A Recombinant Attenuated Yersinia pseudotuberculosis Vaccine Delivering a Y. pestis YopENt138-LcrV Fusion Elicits Broad Protection against Plague and Yersiniosis in Mice.

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.

White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection.

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.

Flagellin-Mediated Protection against Intestinal Yersinia pseudotuberculosis Infection Does Not Require Interleukin-22.

Signaling through Toll-like receptors (TLRs), the main receptors in innate immunity, is essential for the defense of mucosal surfaces. It was previously shown that systemic TLR5 stimulation by bacterial flagellin induces an immediate, transient interleukin-22 (IL-22)-dependent antimicrobial response to bacterial or viral infections of the mucosa. This process was dependent on the activation of type 3 innate lymphoid cells (ILCs). The objective of the present study was to analyze the effects of flagellin treatment in a murine model of oral infection with Yersinia pseudotuberculosis (an invasive, Gram-negative, enteropathogenic bacterium that targets the small intestine). We found that systemic administration of flagellin significantly increased the survival rate after intestinal infection (but not systemic infection) by Y. pseudotuberculosis This protection was associated with a low bacterial count in the gut and the spleen. In contrast, no protection was afforded by administration of the TLR4 agonist lipopolysaccharide, suggesting the presence of a flagellin-specific effect. Lastly, we found that TLR5- and MyD88-mediated signaling was required for the protective effects of flagellin, whereas neither lymphoid cells nor IL-22 was involved.

CD8(+) T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity.

CD8(+) T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8(+) T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8(+) T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8(+) T cells are generated and provide limited protection against challenge with virulent OVA(+)Y. pseudotuberculosis. Perforin expression by OVA-specific CD8(+) T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8(+) T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8(+) T cell status. These studies indicate that CD8(+) T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.

Yersiniosis caused by Yersinia pseudotuberculosis in captive toucans (Ramphastidae) and a Japanese squirrel (Sciurus lis) in zoological gardens in Japan.

Two captive Keel-billed toucans and a Chestnut-mandibled toucan in another zoological garden died suddenly without any pre-existing symptoms, and three months later, a Japanese squirrel died of diarrhea. All these animals showed necrotic enteritis and multifocal necrosis in the liver and spleen with Gram negative bacilli. The bacilli showed strong positive immunolabeling for Yersinia pseudotuberculosis O4 in the Keel-billed toucans, Y. pseudotuberculosis O2 in the Chestnut-mandibled toucan and Y. pseudotuberculosis O1 in the Japanese squirrel, while Y. pseudotuberculosis 4b, 2b and 1b were respectively isolated from the lesions. To our knowledge, this might be the first reported case of fatal yersiniosis in a Japanese squirrel in the world as well as in toucans in Japan.

Influence of PhoP and intra-species variations on virulence of Yersinia pseudotuberculosis during the natural oral infection route.

The two-component regulatory system PhoP/PhoQ has been shown to (i) control expression of virulence-associated traits, (ii) confer survival and growth within macrophages and (iii) play a role in Yersinia infections. However, the influence of PhoP on virulence varied greatly between different murine models of infection and its role in natural oral infections with frequently used representative isolates of Y. pseudotuberculosis was unknown. To address this issue, we constructed an isogenic set of phoP+ and phoP- variants of strain IP32953 and YPIII and analyzed the impact of PhoP using in vitro functionality experiments and a murine oral infection model, whereby we tested for bacterial dissemination and influence on the host immune response. Our results revealed that PhoP has a low impact on virulence, lymphatic and systemic organ colonization, and on immune response modulation by IP32953 and YPIII, indicating that PhoP is not absolutely essential for oral infections but may be involved in fine-tuning the outcome. Our work further revealed certain strain-specific differences in virulence properties, which do not strongly rely on the function of PhoP, but affect tissue colonization, dissemination and/or persistence of the bacteria. Highlighted intra-species variations may provide a potential means to rapidly adjust to environmental changes inside and outside of the host.

Fatal atypical O:3 Yersinia pseudotuberculosis infection in cynomolgus macaques.

Fatal Yersinia pseudotuberculosis infection in cynomolgus macaques was diagnosed based upon pathology, microbiology and PCR for this study. Pathological findings included acute, erosive to ulcerative, necrohemorrhagic enterocolitis. Genotyping by PCR showed an O:3 pattern (gmd-fcl(+), ddhC-prt(+), manB(+), ddhA-B(+)), but an additional gene, wbyK, was detected. This is the second report to identify wbyK+ O:3 genotype as the cause of fatal yersiniosis. The first case was reported in 2008, and involved farm deer in the U.S. As the frequency of wbyK+ O:3 genotype is found more often in different carriers, O:3 genotype is proposed to be divided into two subtypes: O:3a without wbyK and O:3b with wbyK. Virulence gene analysis showed the presence of inv, ypmC, irp1, ybtP-ybtQ, yadA, yopB, yopH, lcrF, and suggested that this O:3b isolate could be a highly pathogenic strain to cynomolgus macaques.

Structure and function of cytidine monophosphate kinase from Yersinia pseudotuberculosis, essential for virulence but not for survival.

The need for new antibiotics has become pressing in light of the emergence of antibiotic-resistant strains of human pathogens. Yersinia pestis, the causative agent of plague, is a public health threat and also an agent of concern in biodefence. It is a recently emerged clonal derivative of the enteric pathogen Yersinia pseudotuberculosis. Previously, we developed a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. One such target was cytidine monophosphate (CMP) kinase, which is an essential gene in some organisms. Previously, we had thought CMP kinase was essential for Y. pseudotuberculosis, but by modification of the mutagenesis approach, we report here the production and characterization of a Δcmk mutant. The isogenic mutant had a growth defect relative to the parental strain, and was highly attenuated in mice. We have also elucidated the structure of the CMP kinase to 2.32 Å, and identified three key residues in the active site that are essential for activity of the enzyme. These findings will have implications for the development of novel CMP kinase inhibitors for therapeutic use.

A protective epitope in type III effector YopE is a major CD8 T cell antigen during primary infection with Yersinia pseudotuberculosis.

Virulence in human-pathogenic Yersinia species is associated with a plasmid-encoded type III secretion system that translocates a set of Yop effector proteins into host cells. One effector, YopE, functions as a Rho GTPase-activating protein (GAP). In addition to acting as a virulence factor, YopE can function as a protective antigen. C57BL/6 mice infected with attenuated Yersinia pestis generate a dominant H2-Kb-restricted CD8 T cell response to an epitope in the N-terminal domain of YopE (YopE69-77), and intranasal vaccination with the YopE69-77 peptide and the mucosal adjuvant cholera toxin (CT) elicits CD8 T cells that are protective against lethal pulmonary challenge with Y. pestis. Because YopE69-77 is conserved in many Yersinia strains, we sought to determine if YopE is a protective antigen for Yersinia pseudotuberculosis and if primary infection with this enteric pathogen elicits a CD8 T cell response to this epitope. Intranasal immunization with the YopE69-77 peptide and CT elicited a CD8 T cell response that was protective against lethal intragastric Y. pseudotuberculosis challenge. The YopE69-77 epitope was a major antigen (∼30% of splenic CD8 T cells were specific for this peptide at the peak of the response) during primary infection with Y. pseudotuberculosis, as shown by flow cytometry tetramer staining. Results of infections with Y. pseudotuberculosis expressing catalytically inactive YopE demonstrated that GAP activity is dispensable for a CD8 T cell response to YopE69-77. Determining the features of YopE that are important for this response will lead to a better understanding of how protective CD8 T cell immunity is generated against Yersinia and other pathogens with type III secretion systems.

Small molecule inhibitors of LcrF, a Yersinia pseudotuberculosis transcription factor, attenuate virulence and limit infection in a murine pneumonia model.

LcrF (VirF), a transcription factor in the multiple adaptational response (MAR) family, regulates expression of the Yersinia type III secretion system (T3SS). Yersinia pseudotuberculosis lcrF-null mutants showed attenuated virulence in tissue culture and animal models of infection. Targeting of LcrF offers a novel, antivirulence strategy for preventing Yersinia infection. A small molecule library was screened for inhibition of LcrF-DNA binding in an in vitro assay. All of the compounds lacked intrinsic antibacterial activity and did not demonstrate toxicity against mammalian cells. A subset of these compounds inhibited T3SS-dependent cytotoxicity of Y. pseudotuberculosis toward macrophages in vitro. In a murine model of Y. pseudotuberculosis pneumonia, two compounds significantly reduced the bacterial burden in the lungs and afforded a dramatic survival advantage. The MAR family of transcription factors is well conserved, with members playing central roles in pathogenesis across bacterial genera; thus, the inhibitors could have broad applicability.

Fatal yersiniosis in farmed deer caused by Yersinia pseudotuberculosis serotype O:3 encoding a mannosyltransferase-like protein WbyK.

Sudden death of 9 deer occurred in a large enclosed deer farm with approximately 400 heads of cervids. Fatal yersiniosis was diagnosed in 2 deer that were submitted for laboratory diagnosis. Histopathologically, the disease was characterized by multifocal pulmonary hemorrhage and mild interstitial pneumonia, marked diffuse cholangiohepatitis, minimal myocarditis with mild myocardial degeneration, and mild multifocal suppurative cystic colitis. Yersinia pseudotuberculosis was isolated from the lungs and colon of the affected animals. The isolates were PCR-positive for genes virF, inv, yopB, and yopH, which are essential for invasion and colonization of host intestine and lung. The isolates reacted with polyclonal antibodies against serotype O:3 antigen. The O-genotyping patterns of the isolates were identical with each other, but different from those of the 21 O-genotypes (or serotypes) reported previously. In addition to the O-antigen genes possessed by classical serotype O:3, a gene (wbyK) encoding a mannosyltransferase-like protein was detected in these isolates. The wbyK gene of the isolates showed 94% of DNA sequence homology with the wbyK gene harbored by Y. pseudotuberculosis O:1b. On the basis of pathology, bacteriology, and serology, the authors concluded that the acute deaths of these deer were caused by Y. pseudotuberculosis infection. Molecular characterization of the isolate revealed a genetic heterogeneity in the O-antigen gene cluster of Y. pseudotuberculosis serotype O:3.

Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia pseudotuberculosis harbouring the pPla plasmid.

Yersinia pestis is a species that emerged recently from Yersinia pseudotuberculosis and gained an exceptional pathogenicity potential. Among the major genetic differences between the plague bacillus and its ancestor is the acquisition of the pPla plasmid, which has been associated with the increased virulence of Y. pestis. In a previous study, introduction of pPla into Y. pseudotuberculosis did not lead to any modification of the virulence of the host bacterium. However, it was subsequently demonstrated that the presence of smooth lipopolysaccharide (LPS) inhibits the activity of Pla. In this study, pPla was introduced into a Y. pseudotuberculosis strain expressing smooth LPS, and into a variant in which a mutation that abrogates the formation of O-antigen (O-Ag) repeats (as in natural isolates of Y. pestis) was generated. It was found that in both strains, Pla was synthesized, exported to the bacterial membrane and processed as in Y. pestis. However, the ability of Pla to activate plasminogen was weak and observed only at 37 degrees C in the smooth strain, while this activity was similar to that of Y. pestis and expressed at both 28 and 37 degrees C in the O-Ag mutant strain. Similarly, Pla-mediated inactivation of the antiprotease alpha2-antiplasmin was not detected in the smooth Y. pseudotuberculosis strain grown at 28 degrees C, but was expressed at both temperatures in the O-Ag mutant strain. Despite the more efficient activity of Pla, the Y. pseudotuberculosis O-Ag mutant strain exhibited a lower pathogenicity upon subcutaneous infection of mice. The results thus indicate that, although abrogation of O side chain synthesis in a Y. pseudotuberculosis strain harbouring pPla potentiates the two proteolytic activities of Pla, this is not sufficient to confer to Y. pseudotuberculosis a higher pathogenicity potential. These results also suggest that acquisition of pPla may not have been sufficient to confer an immediate higher pathogenic potential to the ancestor Y. pestis strain.

The Eyam plague revisited: did the village isolation change transmission from fleas to pulmonary?

Back in the 17th century the Derbyshire village of Eyam fell victim to the Black Death, which is thought to have arrived from London in some old clothes brought by a travelling tailor. The village population was 350 at the commencement of plague, of which only 83 survived. Led by the church leaders, the village community realized that the whole surrounding region was at risk from the epidemic, and therefore decided to seal themselves off from the other surrounding villages. In the first 275 days of the outbreak, transmission was predominantly from infected fleas to susceptible humans. From then onward, mortality sharply increased, which indicates a changing in transmission pattern. We hypothesize that the confinement facilitated the spread of the infection by increasing the contact rate through direct transmission among humans. This would be more consistent with pulmonary plague, a deadlier form of the disease. In order to test the above hypothesis we designed a mathematical model for plague dynamics, incorporating both the indirect (fleas-rats-humans) and direct (human-to-human) transmissions of the infection. Our results show remarkable agreement between data and the model, lending support to our hypotheses. The Eyam plague episode is celebrated as a remarkable act of collective self-sacrifice. However, to the best of our knowledge, there were no evidence before that the confinement actually increased the burden payed by the commoners. In the light of our results, it can be said that the hypothesis that confinement facilitated the spread of the infection by increasing the contact rate through direct transmission is plausible.

LcrV synthesis is altered by DNA adenine methylase overproduction in Yersinia pseudotuberculosis and is required to confer immunity in vaccinated hosts.

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (DamOP Yersinia) are attenuated, confer robust protective immune responses, and synthesize or secrete several Yersinia outer proteins (Yops) under conditions that are nonpermissive for synthesis and secretion in wild-type strains. To understand the molecular basis of immunity elicited by DamOP Yersinia, we investigated the effects of Dam overproduction on the synthesis and localization of a principal Yersinia immunogen, LcrV, a low-calcium-responsive virulence factor involved in Yop synthesis, localization, and suppression of host inflammatory activities. Dam overproduction relaxed the stringent temperature and calcium regulation of LcrV synthesis. Moreover, the LcrV-dependent synthesis and localization of the actin cytotoxin, YopE, were shown to be relaxed in DamOP cells, suggesting that the synthesis and localization of Yops can occur via both LcrV-dependent and -independent mechanisms. Last, the immunity conferred by DamOP Yersinia was strictly dependent on the presence of LcrV, which may result from its role (i) as an immunogen, (ii) as an immunomodulator of host anti-inflammatory activities, or (iii) in the altered synthesis and localization of Yops that could contribute to immunogen repertoire expansion.

[1,3;1,6-beta-D-glucan translam: results of studying and prospects for application]. / Itogi izucheniia i perspektivy primeneniia 1,3;1,6-beta-D-gliukana--translama.

The results of translam chemical structure and biological activity investigation are presented. Translam is a new original semisynthetic polysaccharide of marine origin. The preparation demonstrated potent treatment effect in experimental radiation disease. It had preventing effect at experimental bacterial infections, stimulated hematopoiesis, had effect on humoral and cell immunity and on factors of nonspecific organism resistance.

[The immunomodulating properties of translam]. / Immunomoduliruiushchie svoistva translama.

Effect of translam--beta-1,3;1,6-D-glucan on some of naturally occurring host protective mechanisms was investigated. It was shown that translam has potent biological activity: it has preventive effect on experimental bacterial infections, it stimulates absorbtion and digestion activity of mononuclear phagocytes, it effects on humoral and cell immunity. Translam had mitogenic activity.

Pseudotuberculosis in marmosets, tamarins, and Goeldi's monkeys (Callithrichidae/Callimiconidae) housed at a European zoo.

Yersinia pseudotuberculosis is an insidious bacterial infectious agent distributed worldwide and endemic to European countries. It has caused several animal deaths and may threaten the effectiveness of breeding projects for endangered species. In this retrospective study, we examine the prevalence of pseudotuberculosis in Jersey Zoo (Channel Islands, U.K.) over a period of 16 yr to obtain information that can be applied to prevent the infection. The efforts made to control the disease through vaccination are also explored. Our results show that pseudotuberculosis has been endemic to Jersey Zoo since 1979 and is responsible for significant animal loss in the Callithrichidae/Callimiconidae group. Mortality due to Y. pseudotuberculosis was seasonal; a high percentage of deaths occurred during wet and cold seasons. No significant difference was found in mortality rates of vaccinated versus nonvaccinated animals. Although the efficacy of vaccination has not been confirmed, we believe that an improved vaccination program could be an important tool in controlling outbreaks of infection in marmosets and tamarins.

Characteristics and pathogenicity of non-melibiose-fermenting strains of Yersinia pseudotuberculosis O3.

The biological properties of non-melibiose-fermenting (NMF) strains of Yersinia pseudotuberculosis O3 were investigated. These strains were clearly distinguished from representative melibiose-fermenting (MF) strains of Y. pseudotuberculosis O3 by their pathogenicity in mice, sensitivity to some phages, production of catalase, restriction endonuclease analysis of virulence plasmid DNA with BamHI, detection of specific yersinia outer-membrane proteins with SDS-PAGE, antigenicity of the outer-membrane proteins and neutrophil resistance to phagocytosis. The pathogenicity of NMF strains was clearly less than that of MF strains. In addition, the resistance of NMF strains to phagocytosis and catalase activity was evidently weaker than that of MF strains. These results suggested that the difference of pathogenicity was due to the ability of catalase production. Although the relationship between the above characteristics and melibiose-fermentation was not analysed, the pathogenicity of Y. pseudotuberculosis O3 strains can probably be predicted by testing melibiose-fermentation and catalase production.

[Yersinia pseudotuberculosis sensitivity to fluoroquinolones and the efficacy of pefloxacin in experimental pseudotuberculosis]. / Izuchenie chuvstvitel'nosti Yersinia pseudotuberculosis k ftorkhinolonam i éffektivnosti pefloksatsina pri éksperimental'nom psevdotuberkuleze.

The sensitivity of Y. pseudotuberculosis of different origin and plasmid profile to Russian preparations of the ftoroquinolones group (cyprofloxacin, pefloxacin, norfloxacin) was studied. Of the 3 preparations under study, pefloxacin was found to be most active with respect to Y. pseudotuberculosis. The absence of relationship between antibiotic resistance in 4 Y. pseudotuberculosis strains to cyprofloxacin, pefloxacin and norfloxacin and their additional conjugative R-plasmid with molecular weights of 32 and 38 mD was noted. Pefloxacin was shown to give a good sanitation and protective effect in experimental pseudotuberculosis.

Cytometric haematological analysis of experimental Yersinia pseudotuberculosis infection at temperatures of cultivation of 25 degrees C and 37 degrees C.

The dynamics of acute lethal infection with Yersinia pseudotuberculosis (serovar III) grown at 25 degrees C and 37 degrees C, respectively, were studied in guinea-pigs. Using an automated hematology analyzer, the quantity and quality of the host cellular response was determined at the primary site of infection (the peritoneal cavity) and in parallel in the blood. A much more intensive leucocytic response of monocytic type was observed in the animals infected with microorganism grown at 37 degrees C. This type of reaction was associated with a lower mortality rate and a relatively benign progression of the infection.