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International audience; We aimed to assess the independent effect of clinical spectrum, bacterial inoculum size and physician characteristics on the sensitivity of a rapid antigen detection test (RADT) for group A streptococcus (GAS) in children. Double throat swabs were collected from 1,482 children with pharyngitis and 294 asymptomatic children in a French prospective, office-based, multicenter (n = 17) study, from October 2009 to May 2011. Patient- and physician-level factors potentially affecting RADT sensitivity were studied by univariate and multivariate multilevel analysis, with laboratory throat culture as the reference test. In children with pharyngitis and asymptomatic children, the prevalence of GAS was 38 % (95 % confidence interval 36-41 %) and 11 % (7-14 %), respectively. Overall, RADT sensitivity was 87 % (84-90 %). On stratified and multivariate multilevel analysis, RADT sensitivity was higher for children with pharyngitis than asymptomatic children (89 % vs. 41 %), children 99 %). The sensitivity of the RADT is independently affected by patient- and physician-level factors. Physicians who base their diagnosis of GAS pharyngitis on the results of a RADT alone should consider diagnostic accuracy monitoring and adequate training when needed.

International audience; A new vaccine, 4CMenB, is composed of surface proteins of Neisseria meningitidis and is aimed to target serogroup B (MenB) isolates. The vaccine components are present in meningococcal isolates of other serogroups allowing potential use against meningococcal isolates belonging to non-B serogroups. Isolates of serogroup X (MenX) have been emerged in countries of the African meningitis belt. 4CMenB may offer a vaccine strategy against these isolates as there is no available capsule-based vaccine against MenX. We used the Meningococcal Antigen Typing System (MATS) to determine presence, diversity and levels of expression of 4CMenB antigens among 9 MenX isolates from several African countries in order to estimate the potential coverage of MenX by the 4CMenB vaccine. We performed bactericidal assays against these isolates, using pooled sera from 4CMenB-vaccinated infants, adolescents and adults. The African MenX isolates belonged to the same genotype but showed variation in the vaccine antigens. MATS data and bactericidal assays suggest coverage of the 9 African MenX isolates by 4CMenB but not of two unrelated MenX isolates from France. 4CMenB vaccine can be considered for further investigation to control MenX outbreaks in Africa.

International audience; Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37uC. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20uC and 37uC, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples. Citation: Simon S, Demeure C, Lamourette P, Filali S, Plaisance M, et al. (2013) Fast and Simple Detection of Yersinia pestis Applicable to Field Investigation of Plague Foci.

International audience

International audience; BACKGROUND:Recombinant vaccines containing factor H-binding protein (fHBP) have been developed for the purpose of protection from invasive meningococcal serogroup B disease. Neisseria meningitidis fHBP sequences can be divided into 2 genetically and immunologically distinct subfamilies (A and B); thus, cross protection is conferred within but not between subfamilies. A comprehensive understanding of fHBP epidemiology is required to accurately assess the potential vaccine impact when considering different vaccination implementation strategies.METHODS:Systematically collected invasive meningococcal serogroup B isolates from England, Wales, Northern Ireland, the United States, Norway, France and the Czech Republic were previously characterized for fHBP sequence. This study expanded the evaluation with additional meningococcal serogroup B disease isolates from Spain (n = 346) and Germany (n = 205). This expanded set (n = 1841), collected over a 6-year period (2001 to 2006), was evaluated for fHBP sequence and fHBP subfamily relative to patient age.RESULTS:All 1841 isolates contained fhbp. fHBP sequences from Spain and Germany fell within the previously described subfamilies, with 69% of isolates belonging to subfamily B and 31% to subfamily A; prevalent sequence variants were also similar. Stratification of data by age indicated that disease in infants

International audience; Neisseria meningitidis is a human pathogenic bacterium responsible for life threatening and rapidly evolving invasive infections. Several bacterial virulence factors may play primordial roles during host-bacteria interactions. The meningococcal factor H binding protein, fHbp, interacts with the complement negative regulator, factor H (fH), to enhance meningococcal survival. fHbp is a major component in recombinant vaccines against meningococci that are under development. In 2010, we detected variations in fhbp gene during an outbreak provoked by serogroup C isolates belonging to the clonal complex, ST-11. We therefore explored 680 meningococcal isolates (88% of all invasive isolates in 2009 and 2010) by DNA sequencing of fhbp gene. The level of fHbp at the bacterial surface was determined by ELISA and flow cytometry using anti-fHbp antibodies. We also analyzed the interaction of fHbp with human fH as well as the deposition of C3b complement component. We observed important sequence diversity of fHbp in particular within regions known to interact with fH. The distribution of fhbp alleles differed among meningococcal serogroups and clonal complexes. This diversity affected directly binding of fH to fHbp and seemed to influence the deposition of the complement C3b component on the bacterial surface. However, bacterial killing by anti-fHbp antibodies was still achieved and required a minimum level of fHbp at the bacterial surface regardless the binding to fH or sequence diversity. These data have impacts on our understanding of the role of fHbp in meningococcal pathogenesis. They also provide data on the diversity of fhbp before the introduction of vaccines targeting fHbp and stress the need to include characterization of fHbp in typing schemes of meningococcal isolates.

International audience; BACKGROUND AND AIMS: Helicobacter pylori is a highly diverse pathogen, which encounters epithelial cells as the initial defense barrier during its lifelong infection. The structure of epithelial cells can be disrupted through cleavage of microfilaments. Cytokeratin 18 (CK18) is an intermediate filament, the cleavage of which is considered an early event during apoptosis following activation of effector caspases. METHODS: Helicobacter pylori strains were isolated from 76 dyspeptic patients. cagA 3' variable region and CagA protein status were analyzed by PCR and western blotting, respectively. Eight hours post-co-culture of AGS cells with different H. pylori strains, flow cytometric analysis was performed using M30 monoclonal antibody specific to CK18 cleavage-induced neo-epitope. RESULTS: Higher rates of CK18 cleavage were detected during co-culture of AGS cells with H. pylori strains bearing greater numbers of cagA EPIYA-C and multimerization (CM) motifs. On the other hand, H. pylori strains with greater numbers of EPIYA-B relative to EPIYA-C demonstrated a decrease in CK18 cleavage rate. Thus, H. pylori-mediated cleavage of CK18 appeared proportional to the number of CagA EPIYA-C and CM motifs, which seemed to be downplayed in the presence of EPIYA-B motifs. CONCLUSIONS: Our observation associating the heterogeneity of cagA variants with the potential of H. pylori strains in the induction of CK18 cleavage as an early indication of apoptosis in gastric epithelial cells supports the fact that apoptosis may be a type-specific trait. However, additional cagA-targeted experiments are required to clearly identify the role of EPIYA and CM motifs in apoptosis and/or the responsible effector molecules.

International audience; Lipids are important antigens that induce T cell-mediated specific immune responses. They are presented to T lymphocytes by a specific class of MHC-I like proteins, termed CD1. The majority of the described CD1-presented mycobacterial antigens are presented by the CD1b isoform. We previously demonstrated that the stimulation of CD1b-restricted T cells by the hexamannosylated phosphatidyl-myo-inositol (PIM(6)), a family of mycobacterial antigens, requires a prior partial digestion of the antigen oligomannoside moiety by α-mannosidase and that CD1e is an accessory protein absolutely required for the generation of the lipid immunogenic form. Here, we show that CD1e behaves as a lipid transfer protein influencing lipid immunoediting and membrane transfer of PIM lipids. CD1e selectively assists the α-mannosidase-dependent digestion of PIM(6) species according to their degree of acylation. Moreover, CD1e transfers only diacylated PIM from donor to acceptor liposomes and also from membranes to CD1b. This study provides new insight into the molecular mechanisms by which CD1e contributes to lipid immunoediting and CD1-restricted presentation to T cells.

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag -T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.

International audience; Many bacterial pathogens inject into host cells effector proteins that are substrates for host tyrosine kinases such as Src and Abl family kinases. Phosphorylated effectors eventually subvert host cell signaling, aiding disease development. In the case of the gastric pathogen Helicobacter pylori, which is a major risk factor for the development of gastric cancer, the only known effector protein injected into host cells is the oncoprotein CagA. Here, we followed the hierarchic tyrosine phosphorylation of H. pylori CagA as a model system to study early effector phosphorylation processes. Translocated CagA is phosphorylated on Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs EPIYA-A, EPIYA-B, and EPIYA-C in Western strains of H. pylori and EPIYA-A, EPIYA-B, and EPIYA-D in East Asian strains. We found that c-Src only phosphorylated EPIYA-C and EPIYA-D, whereas c-Abl phosphorylated EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D. Further analysis revealed that CagA molecules were phosphorylated on 1 or 2 EPIYA motifs, but never simultaneously on 3 motifs. Furthermore, none of the phosphorylated EPIYA motifs alone was sufficient for inducing AGS cell scattering and elongation. The preferred combination of phosphorylated EPIYA motifs in Western strains was EPIYA-A and EPIYA-C, either across 2 CagA molecules or simultaneously on 1. Our study thus identifies a tightly regulated hierarchic phosphorylation model for CagA starting at EPIYA-C/D, followed by phosphorylation of EPIYA-A or EPIYA-B. These results provide insight for clinical H. pylori typing and clarify the role of phosphorylated bacterial effector proteins in pathogenesis.

CD8 + T lymphocytes often play a primary role in adaptive immunity to cytosolic microbial pathogens. Surprisingly, CD8 + T cells are not required for protective immunity to the enteric pathogen Shigella flexneri , despite the ability of Shigella to actively secrete proteins into the host cytoplasm, a location from which antigenic peptides are processed for presentation to CD8 + T cells. To determine why CD8 + T cells fail to play a role in adaptive immunity to S. flexneri , we investigated whether antigen-specific CD8 + T cells are primed during infection but are unable to confer protection or, alternatively, whether T cells fail to be primed. To test whether Shigella is capable of stimulating an antigen-specific CD8 + T-cell response, we created an S. flexneri strain that constitutively secretes a viral CD8 + T-cell epitope via the Shigella type III secretion system and characterized the CD8 + T-cell response to this strain both in mice and in cultured cells. Surprisingly, no T cells specific for the viral epitope were stimulated in mice infected with this strain, and cells infected with the recombinant strain were not targeted by epitope-specific T cells. Additionally, we found that the usually robust T-cell response to antigens artificially introduced into the cytoplasm of cultured cells was significantly reduced when the antigen-presenting cell was infected with Shigella . Collectively, these results suggest that antigen-specific CD8 + T cells are not primed during S. flexneri infection and, as a result, afford little protection to the host during primary or subsequent infection.

To the Editor: Streptococcus pyogenes, or group A streptococci (GAS), are human pathogens responsible for pharyngitis as well as skin and soft tissue infections. Invasive GAS diseases, including bacteremia, cellulitis, and necrotizing fasciitis, are life-threatening, especially when associated with toxic shock syndrome. Several risk factors for GAS infections are known, such as diabetes, immunosuppression, drug use, and skin lesions (1,2).

International audience; Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.

Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 μM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.

International audience; Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A(2) (sPLA(2)-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA(2)-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA(2)-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA(2)-IIA expression in AMs indicating that these kinases play a role in sPLA(2)-IIA expression. LT inhibited IL-1beta-induced p38 phosphorylation as well as sPLA(2)-IIA promoter activity in CHO cells. Inhibition of sPLA(2)-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1beta-induced NF-kappaB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IkappaBalpha degradation or NF-kappaB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA(2)-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA(2)-IIA expression is induced via a sequential MAPK-NF-kappaB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-kappaB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.

International audience; Helicobacter pylori (Hp) is a major risk factor for gastrointestinal disorders including gastric cancer. We evaluated host serum antibody responses toward outer membrane protein18 in comparison with Urease A and B subunits. omp18 and ureA-ureB gene fragments were PCR amplified, cloned, and expressed in E. coli expression system. The expressed proteins were visualized on SDS-PAGE and confirmed by immuno-blotting. Purified proteins were applied in western blotting assays in comparison with local and foreign ELISA kits. ROC curve analysis identified the optimum cut-off points for each protein. rOmp18 represented the highest rates of sensitivity (94%), specificity (89%), PPV (97.4%), NPV (77.4%), and accuracy (93.2%) in comparison with urease A and B subunits. These immunologic indices were in "substantial" agreement (Κ = 0.7) with the gold standard tests for Hp detection. This study recommends Hp conserved Omp18 as a reliable serologic marker for accurate detection of Hp infection particularly for application in population screening approaches.

International audience; Bacillus anthracis toxin gene expression requires AtxA, a virulence regulator that also activates capsule gene transcription and controls expression of more than a hundred genes. Here we report that atxA mRNA is 2.7-kb-long and ends, after a 500 nt-long 3' untranslated region, with a stem loop structure followed by a run of U's. The presence of this structure stabilizes atxA mRNA and is necessary for AtxA maximal accumulation, full expression of the PA toxin gene, pagA and optimal PA accumulation. This structure displays terminator activity independently of its orientation when cloned between an inducible promoter and a reporter gene. The 3.6-kb-long DNA fragment carrying both AtxA promoters and the terminator is sufficient for full expression of pagA in the presence of bicarbonate. No pXO1-encoded element other than the DNA fragment encompassing the 2.7 kb atxA transcript and the pagA promoter is required for bicarbonate induction of pagA transcription.

In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis , the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis . Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA , as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally.

Background. Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000–2006. Methods. Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. Results. Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%– 75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. Conclusions. The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST. Invasive disease caused by Neisseria meningitidis is a rapidly progressing, disseminated infection with a case fatality rate of ∼10% [1], and 10%–20% of survivors experience serious permanent sequelae (eg, neurological impairment, digit, limb, or hearing loss). Vaccines for meningococcal serogroups A, C, Y, and W135

International audience; Mycobacteria are characterized by a complex cell wall, the lipid nature of which confers to the bacilli resistance to drying, acid or alkaline conditions, and to chemical disinfectants and therapeutic agents. Pathogenic species, such as Mycobacterium tuberculosis, M. leprae and M. ulcerans, have evolved various strategies to establish residence in their hosts and provoke long-term infections. There is mounting evidence that the unique lipids composing their envelopes, strategically located at the host-pathogen interface, contribute to their escape from immune surveillance. Here, the chemical structure, host cell receptors and biological actions of this emerging class of mycobacterial virulence factors are reviewed.

8 pages; International audience; If immune functions confer obvious benefits to hosts, life-history theory assumes that they also induce costs, leading to trade-offs between immunity and other fitness components. However, whether substantial fitness costs are associated with immune systems in the wild is debatable, as numerous factors may influence the costs and benefits associated with immune activation. Here, we explore the survival cost of immune deployment in postfledging birds. We injected Eurasian collared dove nestlings (Streptopelia decaocto) with antigens from Escherichia coli, and examined whether this immune challenge affected survival after fledging. To assess survival, birds were fitted with radiotags and the fate of each individual was monitored regularly. Our results show that mimicking a bacterial infection in nestlings lowered their survival prospects after fledging, in comparison to controls. The main identified cause of mortality (by examination of dead birds) was presumed to be predation. This study provides experimental evidence that immune activation may entail dramatic survival costs in a free-ranging vertebrate, and emphasizes the potential role that environmental factors such as predation may play in this interaction.

Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis, the etiological agent of anthrax, whose pulmonary form is fatal in the absence of treatment. Inflammatory response is a key process of host defense against invading pathogens. We report here that intranasal instillation of a B. anthracis strain bearing inactive LT stimulates cytokine production and polymorphonuclear (PMN) neutrophils recruitment in lungs. These responses are repressed by a prior instillation of an LT preparation. In contrast, instillation of a B. anthracis strain expressing active LT represses lung inflammation. The inhibitory effects of LT on cytokine production are also observed in vitro using mouse and human pulmonary epithelial cells. These effects are associated with an alteration of ERK and p38-MAPK phosphorylation, but not JNK phosphorylation. We demonstrate that although NF-κB is essential for IL-8 expression, LT downregulates this expression without interfering with NF-κB activation in epithelial cells. Histone modifications are known to induce chromatin remodelling, thereby enhancing NF-κB binding on promoters of a subset of genes involved in immune response. We show that LT selectively prevents histone H3 phosphorylation at Ser 10 and recruitment of the p65 subunit of NF-κB at the IL-8 and KC promoters. Our results suggest that B. anthracis represses the immune response, in part by altering chromatin accessibility of IL-8 promoter to NF-κB in epithelial cells. This epigenetic reprogramming, in addition to previously reported effects of LT, may represent an efficient strategy used by B. anthracis for invading the host., Author Summary Bacillus anthracis, the etiological agent of anthrax, can infect mammals either accidentally or as a potential consequence of a terrorism threat. Pulmonary infection is a life-threatening form of the disease, causing a near 100% mortality rate in the absence of appropriate therapy. Thus, it is important to understand the mechanisms of host defense against B. anthracis. We examined the effects of various B. anthracis strains on lung inflammation in a mouse model of pulmonary anthrax and on human lung epithelial cells, the first barrier of lung against invading pathogens. We showed that a B. anthracis strain expressing lethal toxin inhibits inflammation. In contrast, a strain in which this toxin has been inactivated induces lung inflammation. We next examined the mechanisms involved in the inhibitory effect of lethal toxin. We showed that B. anthracis injects lethal toxin into epithelial cells, blocks the molecules associated on the chromosome, and thus represses production of mediators involved in inflammation. As the latter is a key process in host defense, its alteration by lethal toxin predisposes the host to infection by B. anthracis. This effect on the chromosomal machinery may represent an efficient strategy used by B. anthracis for invading the host.

International audience; The ESX-1 system is responsible for the secretion of the prototypic ESX proteins, namely the 6 kDa early secreted antigenic target (ESAT-6) and the 10 kDa culture filtrate protein (CFP-10). These two proteins, which form a 1:1 heterodimeric complex, are among the most important proteins of Mycobacterium tuberculosis involved in host-pathogen interaction. They induce a strong T cell mediated immune response, are apparently involved in membrane and/or host-cell lysis and represent key virulence factors. There are four other paralogous ESX systems in M. tuberculosis, some of which are essential for in vitro growth. ESX systems also exist in many other actinobacteria and Gram-positive bacteria, and have recently been suggested to be named type VII secretion systems.

Background The increasing number of completely sequenced bacterial genomes allows comparing their architecture and genetic makeup. Such new information highlights the crucial role of lateral genetic exchanges in bacterial evolution and speciation. Results Here we analyzed the twelve sequenced genomes of Streptococcus pyogenes by a naïve approach that examines the preferential nucleotide usage along the chromosome, namely the usage of G versus C (GC-skew) and T versus A (TA-skew). The cumulative GC-skew plot presented an inverted V-shape composed of two symmetrical linear segments, where the minimum and maximum corresponded to the origin and terminus of DNA replication. In contrast, the cumulative TA-skew presented a V-shape, which segments were interrupted by several steep slopes regions (SSRs), indicative of a different nucleotide composition bias. Each S. pyogenes genome contained up to nine individual SSRs, encompassing all described strain-specific prophages. In addition, each genome contained a similar unique non-phage SSR, the core of which consisted of 31 highly homologous genes. This core includes the M-protein, other mga-related factors and other virulence genes, totaling ten intrinsic virulence genes. In addition to a high content in virulence-related genes and to a peculiar nucleotide bias, this SSR, which is 47 kb-long in a M1GAS strain, harbors direct repeats and a tRNA gene, suggesting a mobile element. Moreover, its complete absence in a M-protein negative group A Streptococcus natural isolate demonstrates that it could be spontaneously lost, but in vitro deletion experiments indicates that its excision occurred at very low rate. The stability of this SSR, combined to its presence in all sequenced S. pyogenes sequenced genome, suggests that it results from an ancient acquisition. Conclusion Thus, this non-phagic SSR is compatible with a pathogenicity island, acquired before S. pyogenes speciation. Its potential excision might bear relevance for vaccine development, because vaccines targeting M-protein might select for M-protein-negative variants that still carry other virulence determinants.

The PE and PPE (PE/PPE) multigene families of Mycobacterium tuberculosis are particularly GC-rich and share extensive homologous repetitive sequences. We hypothesized that they may undergo homologous recombination events, a mechanism rarely described in the natural evolution of mycobacteria. To test our hypothesis, we developed a specific oligonucleotide-based microarray targeting nearly all of the PE/PPE genes, aimed at detecting signals for homologous recombination. Such a microarray has never before been reported due to the multiplicity and highly repetitive and homologous nature of these sequences. Application of the microarray to a collection of M. tuberculosis clinical isolates ( n = 33) representing prevalent spoligotype strain families in Tunisia allowed successful detection of six deleted genomic regions involving a total of two PE and seven PPE genes. Some of these deleted genes are known to be immunodominant or involved in virulence. The four precisely determined deletions were flanked by 400- to 500-bp stretches of nearly identical sequences lying mainly at the conserved N-terminal region of the PE/PPE genes. These highly homologous sequences thus serve as substrates to mediate both intergenic and intragenic homologous recombination events, indicating an important function in generating strain variation. Importantly, all recombination events yielded a new in-frame fusion chimeric gene. Hence, homologous recombination within and between PE/PPE genes likely increased their antigenic variability, which may have profound implications in pathogenicity and/or host adaptation. The finding of high prevalence (∼45% and ∼58%) for at least two of the genomic deletions suggests that they likely confer advantageous biological attributes.

International audience; Tuberculosis (TB) is a re-emerging disease that is considered a major human health priority as well as an important disease of livestock. TB is also a zoonosis, and Mycobacterium tuberculosis and M. bovis, the human and bovine causative agents, respectively, are very closely related. Protection against TB is essentially achieved through vaccination with the Bacille Calmetle-Guerin (BCG) strain of M. bovis. Protection is, however, incomplete, and novel improved vaccines are currently under investigation. Production of protective antigens in transgenic plants, or "pharming," is a promising emerging approach, and a zoonosis-like TB is a good model for investigating the potential of this approach. Pharma-Planta, a European Commission-funded project and consortium, was set up to address this topic, within which a component is aimed at assessing the production efficacy and stability of the TB antigens in different compartments of the plant cell. This article is meant to introduce this promising approach for veterinary medicine by describing the ongoing project and its specific genetic engineering strategy.

Summary Objective To evaluate dipstick rapid diagnostic tests (RDTs) for meningococcal meningitis in basic health facilities. Methods Health facility staff received a one-day training. During the meningitis season, they performed RDTs on cerebrospinal fluid (CSF) specimens from suspected cases of meningitis. A frozen aliquot of CSF was later tested using polymerase chain reaction (PCR) to establish the reference diagnosis. RDTs used in health facilities were archived to allow checking the concordance between reported diagnosis and observed results. Reported diagnosis was also compared to PCR diagnosis. A second RDT was performed on each CSF specimen at the reference laboratory. Results Using RDTs, health facilities reported 382 negative results (73.9%), 114 NmA (22.1%), 12 NmW135 (2.3%) and nine uninterpretable results (1.7%), the latter corresponding to the misuse of a reagent by three agents. The agreement between reported diagnosis and archived dipsticks was excellent (kappa = 0.98). The agreement between PCR diagnosis and reported RDTs results was strong (kappa = 0.82). In health facilities, the sensitivity of RDTs for N. meningitidis A was Se = 0.91. The kappa coefficient measuring the agreement between RDTs operated in the reference laboratory and RDTs operated in health facilities was κ = 0.78. Conclusion We confirmed that dipstick RDTs to identify N. meningitidis serogroups A, C, W135 and Y can be reliably operated by non-specialized staff in basic health facilities. RDTs proved very useful to recommend vaccination in NmA epidemics, and also to avoid vaccination in epidemics due to serogroups not included in vaccines (NmX).

International audience; Cystic echinococcosis (CE), caused by Echinococcus granulosus, and alveolar echinococcosis (AE), due to E. multilocularis, are endemic to large areas of north and northwest China. Here we review features of a severe focus of AE and CE in Ningxia Hui Autonomous Region (NHAR), northwest China. We describe for NHAR the results of surveys of hospital surgical and clinical records, including treatment of AE and CE, and active community detection of asymptomatic/early-stage cases and patient follow-up using questionnaire analysis, ultrasound examinations and serology. We compare the key risk factors for both AE and CE in this setting with those reported from other areas. In addition, we document the socio-economic factors impacting on treatment and control of AE and CE. This update indicates that echinococcosis continues to be highly endemic in NHAR and exemplifies the serious public health problem that the disease presents, particularly in poor rural areas. Extensive community surveys of echinococcosis are required throughout northwest China, especially in rural communities. These will enable earlier detection of echinococcosis cases, thereby improving treatment outcomes, and reveal the true epidemiological picture regarding the disease in this region, with a view to developing and implementing new strategies for future control.

The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of ∼15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RD Rio . Using the Brazilian strains, we pinpoint an Ag85C 103 single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RD Rio strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RD Rio strains, were then used to analyze an international collection of M. tuberculosis strains. RD Rio M. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS 6110 -RFLP patterns from representative RD Rio and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C 103 SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS 6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RD Rio strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RD Rio sublineage.

The purpose of the study was to obtain more reliable epidemiological data concerning Entamoeba (E.) histolytica infection in Tunisian food handlers using established molecular tools able to differentiate E. histolytica from E. dispar. From 2002 to 2005, 4,266 fresh stools specimens received in the setting of the National program of food handlers' control were analysed by optical microscopy. Twelve (2.8 per thousand) were positive for the presence of four nuclei cysts identified as E. histolytica/E. dispar. Extraction of DNA from the 12 samples, followed by specific amplifications of E. histolytica and E. dispar SSU rDNA, showed that 11 samples (92%) were positive for E. dispar and negative for E. histolytica. Sequencing analysis of 8 PCR products permitted to verify the results obtained with conventional PCR. The remaining sample was negative by PCR amplifying E. histolytica DNA or E. dispar DNA specifically, although it did not show any inhibition. It probably contains protozoan cysts genetically distinct from these two species but morphological similar. Estimation of relative proportions between E. histolytica and E. dispar in cyst carriers showed that all explored individuals harboured the non pathogenic E. dispar strains. This result highlights the need of use in this population of complementary tests that allow specific diagnosis and obviate unnecessary chemotherapy.; Entre 2002 et 2005, 4 266 manipulateurs de denrées alimentaires de la région de Tunis ont bénéficié d’un examen parasitologique des selles dans le cadre du contrôle systématique. Douze (2,8 ‰) étaient porteurs de kystes d’Entamoeba histolytica/dispar. L’extraction d’ADN directement à partir des échantillons de selles positives, suivie respectivement d’une amplification génique de l’ADNr d ’E. histolytica et d’ E. dispar et d’un séquençage des produits d’amplification a permis d’identifier E. dispar chez 11 porteurs de kystes. Aucun manipulateur n’était porteur de l’espèce pathogène E. histolytica. Ces résultats mettent en exergue la nécessité d’examens complémentaires permettant l’identification des espèces E. histolytica et E. dispar et confirment la faible prévalence de l’amibiase dans le nord de la Tunisie.

International audience; OBJECTIVE: Diagnosis of pulmonary embolism (PE) remains difficult and is often missed in the elderly due to nonspecific and atypical presentation. Diagnostic algorithms able to rule out PE and validated in young adult patients may have reduced applicability in elderly patients, which increases the number of diagnostic tools use and costs. The aim of the present study was to analyze the reported clinical presentation of PE in patients aged 65 and more. MATERIALS AND METHODS: Prospective and retrospective English language studies dealing with the clinical, instrumental and laboratory aspects of PE in patients more than 65 and published after January 1987 and indexed in MEDLINE using keywords as pulmonary embolism, elderly, old, venous thromboembolism (VTE) in the title, abstract or text, were reviewed. RESULTS: Dyspnea (range 59%-91.5%), tachypnea (46%-74%), tachycardia (29%-76%), and chest pain (26%-57%) represented the most common clinical symptoms and signs. Bed rest was the most frequent risk factor for VTE (15%-67%); deep vein thrombosis was detected in 15%-50% of cases. Sinus tachycardia, right bundle branch block, and ST-T abnormalities were the most frequent ECG findings. Abnormalities of chest X-ray varied (less than 50% in one-half of the studies and more than 70% in the other one-half). Arterial blood gas analysis revealed severe hypoxemia and mild hypocapnia as the main findings. D-Dimer was higher than cut-off in 100% of patients in 75% of studies. Clinical usefulness of D-Dimer measurement decreases with age, although the strategies based on D-Dimer seem to be cost-effective at least until 80 years. CONCLUSION: Despite limitations due to pooling data of heterogeneous studies, our review could contribute to the knowledge of the presentation of PE in the elderly with its diagnostic difficulties. A diagnostic strategy based on reviewed data is proposed.

A successful Th cell response to bacterial infections is induced by mature MHC class II molecules presenting specific Ag peptides on the surface of macrophages. In recent studies, we demonstrated that infection with the conventional vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) specifically blocks the surface export of mature class II molecules in human macrophages by a mechanism dependent on inhibition of cathepsin S (Cat S) expression. The present study examined class II expression in macrophages infected with a rBCG strain engineered to express and secrete biologically active human Cat S (rBCG-hcs). Cat S activity was completely restored in cells ingesting rBCG-hcs, which secreted substantial levels of Cat S intracellularly. Thus, infection with rBCG-hcs, but not parental BCG, restored surface expression of mature MHC class II molecules in response to IFN-γ, presumably as result of MHC class II invariant chain degradation dependent on active Cat S secreted by the bacterium. These events correlated with increased class II-directed presentation of mycobacterial Ag85B to a specific CD4+ T cell hybridoma by rBCG-hcs-infected macrophages. Consistent with these findings, rBCG-hcs was found to accelerate the fusion of its phagosome with lysosomes, a process that optimizes Ag processing in infected macrophages. These data demonstrated that intracellular restoration of Cat S activity improves the capacity of BCG-infected macrophages to stimulate CD4+ Th cells. Given that Th cells play a major role in protection against tuberculosis, rBCG-hcs would be a valuable tuberculosis vaccine candidate.

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra “knock-ins” that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis., Author Summary Mycobacterium tuberculosis, the causative agent of human tuberculosis is an extremely successful human pathogen in spite of its lack of classical virulence factors, such as toxins. The pathogenesis of this bacterium is closely linked with its ability to circumvent destruction by the host cell, which depends on a large variety of mycobacterial lipids and secreted proteins. Genome comparison of fully virulent strains with closely related, but attenuated strains that have lost their ability to cause disease is a powerful approach to identify factors contributing to mycobacterial virulence. In this study we have compared the virulent paradigm strain of tuberculosis research M. tuberculosis H37Rv, with the widely used, attenuated laboratory variant, H37Ra. This analysis, combined with gene complementation studies, has identified a mutation in the two-component system regulator PhoP that impacts on the secretion and T cell recognition of the 6-kD early-secreted antigenic target ESAT-6. In previous studies, both PhoP and the ESAT-6 secretion system ESX-1 have been identified independently as major virulence determinants of M. tuberculosis, so the finding that their action is interconnected opens exciting new insights and perspectives into the virulence regulation of tubercle bacilli.

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with the virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter subgroup of proteins. The best characterized of these proteins, antigen 43 (Ag43), is a self-recognizing adhesin that is associated with cell aggregation and biofilm formation in E. coli K-12. The sequenced genome of prototype UPEC strain CFT073 contains two variant Ag43-encoding genes located on pathogenicity islands. The biological significance of both of these genes and their role in UPEC pathogenesis have not been investigated previously. Here we performed a detailed molecular characterization analysis of Ag43a (c3655) and Ag43b (c1273) from UPEC CFT073. Expression of Ag43a and Ag43b in a K-12 background revealed that they possess different functional properties. Ag43a produced a strong aggregation phenotype and promoted significant biofilm growth. Deletion mutants and strains constitutively expressing Ag43a and Ag43b were also constructed using CFT073. When these mutants were analyzed in a mouse model of UTI, Ag43a (but not Ag43b) promoted long-term persistence in the urinary bladder. Our findings demonstrate that Ag43a contributes to UPEC disease pathogenesis and reveal that there are pathogenicity-adapted variants of Ag43 with distinct virulence-related functions.

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using floatation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6·CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.

Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.

International audience; Many bacteria pathogenic for plants or animals, including Shigella spp., which is responsible for shigellosis in humans, use a type III secretion apparatus to inject effector proteins into host cells. Effectors alter cell signaling and host responses induced upon infection; however, their precise biochemical activities have been elucidated in very few cases. Utilizing Saccharomyces cerevisiae as a surrogate host, we show that the Shigella effector IpaH9.8 interrupts pheromone response signaling by promoting the proteasome-dependent destruction of the MAPKK Ste7. In vitro, IpaH9.8 displayed ubiquitin ligase activity toward ubiquitin and Ste7. Replacement of a Cys residue that is invariant among IpaH homologs of plant and animal pathogens abolished the ubiquitin ligase activity of IpaH9.8. We also present evidence that the IpaH homolog SspH1 from Salmonella enterica can ubiquitinate ubiquitin and PKN1, a previously identified SspH1 interaction partner. This study assigns a function for IpaH family members as E3 ubiquitin ligases.

International audience; The Escherichia Coli bacterial extract (OM-89) is used in the treatment of rheumatoid arthritis (RA). We evaluated the immunological changes induced by oral administration of OM-89 in 12 RA patients (polyclonal T cell reactivity to PHA, T cell precursor frequencies specific for OM-89 and Tetanus toxoid (TT), a control antigen and the release of Th1 (IFN-gamma, TNF-alpha), Th2 (IL-4) and T regulatory 1 cell (Tr1) (IL-10) cytokines in the supernatants of PBMC cultures. Stimulation index in response to PHA decreased at month 3 as well as T cell precursor frequencies specific for TT with similar trends for OM-89-specific T cell precursor frequencies. OM-89 induced a strong production of IL-10, a significant decrease in IL-4 production while TNF-alpha and IFN-gamma production tended to decrease during the study. Our results suggest that OM-89 has immunomodulatory properties by inducing changes in PBMC cytokines release suggestive of an induced Tr1 response to OM-89.

International audience; In addition to multidrug therapy, elimination of leprosy requires improved diagnostic methods. Using a comparative genomics approach, 17 potential protein antigens (MLP) that are restricted to Mycobacterium leprae, or of limited distribution, were produced and tested for antigen-specific immune responses on leprosy patients, healthy contacts of leprosy patients, and tuberculosis patients in Mali and Bangladesh, as well as on non-endemic controls. T-cell antigenicity of MLP was confirmed by IFN-gamma production in whole-blood assays with the highest responses observed in paucibacillary leprosy patients and healthy contacts. Four MLP behaved well in both countries and induced significantly different responses between the study groups. Peptides carrying T cell epitopes from one of the antigens gave promising results in restimulation assays in mice and immune responses were not influenced by prior exposure to BCG or environmental mycobacteria. This study provides the immunological framework for the development of a specific, peptide-based immunodiagnostic test for leprosy.

Leprosy is an infectious, neurodegenerative disease of humans caused by Mycobacterium leprae . Despite effective control programs, the incidence of leprosy remains stubbornly high, suggesting that transmission may be more common than expected. The rationale of this work was to use bioinformatics and comparative genomics to identify potentially antigenic proteins for diagnostic purposes. This approach defined three classes of proteins: those restricted to M. leprae (class I), those present in M. leprae with orthologues in other organisms besides mycobacteria (class II), and exported or surface-exposed proteins (class III). Twelve genes (two class I, four class II, and six class III proteins) were cloned in Escherichia coli , and their protein products were purified. Six of these proteins were detected in cell extracts of M. leprae by immunoblotting. The immunogenicity of each recombinant protein was then investigated in leprosy patients by measuring the reactivity of circulating antibody and gamma interferon (IFN-γ) responses in T-cell restimulation assays. Several class II and class III proteins were recognized by circulating antibodies. Importantly, most class II proteins elicited IFN-γ responses that were significantly stronger than those produced by previously identified antigens. Among them, two class II proteins, ML0308 and ML2498, showed marked humoral and cellular immunogenicity, therefore providing promising candidates for the diagnosis of both tuberculoid and lepromatous forms of leprosy.

Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl- myo -inositols (PIM 6 ) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal α-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM 6 . We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.

International audience; Type III secretion (T3S) systems play key roles in pathogenicity of many Gram-negative bacteria and are employed to inject toxins directly into the cytoplasm of target cells. They are composed of over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which toxins travel. The PscF protein is the main component of the Pseudomonas aeruginosa T3S needle. Here we demonstrate that PscF, when purified on its own, is able to form needle-like fibers of 8 nm in width and >1 microm in length. In addition, we demonstrate for the first time that the T3S needle subunit requires two cytoplasmic partners, PscE and PscG, in P. aeruginosa, which trap PscF in a ternary, 1:1:1 complex, thus blocking it in a monomeric state. Knock-out mutants deficient in PscE and PscG are non-cytotoxic, lack PscF, and are unable to export PscF encoded extrachromosomally. Temperature-scanning circular dichroism measurements show that the PscE-PscF-PscG complex is thermally stable and displays a cooperative unfolding/refolding pattern. Thus, PscE and PscG prevent PscF from polymerizing prematurely in the P. aeruginosa cytoplasm and keep it in a secretion prone conformation, strategies which may be shared by other pathogens that employ the T3S system for infection.

The availability of genome sequence offers the opportunity to further expand our knowledge about proteins expressed by Rickettsia conorii, strictly intracellular bacterium responsible for Mediterranean spotted fever. Using two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF mass spectrometry, we established the first reference map of R. conorii proteome. This approach also allowed identification of GroEL as the major antigen recognized by rabbit serum and sera of infected patients. Altogether, this work opens the way to characterize the proteome of R. conorii, to compare protein profiles of different isolates or of bacteria maintained under different experimental conditions and to identify immunogenic proteins as potential vaccine targets.

Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis . It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.

International audience; Phosphorylcholine (ChoP) is commonly expressed at the surface of pathogens of the respiratory tract, including Streptococcus pneumoniae and Neisseria meningitidis. We designed a synthetic hapten comprising ChoP and part of its native carrier structure in S. pneumoniae, i.e. N-acetyl-D-galactosamine (GalNAc). Protein conjugates of this hapten induced GalNAc-ChoP-specific antibodies which recognized ChoP on both S. pneumoniae and N. meningitidis. GalNAc-ChoP could therefore lead to the rational design of a novel multipurpose vaccine against respiratory infections.

There is a considerable body of evidence supporting the role of secretory type II-A phospholipase A2 (sPLA2-IIA) as an effector of the innate immune response. This enzyme also exhibits bactericidal activity especially toward Gram-positive bacteria. In this study we examined the ability of sPLA2-IIA to kill Bacillus anthracis, the etiological agent of anthrax. Our results show that both germinated B. anthracis spores and encapsulated bacilli were sensitive to the bactericidal activity of recombinant sPLA2-IIA in vitro. In contrast, nongerminated spores were resistant. This bactericidal effect was correlated to the ability of sPLA2-IIA to hydrolyze bacterial membrane phospholipids. Guinea pig alveolar macrophages, the major source of sPLA2-IIA in an experimental model of acute lung injury, released enough sPLA2-IIA to kill extracellular B. anthracis. The production of sPLA2-IIA was significantly inhibited by B. anthracis lethal toxin. Human bronchoalveolar lavage fluids from acute respiratory distress syndrome patients are known to contain sPLA2-IIA; bactericidal activity against B. anthracis was detected in a high percentage of these samples. This anthracidal activity was correlated to the levels of sPLA2-IIA and was abolished by an sPLA2-IIA inhibitor. These results suggest that sPLA2-IIA may play a role in innate host defense against B. anthracis infection and that lethal toxin may help the bacteria to escape from the bactericidal action of sPLA2-IIA by inhibiting the production of this enzyme.

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release interferon γ, efficiently recognize M. tuberculosis–infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

International audience; Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens and the molecular basis of its virulence remains poorly understood. Here, we review our current knowledge about the structure and biosynthesis of the mycobacterial cell-wall lipoglycans, lipoarabinomannans (LAM). LAM are ubiquitous of mycobacteria and appear as the most potent non-peptidic molecules to modulate the host immune response. Nevertheless, LAM structure differs according to the mycobacterial species and three types of LAM have been described: mannose-capped LAM (ManLAM), phospho-myo-inositol-capped LAM (PILAM) and non-capped LAM (AraLAM). The type of capping is a major structural feature determining the ability of LAM to modulate the immune response. ManLAM, found in slow-growing mycobacteria, such as M. tuberculosis, have been demonstrated to be powerful anti-inflammatory molecules and emerge as key virulence factors that may be relevant drug targets. LAM-like molecules are not only confined to mycobacteria but are also present in actinomycetes (including the genera Rhodococcus, Corynebacterium or Gordonia). This offers the possibility of comparative studies that should help in deciphering the structure-function relationships and biosynthesis of these complex molecules in the future.

The virulence genotype profile and presence of a pathogenicity island(s) (PAI) were studied in 18 strains of F165-positive Escherichia coli originally isolated from diseased calves or piglets. On the basis of their adhesion phenotypes and genotypes, these extraintestinal pathogenic strains were classified into three groups. The F165 fimbrial complex consists of at least two serologically and genetically distinct fimbriae: F165 1 and F165 2 . F165 1 is encoded by the foo operon ( pap -like), and F165 2 is encoded by fot ( sfa related). Strains in group 1 were foo and fot positive, strains in group 2 were foo and afa positive, and strains in group 3 were foo positive only. The strains were tested for the presence of virulence genes found mainly in extraintestinal pathogenic E. coli (ExPEC) strains. Although all the strains were positive for the papA variant encoding F11 fimbriae incD , traT , and papC , the prevalence of virulence genes commonly found in PAIs associated with ExPEC strains was highly variable, with strains of group 2 harboring most of the virulence genes tested. papG allele III was detected in all strains in group 1 and in one strain in group 3. All other strains were negative for the known alleles encoding PapG adhesins. The association of virulence genes with tRNA genes was characterized in these strains by using pulsed-field gel electrophoresis and DNA hybridization. The insertion site of the foo operon was found at the pheU tRNA locus in 16 of the 18 strains and at the selC tRNA locus in the other 2 strains. Furthermore, 8 of the 18 strains harbored a high-pathogenicity island which was inserted in either the asnT or the asnV / U tRNA locus. These results suggest the presence of one or more PAIs in septicemic strains from animals and the association of the foo operon with at least one of these islands. F165-positive strains share certain virulence traits with ExPEC, and most of them are pathogenic in piglets, as tested in experimental infections.

The Ysc-Yop type III secretion (TTS) system allows extracellular Yersinia bacteria, adhering to eukaryotic target cells, to inject Yop effector proteins in the cytosol of these cells. The secretion apparatus, called the injectisome, ends up with a needle-like structure made of YscF. YopN, one of the proteins secreted by the injectisome is thought to act as a plug. YopB, YopD and LcrV, three other proteins secreted by the injectisome and called 'translocators' form a pore allowing translocation of the Yop effectors across the target cell plasma membrane. Here, we tested the role of LcrV, YscF and YopN in the formation of this pore in macrophages by monitoring the release of the low-molecular-weight fluorescent dye BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, 623Da) and of the high-molecular-weight lactate dehydrogenase (LDH, 135 kDa). BCECF is released through the translocation pore itself provided no Yop effector is trafficking through the channel. In contrast, LDH is released by the osmotic lysis of the target cell that occurs after pore formation. This release is reduced by the GAP activity of YopE. In order to study the role of LcrV, one has to circumvent the regulatory effect of LcrV on the synthesis of YopB and YopD. We observed here that this regulatory role of LcrV is lost in a yopQ mutant and hence we studied the role of LcrV in a yopQ mutant background. A lcrV, yopQ double mutant was deficient in pore formation while able to produce YopB and YopD. Pore formation was restored by the introduction of lcrV(+) but not yopQ(+) confirming that LcrV itself is directly required for pore formation. Bacteria secreting only YopB, YopD and LcrV could form pores, showing that YopB, YopD and LcrV are sufficient for pore formation provided they are secreted by the same bacterium. LcrV is not involved in secretion of YopB and YopD as suggested previously. Bacteria producing normal Ysc injectisomes, including the YscF needle but no translocators did not form pores, indicating that the needle is not sufficient by itself for pore formation, as was also suggested. yopN mutant bacteria formed needles and released BCECF even if they secreted the effectors. This observation suggests that many translocation pores are not filled in the absence of YopN and thus that YopN might form a link between the needle and the pore, guiding the effectors.