Search

Mycobacterium tuberculosis and its close relative Mycobacterium marinum infect macrophages and induce the formation of granulomas, organized macrophage-rich immune aggregates. These mycobacterial pathogens can accelerate and co-opt granuloma formation for their benefit, using the specialized secretion system ESX-1, a key virulence determinant. ESX-1-mediated virulence is attributed to the damage it causes to the membranes of macrophage phagosomal compartments, within which the bacteria reside. This phagosomal damage, in turn, has been attributed to the membranolytic activity of ESAT-6, the major secreted substrate of ESX-1. However, mutations that perturb ESAT- 6’s membranolytic activity often result in global impairment of ESX-1 secretion. This has precluded an understanding of the causal and mechanistic relationships between ESAT-6 membranolysis and ESX-1-mediated virulence. Here, we identify two conserved residues in the unstructured C-terminal tail of ESAT-6 required for phagosomal damage, granuloma formation and virulence. Importantly, these ESAT-6 mutants have near- normal levels of secretion, far higher than the minimal threshold we establish is needed for ESX-1-mediated virulence early in infection. Unexpectedly, these loss-of-function ESAT-6 mutants retain the ability to lyse acidified liposomes. Thus, ESAT-6’s virulence functions in vivo can be uncoupled from this in vitro surrogate assay. These uncoupling mutants highlight an enigmatic functional domain of ESAT-6 and provide key tools to investigate the mechanism of phagosomal damage and virulence.Significance StatementTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis, and its close pathogenic relative Mycobacterium marinum, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes.Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C-terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.

International audience; Background: The multicomponent meningococcal serogroup B vaccine (4CMenB) is currently indicated for active immunization against invasive meningococcal disease caused by Neisseria meningitidis serogroup B (MenB). However, genes encoding the 4CMenB antigens are also variably present and expressed in strains belonging to other meningococcal serogroups. In this study, we evaluated the ability of antibodies raised by 4CMenB immunisation to induce complement-mediated bactericidal killing of non-MenB strains. Methods: A total of 227 invasive non-MenB disease isolates were collected between 1 July 2007 and 30 June 2008 from England and Wales, France, and Germany; 41 isolates were collected during 2012 from Brazil. The isolates were subjected to genotypic analyses. A subset of 147 isolates (MenC, MenW and MenY) representative of the meningococcal genetic diversity of the total sample were tested in the human complement serum bactericidal antibody assay (hSBA) using sera from infants immunised with 4CMenB. Results: Serogroup and clonal complex repertoires of non-MenB isolates were different for each country. For the European panel, MenC, MenW and MenY isolates belonged mainly to ST-11, ST-22 and ST-23 complexes, respectively. For the Brazilian panel, most MenC and MenW isolates belonged to the ST-103 and ST-11 complexes, respectively, and most MenY isolates were not assigned to clonal complexes. Of the 147 non-MenB isolates, 109 were killed in hSBA, resulting in an overall coverage of 74%. Conclusion: This is the first study in which 147 non-MenB serogroup isolates have been analysed in hSBA to evaluate the potential of a MenB vaccine to cover strains belonging to other serogroups. These data demonstrate that antibodies raised by 4CMenB are able to induce bactericidal killing of 109 non-MenB isolates, representative of non-MenB genetic and geographic diversity. These findings support previous evidence that 4CMenB immunisation can provide cross-protection against non-MenB strains in infants, which represents an added benefit of 4CMenB vaccination.

International audience; Neisseria meningitidis serogroup B (MenB) has recently become the major cause of invasive meningococcal disease in Poland. Therefore, the purpose of this study was to characterize MenB isolates, responsible for invasive meningococcal disease in 2010-2016, by MLST and sequencing of genes encoding proteins used as 4CMenB vaccine antigens. Two methods of coverage estimation were performed: extrapolation of MATS results of Polish meningococci 2010-2011 (exMATS) and gMATS, which combines genotyping and MATS results. Among 662 isolates 20 clonal complexes (CC) were detected, of which the most frequent were CC32, CC41/44 and CC18, accounting for 31.9%, 16.5% and 12.7%, respectively. A total of 111 combinations of PorA variable regions (VR1/VR2) were found, with P1.7,16 (15.0%) and P1.22,14 (13.6%) being prevalent. Vaccine variant VR2:4 was detected in 7.3% of isolates, mainly representing CC41/44 and non-assigned CC. Eighty five fHbp alleles encoding 74 peptide subvariants were revealed. Subvariant 1.1, a component of 4CMenB, was prevalent (24.2%) and found generally in CC32. Typing of the nhba gene revealed 102 alleles encoding 87 peptides. The most frequent was peptide 3 (22.4%), whereas vaccine peptide 2 was detected in 9.8%, mostly among CC41/44. The nadA gene was detected in 34.0% of isolates and the most prevalent was peptide 1 (variant NadA-1; 71.6%), found almost exclusively in CC32 meningococci. Vaccine peptide 8 (variant NadA-2/3) was identified once. Consequently, 292 completed BAST profiles were revealed. Regarding vaccine coverage, 39.7% of isolates had at least one 4CMenB vaccine variant, but according to exMATS and gMATS the coverage was 83.3% and 86.6%, respectively. In conclusion, Polish MenB (2010-2016) was highly diverse according to MLST and gene alleles encoding 4CMenB vaccine antigens. Some correlations between clonal complexes and variants of examined proteins/BAST profiles were revealed and a high coverage of 4CMenB vaccine was estimated.

International audience; Invasive meningococcal disease (IMD), caused by Neisseria meningitidis (Nm) strains, is a life-threatening but vaccine-preventable condition. Bexsero is a four-component vaccine that offers broad protection against Nm of serogroup B (NmB), particularly common in Europe. In Switzerland, Bexsero has not yet been licensed and no information is available concerning the predicted vaccine coverage on isolates of circulating Nm. We performed genotyping of Bexsero antigen loci by whole-genome sequencing (WGS) on 104 NmB collected in Switzerland in the 2010-2015 period. We searched for antigen variants previously defined as predictors of strain coverage and estimated that 50% of IMD NmB strains were potentially covered by the vaccine. Clonal complexes (cc) 32, 41/44 and 269, considered the best covered lineages, were further sub-typed according to Bexsero Antigen Sequence Type (BAST) scheme. We also genotyped by WGS 40 Nm of serogroup W (NmW) collected in the country between 2010 and 2016. NmW cc22 isolates appeared to be covered by the vaccine, which was not the case for cc11 isolates, whose incidence has recently increased in Switzerland and all over Europe. Our work underlines the benefit of using WGS for surveillance of vaccine antigen variant distribution in local Nm population and taking proper measures to prevent the spread of NmB.

The genomic sequence data generated in this work were submitted to the European Nucleotide Archive and are available from the International Nucleotide Sequence Database Collaboration (NCBI/ENA/DDBJ) databases under project accession number PRJEB27412 and run data accession numbers ERS2572942 to ERS2572951.; International audience; The recent increase in pertussis cases observed in some countries may have several causes, including the evolution of Bordetella pertussis populations towards escape of vaccine-induced immunity. Most genomic studies of B. pertussis isolates performed so far are from countries that use acellular vaccines. The objective was to analyse genomic sequences of isolates collected during the 2014 whooping cough epidemic in Tunisia, a country where whole-cell vaccines are used. Ten Tunisian isolates and four vaccine strains were sequenced and compared to 169 isolates from countries where acellular vaccines are used. Phylogenetic analysis showed that Tunisian isolates are diverse, demonstrating a multi-strain 2014 epidemic peak, and are intermixed with those circulating in other world regions, showing inter-country transmission. Consistently, Tunisian isolates have antigen variant composition observed in other world regions. No pertactin-deficient strain was observed. The Tunisian B. pertussis population appears to be largely connected with populations from other countries.

Inhalational anthrax caused by Bacillus anthracis, a spore-forming Gram-positive bacterium, is a highly lethal infection. Antibodies targeting the protective antigen (PA) binding component of the toxins have recently been authorized as an adjunct to antibiotics, although no conclusive evidence demonstrates that anthrax antitoxin therapy has any significant benefit. We discuss here the rational basis of anti-PA development regarding the pathogenesis of the disease., Inhalational anthrax caused by Bacillus anthracis, a spore-forming Gram-positive bacterium, is a highly lethal infection. Antibodies targeting the protective antigen (PA) binding component of the toxins have recently been authorized as an adjunct to antibiotics, although no conclusive evidence demonstrates that anthrax antitoxin therapy has any significant benefit. We discuss here the rational basis of anti-PA development regarding the pathogenesis of the disease. We argue that inductive reasoning may induce therapeutic bias. We identified anthrax animal model analysis as another bias. Further studies are needed to assess the benefit of anti-PA antibodies in the treatment of inhalational anthrax, while a clearer consensus should be established around what evidence should be proven in an anthrax model.

Background Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. Methods To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. Results One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. Conclusions In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax., The enzymatically active components of B anthracis toxins and bacteria can diffuse very early in the blood during inhalational anthrax model, paving the way for an ultra-precocious diagnosis and the setup of an early and effective treatment.

International audience; Shigella are gram-negative bacteria that cause severe diarrhea and dysentery, with a high level of antimicrobial resistance. Disease-induced protection against reinfection in Shigella-endemic areas provides convincing evidence on the feasibility of a vaccine and on the importance of Shigella lipopolysaccharides as targets of the host humoral protective immune response against disease. This article provides an overview of the original and current strategies toward the development of a Shigella glycan-protein conjugate vaccine that would cover the most commonly detected strains. Going beyond pioneering “lattice”-type polysaccharide-protein conjugates, progress, and challenges are addressed with focus on promising alternatives, which have reached phases I and II clinical trial. Glycoengineered bioconjugates and “sun”-type conjugates featuring well-defined synthetic carbohydrate antigens are discussed with insights on the molecular parameters governing the rational design of a cost-effective glycoconjugate vaccine efficacious in preventing diseases caused by Shigella in the most at risk populations, young children living in endemic areas.

Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation., Author summary One of the major findings of the pioneering Mycobacterium tuberculosis H37Rv genome sequencing project was the identification of the highly abundant PE and PPE proteins, named after their N-terminal motifs Pro–Glu (PE) or Pro–Pro–Glu (PPE). Within the 20 years of research since then, many claims were made that PE/PPE proteins, including the two large subgroups encoded by repetitive sequences with very high GC content (PE_PGRS and PPE-MPTR families), are exported to the bacterial surface or beyond, and show broad immunomodulatory impact on host-pathogen interaction. We thus screened strains from different branches of the M. tuberculosis complex, including the attenuated Mycobacterium bovis BCG vaccine strains, for their capacity to export PE_PGRS/PPE-MPTR proteins. Strikingly, we found that BCG strains were unable to export the plethora of PE_PGRS/PPE-MPTR proteins due to the absence of the region of difference RD5, which in M. tuberculosis encodes PPE38, required for PE_PGRS/PPE-MPTR export. Surprisingly, the restoration of PE_PGRS/PPE-MPTR export by genetic complementation in recombinant BCG did not result in immunomodulatory changes or altered protection in mouse models. Our results thus put into perspective the numerous reports on virulence-associated immunomodulatory impact of individual PE_PGRS and PPE-MPTR proteins and open novel questions on their biological function(s).

Recent studies highlight that bacterial pathogens can reprogram target cells by influencing epigenetic factors. The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS ‘needle’ delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol. Here we show that Pseudomonas aeruginosa induces early T3SS-dependent dephosphorylation and deacetylation of histone H3 in eukaryotic cells. This is not triggered by any of the P. aeruginosa T3SS effectors, but results from the insertion of the PopB–PopD translocon into the membrane. This suggests that the P. aeruginosa translocon is a genuine T3SS effector acting as a pore-forming toxin. We visualized the translocon plugged into the host cell membrane after the bacterium has left the site of contact, and demonstrate that subsequent ion exchange through this pore is responsible for histone H3 modifications and host cell subversion. The pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon serves as a virulence factor to induce host epigenome modification and promote infection.

Background Capsular group X N. meningitidis (MenX) has emerged as a cause of localized disease outbreaks in sub-Saharan Africa, but the human immune response following exposure to MenX antigens is poorly described. We therefore assessed the natural immunity against MenX in individuals who were living in an area affected by a MenX outbreak during 2007 in Togo, West Africa. During 2009, 300 healthy individuals (100 aged 3–5 years, 100 aged 13–19 years and 100 aged 20–25 years) were included in the study, and serum responses were compared with sera from age-matched controls from the U.K. and Burkina Faso. Methods MenX serum bactericidal antibody (SBA) was measured using rabbit complement, and antibodies against MenX polysaccharide (XPS) and outer membrane vesicles (XOMVs) were quantified by ELISA. Results The proportion of Togolese individuals with an SBA titer of ≥8 against the MenX strain was 29% (95% confidence interval (CI) 18–41) among those aged 3–5 years, 34% (95% CI 9–60) among those aged 13–19 years and 32% (95% CI 24–40) among those aged 20–25 years. These were significantly higher than observed in the control populations from the U.K (range 13–16%) and Burkina Faso (range 2–6%). Conclusion In Togolese individuals, the concentration of serum IgG against XPS was higher among the two older age groups as compared to the youngest age group. Antibody concentrations against MenX PS correlated significantly with SBA titers. This supports further development of a MenX PS based conjugate vaccine. Further studies are needed to verify the ability of MenX PS to induce SBA in humans.

International audience; We previously reported a shift in the electrophoretic type (ET) of invasive MenC in Canada from predominantly ET-15 to ET-37 in the post-MenC conjugate vaccine period. This study sought to confirm this trend by examining all culture-confirmed invasive MenC case isolates in Canada in the period from 1 January 2009 to 31 December 2013. Of the 50 MenC isolates, 18 belonged to ET-15, 28 belonged to ET-37 (but not ET-15), and four belonged to other clonal types. Analysis of the serotype and serosubtype antigens, porA and fetA gene sequences provided data to show that invasive MenC belonging to ET-15 and ET-37 were two very different subpopulations within the ST-11 clonal complex. Sequence analysis of the fHbp genes suggested that 12 different types of factor H-binding protein were found among the ET-15 isolates while 86 % of ET-37 isolates were found to have fHbp genes predicted to encode peptide 22. The nadA gene in 12 MenC isolates was disrupted due to IS1301 insertion and 11 of these 12 isolates belonged to ET-15. Ten per cent of the invasive MenC were found to have a frame-shift mutation in their fHbp genes that predicted no fHbp produced. Significant diversity and frame-shift mutations of fHbp genes were found in invasive MenC strains in Canada.

Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses., Author summary Leptospirosis is a widespread zoonosis caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). L. interrogans are primarily extracellular pathogens although some reports suggest they may replicate within macrophages. In humans, leptospirosis can cause mild or severe disease, potentially leading to death, although rats or mice, which constitute the reservoir, are asymptomatic carriers. Host defense mechanisms involved in leptospirosis remain poorly understood. Toll-Like Receptor (TLR)2 and TLR4 are crucial for the clearance of L. interrogans, but the role of the cytosolic NOD receptors in leptospirosis is unknown. Here, we report that pathogenic leptospires escape the sensing of bacterial peptidoglycan through the NOD response. We found that an outer membrane lipoprotein of L. interrogans binds to and protects the peptidoglycan from degradation into muropeptides, thereby blocking signaling through NOD proteins. Moreover, in absence of this lipoprotein, the peptidoglycan of L. interrogans is properly sensed by human NOD1 but not by murine NOD1. This is due to the near absence of muramyl tetrapeptide, the murine NOD1 agonist, in the peptidoglycan of pathogenic leptospires. These novel mechanisms of NOD avoidance may facilitate the escape of leptospires from the innate immune system of their hosts.

Item does not contain fulltext Streptococcus gallolyticus subspecies gallolyticus (SGG) is potentially associated with colorectal cancer (CRC) and its precursors. A previous case-control study measured antibody responses to SGG pilus proteins Gallo2178 and Gallo2179 and identified significant associations with a small fraction of CRC cases. We aimed at replicating and expanding these findings in an independent study including additional SGG antigens and explored the association with precancerous lesions. We applied multiplex serology to measure antibodies to eleven SGG proteins in serum samples of a screening colonoscopy trial (BliTz study) including participants diagnosed with either non-advanced adenoma (NAA, n = 30), advanced adenoma (AA, n = 100), CRC (n = 50) or controls (n = 228). In addition, we analyzed CRC samples (n = 318) from patients recruited in a clinical setting (DACHSplus study). The association of antibody responses to SGG pilus proteins Gallo2178 and Gallo2179 with CRC was replicated with 4% positive DACHSplus cases compared to 0% positive BliTz controls. Positivity to two or more proteins of a newly defined panel of six SGG markers was significantly associated with CRC in the DACHSplus study (OR: 1.81, 95% CI: 1.07-3.06). Odds for CRC, AA and NAA in the BliTz study were also increased with antibody responses to SGG, and the association was significant for NAA (OR: 2.98, 95% CI: 1.18-7.57). Antibody responses to SGG are associated with CRC and its precursors. The newly identified SGG six-marker panel and associations found with precancerous lesions should be further explored.

Leprosy is a human infectious disease caused by Mycobacterium leprae. A strong host genetic contribution to leprosy susceptibility is well established. However, the modulation of the transcriptional response to infection and the mechanism(s) of disease control are poorly understood. To address this gap in knowledge of leprosy pathogenicity, we conducted a genome-wide search for expression quantitative trait loci (eQTL) that are associated with transcript variation before and after stimulation with M. leprae sonicate in whole blood cells. We show that M. leprae antigen stimulation mainly triggered the upregulation of immune related genes and that a substantial proportion of the differential gene expression is genetically controlled. Indeed, using stringent criteria, we identified 318 genes displaying cis-eQTL at an FDR of 0.01, including 66 genes displaying response-eQTL (reQTL), i.e. cis-eQTL that showed significant evidence for interaction with the M. leprae stimulus. Such reQTL correspond to regulatory variations that affect the interaction between human whole blood cells and M. leprae sonicate and, thus, likely between the human host and M. leprae bacilli. We found that reQTL were significantly enriched among binding sites of transcription factors that are activated in response to infection, and that they were enriched among single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy per se and Type-I Reaction, and seven of them have been targeted by recent positive selection. Our study suggested that natural selection shaped our genomic diversity to face pathogen exposure including M. leprae infection., Author summary Each year, 200,000 new leprosy cases are reported worldwide. While there is unambiguous evidence for a role of host genetics in leprosy pathogenesis, the mechanisms by which the human host fights the infection are poorly understood. Here, we highlight the search for naturally occurring genetic variations that modulate gene expression levels following exposure to sonicate of Mycobacterium leprae, the bacterium causing the disease. Because M. leprae is not cultivable and the genuine immune cells involved in the host response during infection are still unknown, we performed a genome-wide search for such genetic variations after stimulation of whole-blood from leprosy patients with M. leprae sonicate. This design allowed to provide a general framework for the genetic control of host responses to M. leprae and outlined the contribution of host genetics to leprosy pathogenesis. Among the M. leprae-dependent genetic regulators of gene expression levels there was an enrichment of variants (i) associated with leprosy, (ii) located in transcription factor binding sites and (iii) targeted by recent positive selection.

International audience; Tuberculosis is a worldwide disease considered as a major health problem with high morbidity and mortality rates. Poor detection of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis remains a major obstacle to the global control of this disease. Here we report the development of a new test based on the detection of the major virulent factor of Mtb, namely the early secreted antigenic target 6-kDa protein or ESAT-6. A label free electrochemical immunosensor using an anti-ESAT-6 monoclonal antibody as a bio-receptor is described herein. Anti-ESAT-6 antibodies were first covalently immobilized on the surface of a gold screen-printed electrode functionalized via a self-assembled thiol monolayer. Interaction between the bio-receptor and ESAT-6 antigen was evaluated by square wave voltammetry method using [Fe(CN)6](3-/4-) as redox probe. The detection limit of ESAT-6 antigen was 7ng/ml. The immunosensor has also been able to detect native ESAT-6 antigen secreted in cell culture filtrates of three pathogenic strains of Mtb (CDC1551, H37RV and H8N8). Overall, this work describes an immune-electrochemical biosensor, based on ESAT-6 antigen detection, as a useful diagnostic tool for tuberculosis.

International audience; Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an efficacious pneumococcal protein vaccine are universal presence and minimal variation of the target antigen in the pneumococcal population, and the capability to induce a robust human immune response. We used in silico analysis to assess the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries, across four continents. CbpA (76%), PspA (68%), PhtD (28%), PcpA (11%) were not universally encoded in the study population, and would not provide full coverage against serotype 1. PcpA was widely present in the European (82%), but not in the African (2%) population. A multi-valent vaccine incorporating CbpA, PcpA, PhtD and PspA was predicted to provide coverage against 86% of the global population. SP0148, SP1912 and SP2108 were universally encoded and we further assessed their predicted amino acid, antigenic and structural variation. Multiple allelic variants of these proteins were identified, different allelic variants dominated in different continents; the observed variation was predicted to impact the antigenicity and structure of two SP0148 variants, one SP1912 variant and four SP2108 variants, however these variants were each only present in a small fraction of the global population (

International audience; Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH.; A selection of 34 peptides, from the MiDLDH and LcDLDH, able to interact with Major Histocompatibility Complex (MHC) 1 and MHC 2, was obtained using three different epitope prediction websites. By means of ELISPOT assays, we compared the frequency of Interferon gamma (IFNγ) secreting peripheral blood mononuclear cells (PBMC) after stimulation with overlapping peptide pools. Tests were performed using cells from 35 healthy blood donors.; One peptide pool containing five peptides from MiDLDH and able to interact with MHC 2 induced a marked IFNγ specific immune response (Pool F, p

International audience; Despite the recent description of the mucosal vaccine-induced reduction of Helicobacter pylori natural infection in a phase 3 clinical trial, the absence of immune correlates of protection slows the final development of the vaccine. In this study, we evaluated the role of interleukin (IL)-22 in mucosal vaccine-induced protection. Gastric IL-22 levels were increased in mice intranasally immunized with urease þ cholera toxin and challenged with H. felis, as compared with controls. Flow cytometry analysis showed that a peak of CD4 þ IL-22 þ IL-17 þ Tcells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice. The inhibition of the IL-22 biological activity prevented the vaccine-induced reduction of H. pylori infection. Remarkably, anti-microbial peptides (AMPs) extracted from the stomachs of vaccinated mice, but not from the stomachs of non-immunized or immunized mice, injected with anti-IL-22 antibodies efficiently killed H. pylori in vitro. Finally, H. pylori infection in vaccinated RegIIIb-deficient mice was not reduced as efficiently as in wild-type mice. These results demonstrate that IL-22 has a critical role in vaccine-induced protection, by promoting the expression of AMPs, such as RegIIIb, capable of killing Helicobacter. Therefore, it can be concluded that urease-specific memory Th17/Th22 cells could constitute immune correlates of vaccine protection in humans.

The ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors of Mycobacterium tuberculosis , given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates of M. tuberculosis produce and secrete larger amounts of ESAT-6 than the widely used M. tuberculosis H37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed that whiB6 ( rv3862c ), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly available M. tuberculosis complex genomes or in any of the 76 clinical M. tuberculosis isolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulates whiB6 expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy of whiB6 in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that in M. tuberculosis —with the exception of the H37Rv strain—ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.

International audience; Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6110 element upstream of the phoPR locus may completely revert the phoPR-bovis-associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.

International audience; Borrelia persica is a strain seen only in the Middle East and responsible for relapsing fever. These spirochetes are notable for multiphasic antigenic variation of polymorphic outer membrane lipoproteins, a phenomenon responsible for immune evasion. Diagnosis of the disease is a problem and requires a fixed antigen like the flagellar antigen. In vitro culture of B. persica was carried out for the first time and flagellar antigen was purified from culture. 10% SDS was added to the mixture to dissolve the cell wall and then the solution was sheared in an Omni mixer. Electron microscopy confirmed the purity of a 42 KDa periplasmic antigen as revealed by SDS-PAGE. Indirect haemagglutination kits were designed using the pure flagella and tested for cross reactivity with another relapsing fever spirochaete Borrelia microtii positive serum. The kit showed 98% sensitivity and 95% specificity.

Upon priming with Candida albicans or with the fungal cell wall component β-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed “trained immunity.” In contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes.

Summary Background A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. Methods We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. Findings 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63–90, range of point estimates 73–87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. Interpretation MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. Funding Novartis Vaccines and Diagnostics.

International audience; Mycobacterium tuberculosis uses sophisticated secretion systems, named 6 kDa early secretory antigenic target (ESAT6) protein family secretion (ESX) systems (also known as type VII secretion systems), to export a set of effector proteins that helps the pathogen to resist or evade the host immune response. Since the discovery of the esx loci during the M. tuberculosis H37Rv genome project, structural biology, cell biology and evolutionary analyses have advanced our knowledge of the function of these systems. In this Review, we highlight the intriguing roles that these studies have revealed for ESX systems in bacterial survival and pathogenicity during infection with M. tuberculosis. Furthermore, we discuss the diversity of ESX systems that has been described among mycobacteria and selected non-mycobacterial species. Finally, we consider how our knowledge of ESX systems might be applied to the development of novel strategies for the treatment and prevention of disease.

International audience; OBJECTIVES:Bivalent rLP2086 (Trumenba®; MenB-FHbp), composed of two factor H binding proteins (FHbps), is a vaccine approved in the United States for prevention of Neisseria meningitidis serogroup B (MnB) invasive meningococcal disease (IMD). Bactericidal activity of sera from subjects vaccinated with bivalent rLP2086 was assessed against MnB isolates from recent disease outbreaks in France.METHODS:MnB isolates from IMD cases were characterized by whole genome sequencing and FHbp expression was assessed using a flow cytometry-based assay. Sera from subjects (11

Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia ( P −3 ). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC 50 ) of PP were independent of treatment outcomes, in contrast to median mefloquine IC 50 , which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P = 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P = 0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.)

Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens., Author Summary Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is one of the most widely spread human pathogens, responsible for more than 9.6 million of new tuberculosis cases and 1.5 million deaths, annually. The resurgence of pulmonary tuberculosis in immuno-compromised patients, including HIV-co-infected populations, and increasing spread of drug-resistant Mtb strains are worrying. Given the estimated 2 billion cases of latent Mtb infections and the only partial efficacy of the unique, currently available tuberculosis-vaccine Mycobacterium bovis BCG (Bacille Calmette-Guerin) it is necessary to develop improved vaccines. Here, we demonstrate that the host cellular immunity, mediated by CD4+ T lymphocytes, specific to the “PE/PPE” families of mycobacterial antigens, contribute to the protection against Mtb-induced disease. We revealed the fine composition of the PE/PPE-specific T cells by characterizing their effector functions and differentiation status. We previously described a live attenuated mycobacterial strain as a vaccine candidate that is able to induce such CD4+ T cells and which displays particular properties at stimulating the cells of the innate immune system. These responses play a central role in the initiation of the host defense and in the protection against tuberculosis. Our results pave the way for further development of candidates in preclinical models of anti-tuberculosis vaccination.

Edema Factor (EF) is a component of Bacillus anthracis toxin essential for virulence. Its adenylyl cyclase activity is induced by complexation with the ubiquitous eukaryotic cellular protein, calmodulin (CaM). EF and its complexes with CaM, nucleotides and/or ions, have been extensively characterized by X-ray crystallography. Those structural data allowed molecular simulations analysis of various aspects of EF action mechanism, including the delineation of EF and CaM domains through their association energetics, the impact of calcium binding on CaM, and the role of catalytic site ions. Furthermore, a transition path connecting the free inactive form to the CaM-complexed active form of EF was built to model the activation mechanism in an attempt to define an inhibition strategy. The cavities at the surface of EF were determined for each path intermediate to identify potential sites where the binding of a ligand could block activation. A non-catalytic cavity (allosteric) was found to shrink rapidly at early stages of the path and was chosen to perform virtual screening. Amongst 18 compounds selected in silico and tested in an enzymatic assay, 6 thiophen ureidoacid derivatives formed a new family of EF allosteric inhibitors with IC50 as low as 2 micromolars.

International audience; Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli.

International audience; The aim of this study was to investigate the antiproliferative proteins that probably have a role in Helicobacter pylori evade of immune response and cause chronic infection disease and also to see if coccoid form had a role in its chronicity. H. pylori strain VacA s2/m2 positive and CagA negative, from a gastric biopsy of a patient with peptic ulcer disease, was isolated and cultured in brucella agar. Both spiral and coccoid forms were harvested and ruptured by sonication. The cytoplasmic solutions of both forms were collected and their fractions obtained by gel chromatography and preparative polyacrylamide gel electrophoresis. The fractions were analyzed by MTT assay for their antiproliferative activity. We isolated two proteins with a significant dose dependent antiproliferative activity that analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one of them that was urease positive showed two bands with 61 and 27 kDa, which is resumed to urease of H. pylori, another consist of 57 and 63 kDa. Helicobacterpylori secret some proteins like urease that inhibit immune cells proliferation response against its antigens.

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.

International audience; The mammalian innate immune response provides a barrier against invading pathogens. Innate immune mechanisms are used by the host to respond to a range of bacterial pathogens in an acute and conserved fashion. Host cells express pattern recognition receptors that sense pathogen-associated molecular patterns. After detection, an arsenal of antimicrobial mechanisms is deployed to kill bacteria in infected cells. Innate immunity also stimulates antigen-specific responses mediated by the adaptive immune system. In response, pathogens manipulate host defence mechanisms to survive and eventually replicate. This Review focuses on the control of host innate immune responses by pathogenic intracellular bacteria.

International audience; Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming the identification of secreted proteins that are differently expressed at 30 degrees C versus 37 degrees C and at mid-exponential versus early-stationary growth phase and antigenic proteins from R. equi ATCC 33701. A total of 48 proteins was identified regardless of growth conditions. The cholesterol oxidase ChoE appears to be the major secretory protein. Moreover, four proteins revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. The sequence analysis predicted that 24 proteins are transported by a signal peptide-dependent pathway. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate for developing vaccines against R. equi infection in horse.

pas de résumé

International audience; Neisseria meningitidis of serogroup B (MenB) is currently responsible for more than 70% of cases of invasive meningococcal disease (IMD) in Poland and Europe as a whole. The aim of this study was to estimate strain coverage of a multicomponent meningococcal serogroup B vaccine (4CMenB) in Poland; the meningococcal antigen typing system (MATS) was used to test a panel of 196 invasive MenB strains isolated in Poland in 2010 and 2011. The strains were also characterized by MLST and sequencing of porA, factor H-binding protein (fHbp), Neisserial heparin-binding antigen (nhba) and Neisserial adhesin A (nadA) genes. MATS and molecular data were analyzed independently and in combination. The MATS results predicted that 83.7% (95% CI: 78.6-91.0%) of isolates would be covered by the 4CMenB vaccine; 59.2% by one vaccine antigen, 19.9% by two and 4.6% by three antigens. Coverage by each antigen was as follows: fHbp 73.0% (95% CI: 68.9-77.5%), NHBA 28.6% (95% CI: 13.3-47.4%), NadA 1.0% (95% CI: 1.0-2.0%) and PorA 10.2%. Molecular analysis revealed that the most frequent clonal complexes (ccs) were cc32 (33.2%), cc18 (17.9%) and cc41/44 (15.8%) with estimated coverage of 98.5%, 88.6% and 93.5%, respectively. Consistent with findings for other European countries, our study predicts high coverage by the 4CMenB vaccine in Poland.

The emergence of Neisseria meningitidis serogroup X (NmX) in the African meningitis belt has urged the development of diagnostic tools and vaccines for this serogroup, especially following the introduction of a conjugate vaccine against N. meningitidis serogroup A (NmA). We have developed and evaluated a new rapid diagnostic test (RDT) for detecting the capsular polysaccharide (cps) antigen of this emerging serogroup. Whole inactivated NmX bacteria were used to immunize rabbits. Following purification by affinity chromatography, the cpsX-specific IgG antibodies were utilized to develop an NmX-specific immunochromatography dipstick RDT. The test was validated against purified cpsX and meningococcal strains of different serogroups. Its performance was evaluated against that of PCR on a collection of 369 cerebrospinal fluid (CSF) samples obtained from patients living in countries within the meningitis belt (Cameroon, Côte d'Ivoire, and Niger) or in France. The RDT was highly specific for NmX strains. Cutoffs of 10 5 CFU/ml and 1 ng/ml were observed for the reference NmX strain and purified cpsX, respectively. Sensitivity and specificity were 100% and 94%, respectively. A high agreement between PCR and RDT (Kappa coefficient, 0.98) was observed. The RDT gave a high positive likelihood ratio and a low negative likelihood (0.07), indicating almost 100% probability of declaring disease or not when the test is positive or negative, respectively. This unique NmX-specific test could be added to the available set of RDT for the detection of meningococcal meningitis in Africa as a major tool to reinforce epidemiological surveillance after the introduction of the NmA conjugate vaccine.

PubMedID: 25724123 Background Mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency (CID) also classified as autosomal recessive (AR) hyper-IgE syndrome (HIES). Recognizing patients with CID/HIES is of clinical importance because of the difference in prognosis and management. Objectives We sought to define the clinical features that distinguish DOCK8 deficiency from other forms of HIES and CIDs, study the mutational spectrum of DOCK8 deficiency, and report on the frequency of specific clinical findings. Methods Eighty-two patients from 60 families with CID and the phenotype of AR-HIES with (64 patients) and without (18 patients) DOCK8 mutations were studied. Support vector machines were used to compare clinical data from 35 patients with DOCK8 deficiency with those from 10 patients with AR-HIES without a DOCK8 mutation and 64 patients with signal transducer and activator of transcription 3 (STAT3) mutations. Results DOCK8-deficient patients had median IgE levels of 5201 IU, high eosinophil levels of usually at least 800/µL (92% of patients), and low IgM levels (62%). About 20% of patients were lymphopenic, mainly because of low CD4+ and CD8+ T-cell counts. Fewer than half of the patients tested produced normal specific antibody responses to recall antigens. Bacterial (84%), viral (78%), and fungal (70%) infections were frequently observed. Skin abscesses (60%) and allergies (73%) were common clinical problems. In contrast to STAT3 deficiency, there were few pneumatoceles, bone fractures, and teething problems. Mortality was high (34%). A combination of 5 clinical features was helpful in distinguishing patients with DOCK8 mutations from those with STAT3 mutations. Conclusions DOCK8 deficiency is likely in patients with severe viral infections, allergies, and/or low IgM levels who have a diagnosis of HIES plus hypereosinophilia and upper respiratory tract infections in the absence of parenchymal lung abnormalities, retained primary teeth, and minimal trauma fractures. © 2015 American Academy of Allergy, Asthma & Immunology. National Institutes of Health: R01AI065617 National Institutes of Health: R21AI087627

International audience; Pathogenesis of Mycobacterium tuberculosis depends on the secretion of key virulence factors, such as the 6 kDa early secreted antigenic target ESAT-6 (EsxA) and its protein partner, the 10 kDa culture filtrate protein CFP-10 (EsxB), via the ESX-1 secretion system. ESX-1 represents the prototype system of the recently named type VII secretion systems that exist in a range of actinobacteria. The M. tuberculosis genome harbours a total of five gene clusters potentially coding for type VII secretion systems, designated ESX-1 - ESX-5, with ESX-4 being the most ancient system from which other ESX systems seem to have evolved by gene duplication and gene insertion events. The five ESX systems show similarity in gene content and gene order but differ in function. ESX-1 and ESX-5 are both crucial virulence determinants of M. tuberculosis, but with different mechanisms. While ESX-1 is implicated in the lysis of the host cell phagosomes, ESX-5 is involved in secretion of the mycobacteria specific PE and PPE proteins and cell wall stability. Research on type VII secretion systems has thus become a large and competitive research topic that is tightly linked to studies of host-pathogen interaction of pathogenic mycobacteria. Insights into this matter are of relevance for redrawing the patho-evolution of M. tuberculosis, which might help improving current strategies for prevention, diagnostics and therapy of tuberculosis as well as elucidating the virulence mechanisms employed by this important human pathogen.

Human immunodeficiency virus/simian immunodeficiency virus (SIV) diversification is a direct consequence of viral replication and occurs principally in secondary lymphoid organs where CD4 + T cells are activated and proliferate. However, the evolution of viral quasispecies may also be driven by various nonexclusive mechanisms, including adaptation to specific immune responses and modification of viral fitness. Analysis of viral quasispecies in SIV-infected macaques subjected to repeated antigenic stimulations allowed us to demonstrate transient expansions of SIV populations that were highly dependent upon activation of antigen-specific T cells. T-cell clones expanded in response to a particular antigen were infected by a specific viral population and persisted for prolonged periods. Upon a second stimulation by encounter with the same antigen, these specific genomes were at the origin of a new burst of replication, leading to rapid but transient replacement of the viral quasispecies in blood. Finally, longitudinal analysis of SIV sequence variation during and between antigenic stimulations revealed that viral evolution is mostly constrained to periods of strong immunological activity.

International audience; The molecular bases of Mycobacterium tuberculosis pathogenicity remain unclear. We report here how M. tuberculosis mannosylated lipoarabinomannans contribute to the survival of bacilli in the human reservoir by (i) inhibiting IL-12 production by macrophages and dendritic cells and (ii) modulating M. tuberculosis-induced macrophage apoptosis.

International audience; The prevention of meningococcal disease may be improved by recombinant vaccines such as 4CMenB and rLP2086 that target the factor H binding protein (fHbp), an immunogenic surface component of Neisseria meningitidis present as one of three variants. Whether such vaccines decrease carriage of invasive isolates and thus induce herd immunity is unknown. We analyzed the genetic diversity and levels of expression of fHbp among 268 carriage strains and compare them to those of 467 invasive strains. fhbp gene sequencing showed higher proportions of variants 2 and 3 among carriage isolates (p, 0.0001). Carriage isolates expressed lower levels of fHbp (p,0.01) but that remain high enough to predict targeting by antibodies against fHbp particularly in group B isolates belonging to the frequent hypervirulent clonal complexes in Europe and North America (cc32, cc41/44, cc269). This suggests that fHbp targeting meningococcal vaccines might reduce, at least in part, the acquisition of some hyperinvasive isolates.

Shigella flexneri interacts with B cells and induces apoptosis via IpaD binding to TLR2., Antibody-mediated immunity to Shigella, the causative agent of bacillary dysentery, requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. We show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and occasionally invades B lymphocytes. The induction of a type three secretion apparatus (T3SA)–dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo. In addition to cell death occurring in Shigella-invaded CL-01 B lymphocytes, we provide evidence that the T3SA needle tip protein IpaD can induce cell death in noninvaded cells. IpaD binds to and induces B cell apoptosis via TLR2, a signaling receptor thus far considered to result in activation of B lymphocytes. The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding. Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals. This study therefore adds direct B lymphocyte targeting to the diversity of mechanisms used by Shigella to dampen the host immune response.

International audience; The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.

International audience; New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects 25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.

International audience; The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.

Polymorphic variability in Helicobacter pylori factors CagA and VacA contributes to bacterial virulence. The presence of one CagA EPIYA-C site is an independent risk factor for gastroduodenal ulceration (odds ratio [OR], 4.647; 95% confidence interval [CI], 2.037 to 10.602), while the presence of the vacA i1 allele is a risk factor for increased activity (OR, 5.310; 95% CI, 2.295 to 12.287) and severity of gastritis (OR, 3.862; 95% CI, 1.728 to 8.632).

International audience; We report the nearly complete 1H, 13C, and 15N resonance assignments of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitides. Secondary structure determination using CSI method leads to the prediction of nine beta-sheet parts.

Background Meningococcocal meningitis represents an important cause of mortality and morbidity in sub-Saharan countries. Confirmatory bacteriological or molecular diagnosis is essential for patient management/treatment and meningitis surveillance, but many laboratory tests are expensive and rarely available for low-income countries. A rapid diagnostic test (RDT) represents a valuable alternative to improve case management and surveillance. Methods A dipstick RDT developed in early 2000s that detects Neisseria meningitidis serogroups A, C, W and Y but for which a new conjugated antibody (L4-8) for the detection of serogroup A replaced the original K15-2 was assessed in the field by trained staff from health centres and district hospitals in Niger. The results were compared to those obtained in the reference laboratory and the sensitivity and specificity of RDTs were determined using conventional and real-time PCR assays as a gold standard. Results RDT results from field staff and the reference laboratory obtained for 2095 cerebrospinal fluid (CSF) specimens presented a strong concordance of 94% with Cohen's κ coefficient of 0.88. The observed concordance between RDTs operated by staff from the reference laboratory vs combination of conventional and real-time PCR assays was 89% with Cohen's κ coefficient of 0.76 indicating very good agreement. The theoretical overall sensitivity for RDT was 91.5% and the specificity 84.6%. Conclusions RDT has proven to be relatively sensitive and specific for the detection of meningococcal serogroups A/C/Y/W. We confirmed that these RDTs can be reliably operated by trained but non-specialised staff in basic health facilities.

International audience; We investigated mechanisms of the false-positive test results on rapid-antigen detection test (RADT) for group A Streptococcal (GAS) pharyngitis. Most RADT false-positives (76%) were associated with polymerase chain reaction-positive GAS results, suggesting that RADT specificity could be considered close to 100%. Finding that 61% of GAS culture-negative but RADT-positive cases were positive on both GAS polymerase chain reaction and Staphylococcus aureus testing, we posit bacterial inhibition as causative.