhibitory concentration (MIC) values and antibiotic susceptibility categories for various Y. pestis therapeutic antibiotics. We confirmed the assay by testing several nonvirulent Y. pestis isolates with reduced susceptibility to doxycycline or ciprofloxacin. Moreover, the assay can be performed directly on positive human blood cultures. Furthermore, as Y. pestis may naturally or deliberately be spread in the environment, we demonstrate the compatibility of this direct method for this scenario. This direct phage-based ID-AST shortens the time needed for standard AST to less than a day, enabling rapid and correct treatment, which may also prevent the spread of the disease.

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.

Great efforts are being made to develop new rapid antibiotic susceptibility tests to meet the demand for clinical relevance versus disease progression. This is important especially in diseases caused by bacteria such as Yersinia pestis, the causative agent of plague, which grows rapidly in vivo but relatively slow in vitro. This compromises the ability to use standard growth-based susceptibility tests to obtain rapid and proper antibiotic treatment guidance. Using our previously described platform of quantifying antibiotic-specific transcriptional changes, we developed a molecular test based on changes in expression levels of doxycycline response-dependent marker genes that we identified by transcriptomic analysis. This enabled us to determine the minimal inhibitory concentration of doxycycline within 7 h compared to the 24 h required by the standard CLSI test. This assay was validated with various Y. pestis strains. Moreover, we demonstrated the applicability of the molecular test, combined with a new rapid bacterial isolation step from blood cultures, and show its relevance as a rapid test in clinical settings.

Rapid determination of bacterial antibiotic susceptibility is important for proper treatment of infections. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently published guidelines for rapid antimicrobial susceptibility testing (RAST) performed directly from positive blood culture vials. These guidelines, however, were only published for a limited number of common pathogenic bacteria. In this study, we evaluated the applicability of these guidelines to three Tier 1 bioterror agents (Bacillus anthracis, Yersinia pestis and Francisella tularensis) that require prompt antibiotic treatment to mitigate morbidity and mortality. We used spiked-in human blood incubated in a BACTEC™ FX40 system to determine the proper conditions for RAST using disc-diffusion and Etest assays. We found that reliable disc-diffusion inhibition diameters and Etest MIC values could be obtained in remarkably short times. Compared to the EUCAST-recommended disc-diffusion assays that will require adjusted clinical breakpoint tables, Etest-based RAST was advantageous, as the obtained MIC values were similar to the standard MIC values, enabling the use of established category breakpoint tables. Our results demonstrate the promising applicability of the EUCAST RAST for B. anthracis-, Y. pestis- or F. tularensis-positive blood cultures, which can lead to shorter diagnostics and prompt antibiotic treatment of these dangerous pathogens.

Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response—LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as “oligo-clonal” reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.

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.

The global increase in multidrug-resistant (MDR) pathogenic bacteria has led to growing interest in bacteriophage (“phage”) therapy. Therapeutic phages are usually selected based on their ability to infect and lyse target bacteria, using in vitro assays. In these assays, phage infection is determined using target bacteria grown in standard commercial rich media, while evaluation of the actual therapeutic activity requires the presence of human blood. In the present work, we characterized the ability of two different Yersinia pestis lytic phages (ϕA1122 and PST) to infect and kill a luminescent Y. pestis EV76 strain suspended in Brain Heart Infusion (BHI)-rich medium or in human whole blood, simulating the host environment. We found that the ability of the phages to infect and lyse blood-suspended Y. pestis was not correlated with their ability to infect and lyse BHI-suspended bacteria. While the two different phages exhibited efficient infective capacity in a BHI-suspended culture, only the PST phage showed efficient lysis ability against blood-suspended bacteria. Therefore, we recommend that for personalized phage therapy, selection of phage(s) for efficient treatment of patients suffering from MDR bacterial infections should include prior testing of the candidate phage(s) for their lysis ability in the presence of human blood.

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.

Prompt and effective elicitation of protective immunity is highly relevant for cases of rapidly deteriorating fatal diseases, such as plague, which is caused by Yersinia pestis. Here, we assessed the potential of a live vaccine to induce rapid protection against this infection. We demonstrated that the Y. pestis EV76 live vaccine protected mice against an immediate lethal challenge, limiting the multiplication of the virulent pathogen and its dissemination into circulation. Ex vivo analysis of Y. pestis growth in serum derived from EV76-immunized mice revealed that an antibacterial activity was produced rapidly. This activity was mediated by the host heme- and iron-binding proteins hemopexin and transferrin, and it occurred in strong correlation with the kinetics of hemopexin induction in vivo. We suggest a new concept in which a live vaccine is capable of rapidly inducing iron nutritional immunity, thus limiting the propagation of pathogens. This concept could be exploited to design novel therapeutic interventions.

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.

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.

Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53ΔJ+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53ΔJ+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-γ dependent. Moreover, inoculation of mice with Y. pestis Kim53ΔJ+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment.

Yersinia pestis is the causative agent of plague. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the pcm gene was disrupted. In the present study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis using a set of isogenic surE, pcm, nlpD and rpoS mutants of the fully virulent Kimberley53 strain. We show that in Y. pestis, nlpD expression is controlled from elements residing within the upstream genes surE and pcm. The NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD(50) of at least 10(7) cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The filamented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrated that subcutaneous administration of the nlpD mutant could protect animals against bubonic and primary pneumonic plague. Taken together, these results demonstrate that Y. pestis NlpD is a novel virulence factor essential for the development of bubonic and pneumonic plague. Further, the nlpD mutant is superior to the EV76 prototype live vaccine strain in immunogenicity and in conferring effective protective immunity. Thus it could serve as a basis for a very potent live vaccine against bubonic and pneumonic plague.

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50) of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.