Polymorphism in the Yersinia LcrV Antigen Enables Immune Escape From the Protection Conferred by an LcrV-Secreting Lactococcus Lactis in a Pseudotuberculosis Mouse Model.

Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria.IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

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.

Characterization of the protective immune response to Yersinia pseudotuberculosis infection in mice vaccinated with an LcrV-secreting strain of Lactococcus lactis.

BACKGROUND: Pseudotuberculosis is an infection caused by the bacterial enteropathogen Yersinia pseudotuberculosis and is considered to be a significant problem in veterinary medicine. We previously found that intranasal administration of a recombinant Lactococcus lactis strain that secretes the low-calcium response V (LcrV) antigen from Y. pseudotuberculosis (Ll-LcrV) confers protection against a lethal Y. pseudotuberculosis infection. Here, we aimed at characterizing the immunological basis of this LcrV-elicited protective response and at determining the duration of vaccine-induced immunity. METHODS: Splenocytes from BALB/c mice intranasally immunized with Ll-LcrV or Ll as control were immunostained then analyzed by flow cytometry. Protection against a lethal intravenous injection of Y. pseudotuberculosis was also determined (i) in immunized BALB/c mice depleted or not of CD4+, CD8+ or CD25+ cells and (ii) in naïve BALB/c mice receiving serum from immunized mice by counting the number of bacteria in liver and spleen. Lastly, survival rate of immunized BALB/c mice following a lethal intravenous injection of Y. pseudotuberculosis was followed up to 9-months. RESULTS: We found that T and B lymphocytes but not non-conventional lymphoid cells were affected by Ll-LcrV immunization. We also observed that depletion of CD4+ and CD25+ but not CD8+ cells in immunized mice eradicated protection against a lethal systemic Y. pseudotuberculosis infection, suggesting that activated CD4+ T lymphocytes are required for vaccine-induced protection. Adoptive transfer of LcrV-specific antibodies from Ll-LcrV-immunized animals significantly reduced the bacterial counts in the liver compared to non-vaccinated mice. Lastly, the protective immunity conferred by Ll-LcrV decreased slightly over time; nevertheless almost 60% of the mice survived a lethal bacterial challenge at 9months post-vaccination. CONCLUSION: Mucosal vaccination of mice with Ll-LcrV induced cell- and antibody-mediated protective immunity against Y. pseudotuberculosis infection in the mouse and the protection is long-lasting.

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis.

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥ 37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.

Superantigenic Yersinia pseudotuberculosis induces the expression of granzymes and perforin by CD4+ T cells.

Bacterial superantigens (SAgs) are immunostimulatory toxins that induce acute diseases mainly through the massive release of inflammatory cytokines. Yersinia pseudotuberculosis is the only Gram-negative bacterium known to produce a SAg (Y. pseudotuberculosis-derived mitogen [YPM]). This SAg binds major histocompatibility complex class II molecules on antigen-presenting cells and T cell receptors (TcR) bearing the variable region Vß3, Vß9, Vß13.1, or Vß13.2 (in humans) and Vß7 or Vß8 (in mice). We have previously shown that YPM exacerbates the virulence of Y. pseudotuberculosis in mice. With a view to understanding the mechanism of YPM's toxicity, we compared the immune response in BALB/c mice infected with a YPM-producing Y. pseudotuberculosis or the corresponding isogenic, SAg-deficient mutant. Five days after infection, we observed strong CD4(+) Vß7(+) T cell expansion and marked interleukin-4 (IL-4) production in mice inoculated with SAg-producing Y. pseudotuberculosis. These phenomena were correlated with the activation of ypm gene transcription in liver and spleen. A transcriptomic analysis revealed that the presence of YPM also increased expression of granzyme and perforin genes in the host's liver and spleen. This expression was attributed to a CD4(+) T cell subset, rather than to natural killer T (NKT) cells that display a TcR with a Vß region that is potentially recognized by YPM. Increased production of cytotoxic molecules was correlated with hepatotoxicity, as demonstrated by an increase in plasma alanine aminotransferase activity. Our results demonstrate that YPM activates a potentially hepatotoxic CD4(+) T cell population.

Proposed reclassification of Pasteurella lymphangitidis Sneath & Stevens 1990 as Yersinia pseudotuberculosis.

The 16S rRNA gene sequences of Pasteurella lymphangitidis, Yersinia pseudotuberculosis and Yersinia pestis were found to be identical and multilocus sequence analysis could not discriminate between the three species. The susceptibility to a Y. pseudotuberculosis phage and the presence of the Y. pseudotuberculosis-specific invasin gene in P. lymphangitidis indicate that the latter should be reclassified as Y. pseudotuberculosis.

CHROMagar Yersinia, a new chromogenic agar for screening of potentially pathogenic Yersinia enterocolitica isolates in stools.

CHROMagar Yersinia (CAY) is a new chromogenic medium for the presumptive detection of virulent Yersinia enterocolitica in stools. Based on a comparative analysis of 1,494 consecutive stools from hospitalized patients, CAY was found to be just as sensitive as the reference medium (cefsulodin-irgasan-novobiocin agar) but was significantly more specific and had a very low false-positive rate. CAY reduces the workload (and thus costs) for stool analysis and can therefore be recommended for routine laboratory use.

Efficacy of ciprofloxacin-gentamicin combination therapy in murine bubonic plague.

Potential benefits of combination antibiotic therapy for the treatment of plague have never been evaluated. We compared the efficacy of a ciprofloxacin (CIN) and gentamicin (GEN) combination therapy with that of each antibiotic administered alone (i) against Yersinia pestis in vitro and (ii) in a mouse model of bubonic plague in which animals were intravenously injected with antibiotics for five days, starting at two different times after infection (44 h and 56 h). In vitro, the CIN+GEN combination was synergistic at 0.5x the individual drugs' MICs and indifferent at 1x- or 2x MIC. In vivo, the survival rate for mice treated with CIN+GEN was similar to that observed with CIN alone and slightly higher than that observed for GEN alone 100, 100 and 85%, respectively when treatment was started 44 h post challenge. 100% of survivors were recorded in the CIN+GEN group vs 86 and 83% in the CIN and GEN groups, respectively when treatment was delayed to 56 h post-challenge. However, these differences were not statistically significant. Five days after the end of treatment, Y. pestis were observed in lymph nodes draining the inoculation site (but not in the spleen) in surviving mice in each of the three groups. The median lymph node log(10) CFU recovered from persistently infected lymph nodes was significantly higher with GEN than with CIN (5.8 vs. 3.2, p = 0.04) or CIN+GEN (5.8 vs. 2.8, p = 0.01). Taken as the whole, our data show that CIN+GEN combination is as effective as CIN alone but, regimens containing CIN are more effective to eradicate Y. pestis from the draining lymph node than the recommended GEN monotherapy. Moreover, draining lymph nodes may serve as a reservoir for the continued release of Y. pestis into the blood - even after five days of intravenous antibiotic treatment.

In silico comparison of Yersinia pestis and Yersinia pseudotuberculosis transcriptomes reveals a higher expression level of crucial virulence determinants in the plague bacillus.

Although Yersinia pestis and Yersinia pseudotuberculosis are genetically very similar (97% nucleotide sequence identity for most of the chromosomal genes), they exhibit very different patterns of infection. Y. pestis causes plague which is usually fatal in the absence of treatment, whereas Y. pseudotuberculosis generally triggers non-life-threatening intestinal symptoms. This drastic difference in pathogenicity may result from the acquisition of a few species-specific genes, but also from differences in their transcriptional regulation networks. In this study, we performed an in silico comparative whole-genome transcriptome analysis of Y. pestis and Y. pseudotuberculosis grown in parallel under 8 distinct conditions to determine whether they exhibit differences in their regulatory networks. In this analysis, 304 genes common to both species were found to display significant inter-species differences in transcriptional levels, with 91% of them being more expressed in Y. pestis. Remarkably, 3 major virulence determinants conserved in the 2 species (the pYV virulence plasmid, the High Pathogenicity Island, and the ail locus) were among the genes more expressed in Y. pestis. Furthermore, the induction at 37°C of pYV-borne genes was considerably greater in Y. pestis than in Y. pseudotuberculosis. Conversely, the rovA transcriptional regulator gene was more transcribed in Y. pseudotuberculosis. We also performed a clustering analysis of the transcriptome data of both Y. pestis and Y. pseudotuberculosis, which allowed to group genes according to their expression profiles. This analysis identified groups of genes with unknown functions which, based on regulation patterns similar to those of known virulence genes, are potential new virulence determinants in Y. pestis. In conclusion, this is the first comparative analysis at the whole-genome level of the transcription profiles of Y. pestis and Y. pseudotuberculosis. Our results suggest that the higher pathogenicity of the plague bacillus may not only result from the acquisition of new genetic material, but also from a higher expression level of common crucial virulence genes. This in silico analysis thus opens new avenues for investigating Y. pestis gain of pathogenicity and new potential virulence factors.

Characterizing consumer health terminology in the breast cancer field.

Despite the large availability of medical information on the Internet, health consumers still encounter problems to find, interpret and understand this information. These problems are mainly due to their lack in medical knowledge and the difference between their language and the language of health professionals. In order to propose information retrieval services more adapted to health consumers language and knowledge, we have developed techniques to collect, identify and analyze the terms and the expressions used by lay persons to talk about breast cancer. The study of health consumers' language is a relatively recent research field. Many studies have been conducted to analyze and characterize the vocabulary used by health consumers to talk about medical subjects in English. We have conducted the same study for the French language in the breast cancer field. We have gathered a corpus of texts to identify terms and expressions used by health consumers who talk about breast cancer in French. The terms have been organized in a concept-based terminology. This terminology has been analyzed on several levels: concept level, term level, term-concept level and finally relation level.

Autophagosomes can support Yersinia pseudotuberculosis replication in macrophages.

Yersinia pseudotuberculosis is able to replicate inside macrophages. However, the intracellular trafficking of the pathogen after its entry into the macrophage remains poorly understood. Using in vitro infected bone marrow-derived macrophages, we show that Y. pseudotuberculosis activates the autophagy pathway. Host cell autophagosomes subverted by bacteria do not become acidified and sustain bacteria replication. Moreover, we report that autophagy inhibition correlated with bacterial trafficking inside an acidic compartment. This study indicates that Y. pseudotuberculosis hijacks the autophagy pathway for its replication and also opens up new opportunities for deciphering the molecular basis of the host cell signalling response to intracellular Yersinia infection.

Hypoxic single-pass isolated hepatic perfusion of hypotonic Cisplatin: safety study in the pig.

BACKGROUND: Isolated hepatic perfusion (IHP) of chemotherapy has been proposed to deliver high doses of drug while avoiding systemic toxicity. Hypotonic cisplatin has a high in vitro activity on human colon cancer cells. We studied the safety of a 30-min hypoxic single-pass IHP with hypotonic cisplatin. METHODS: A preliminary in vitro assay was performed to compare the cytotoxicity of cisplatin and oxaliplatin, in either a normotonic or hypotonic medium. Cisplatin in hypotonic medium was then chosen for the in vivo IHP. Eleven pigs underwent 30 min of IHP with 0, 50, 75, or 100 mg/L of cisplatin in a hypotonic solution under total vascular exclusion of the liver. Clinical and biological parameters were recorded for 30 days, and liver histology was performed at necropsy. The cytotoxic activity of the effluent against resistant human colon cancer cells was tested in vitro. RESULTS: No hepatic failure was recorded after IHP with cisplatin, but limited foci of necrosis were found at necropsy in animals receiving 75 or 100 mg/L of cisplatin. No clinical, biological, macroscopic, or microscopic toxicity was observed after IHP with 50 mg/L of hypotonic cisplatin. The liver effluent showed high in vitro cytotoxic activity against colon cancer cells. CONCLUSIONS: A hypoxic single-pass isolated liver perfusion with hypotonic cisplatin is feasible and safe. Effluent from the liver is highly cytotoxic on cancer cells. A clinical study with 50 mg/L of hypotonic cisplatin is warranted in patients with unresectable liver metastases from colon cancer.

An iron-regulated LysR-type element mediates antimicrobial peptide resistance and virulence in Yersinia pseudotuberculosis.

During the course of its infection of the mammalian digestive tract, the entero-invasive, Gram-negative bacterium Yersinia pseudotuberculosis must overcome various hostile living conditions (notably, iron starvation and the presence of antimicrobial compounds produced in situ). We have previously reported that in vitro bacterial growth during iron deprivation raises resistance to the antimicrobial peptide polymyxin B; here, we show that this phenotype is mediated by a chromosomal gene (YPTB0333) encoding a transcriptional regulator from the LysR family. We determined that the product of YPTB0333 is a pleiotropic regulator which controls (in addition to its own expression) genes encoding the Yfe iron-uptake system and polymyxin B resistance. Lastly, by using a mouse model of oral infection, we demonstrated that YPTB0333 is required for colonization of Peyer's patches and mesenteric lymph nodes by Y. pseudotuberculosis.

Protection against Yersinia pseudotuberculosis infection conferred by a Lactococcus lactis mucosal delivery vector secreting LcrV.

Herein, we sought to evaluate the potential of a recombinant Lactococcus lactis strain secreting the Yersinia pseudotuberculosis low-calcium response V (LcrV) antigen for mucosal vaccination against Yersinia infections. We showed that the recombinant strain induced specific systemic and mucosal antibody and cellular immune responses after intranasal immunization and protected mice against both oral and systemic Y. pseudotuberculosis infections. This constitutes the first proof of principle for a novel anti-Yersinia mucosal vaccination strategy using recombinant lactic acid bacteria.

Growth of Yersinia pseudotuberculosis in human plasma: impacts on virulence and metabolic gene expression.

BACKGROUND: In man, infection by the Gram-negative enteropathogen Yersinia pseudotuberculosis is usually limited to the terminal ileum. However, in immunocompromised patients, the microorganism may disseminate from the digestive tract and thus cause a systemic infection with septicemia. RESULTS: To gain insight into the metabolic pathways and virulence factors expressed by the bacterium at the blood stage of pseudotuberculosis, we compared the overall gene transcription patterns (the transcriptome) of bacterial cells cultured in either human plasma or Luria-Bertani medium. The most marked plasma-triggered metabolic consequence in Y. pseudotuberculosis was the switch to high glucose consumption, which is reminiscent of the acetogenic pathway (known as "glucose overflow") in Escherichia coli. However, upregulation of the glyoxylate shunt enzymes suggests that (in contrast to E. coli) acetate may be further metabolized in Y. pseudotuberculosis. Our data also indicate that the bloodstream environment can regulate major virulence genes (positively or negatively); the yadA adhesin gene and most of the transcriptional units of the pYV-encoded type III secretion apparatus were found to be upregulated, whereas transcription of the pH6 antigen locus was strongly repressed. CONCLUSION: Our results suggest that plasma growth of Y. pseudotuberculosis is responsible for major transcriptional regulatory events and prompts key metabolic reorientations within the bacterium, which may in turn have an impact on virulence.

Sudden onset of pseudotuberculosis in humans, France, 2004-05.

Cases of Yersinia pseudotuberculosis infection increased in France during the winter of 2004-05 in the absence of epidemiologic links between patients or strains. This increase represents transient amplification of a pathogen endemic to the area and may be related to increased prevalence of the pathogen in rodent reservoirs.

Phenotypic analysis of Yersinia pseudotuberculosis 32777 response regulator mutants: new insights into two-component system regulon plasticity in bacteria.

Two-component regulatory systems (2CSs) typically comprise a sensor kinase and a response regulator that, in concert, monitor the concentration of particular extracellular factors and mediate the transcription of specific genes accordingly. As such, 2CSs play an important role in the regulation of bacterial pathogenesis. On the basis of genome-wide in silico analysis, the Gram-negative enteropathogenic bacterium Yersinia pseudotuberculosis is thought to encode 24 complete 2CSs. In the present work, we mutated the corresponding 2CS response regulator-encoding genes in Y. pseudotuberculosis strain 32777 and assessed the in vitro resistance of each mutant to the various types of stress encountered by Yersinia cells in the digestive tract. Eight of the generated regulatory mutants (phoP, ompR, pmrA, ntrC-, arcA-, rstA-, rcsB-, and yfhA-like mutants) showed significant changes in tolerance towards at least one type of stress, when compared with the wild-type strain. Of these eight, four (ompR, phoP, rstA-, and yfhA-like mutants) were found to be less virulent than the wild type in the BALB/c mouse model. Although some mutant phenotypes were consistent with those (when known) of the corresponding, putative ortholog mutants in other pathogenic species, several response regulators behaved differently in Y. pseudotuberculosis; these included the PmrA, PhoP, and ArcA-like response regulators, which were found to control bile salt resistance in a manner different from that observed in Salmonella. Hence, in addition to genome evolution, transcriptional network remodeling may be a major cause of phenotypic adaptation (and thus species divergence) in Y. pseudotuberculosis.

Two-component system regulon plasticity in bacteria: a concept emerging from phenotypic analysis of Yersinia pseudotuberculosis response regulator mutants.

In bacteria, the most rapid and efficient means of adapting gene transcription to extracellular stresses often involves sophisticated systems referred to as two-component systems (2CSs). Although highly conserved throughout the bacterial world, some of these systems may control distinct cell events and have differing contributions to virulence, depending on the species considered. This chapter summarizes the work performed by our group--from the initial PhoP-PhoQ and PmrA-PmrB studies to the most recent genome-scale preliminary analyses--in an attempt to highlight the contribution of 2CS regulon plasticity to the acquisition of some of Yersinia pseudotuberculosis' specific features.

Therapeutic potential of Yersinia anti-inflammatory components.

Microbial pathogens have developed various stratagems for modulating and/or circumventing the host's innate and adaptive immunity. Hence, certain virulence factors can be viewed as potential therapeutic agents for human immunopathological diseases. This is the case for virulence plasmid-encoded proteins from pathogenic Yersiniae that inhibit the host's inflammatory response by interfering with various cellular signaling pathways.