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Antigenic variation, the capacity to produce a range of variable antigens, is a well-described strategy of Plasmodium and other parasites to evade host immunity. Here, we show that gene amplification is an additional evasion mechanism used by Plasmodium vivax to escape humoral immunity targeting PvDBP, the key ligand involved in reticulocyte invasion. PvDBP gene amplification leads to increased mRNA levels and protects P. vivax in vitro against invasion inhibitory human monoclonal antibodies targeting a conserved binding domain of DBP. Patient samples suggest that parasites with increased pvdbp copy number are able to infect individuals with naturally acquired antibodies highly blocking the binding of PvDBP to the Duffy receptor. These results show that gene copy number variation affect the parasite’s ability to evade anti-PvDBP humoral immunity., Duffy binding protein (DBP) of Plasmodium vivax is important for invasion and is a potential vaccine candidate. Here, the authors show that PvDBP gene amplification protects P vivax in vitro against invasion inhibitory human monoclonal antibodies and is associated to infection of patients with PvDBP binding inhibitory antibodies.

BackgroundSARS-CoV-2 breakthrough infections after complete vaccination are increasing whereas their determinants remain uncharacterized.MethodsWe analyzed two cases of post-vaccination SARS-CoV-2 infections by α and β variants, respectively. For each participant both humoral (binding and neutralizing antibodies) and cellular (activation markers and cytokine expression) immune responses were characterized longitudinally.ResultsThe first participant (P1) was infected by an α variant and displayed an extended and short period of viral excretion and symptom. Analysis of cellular and humoral response 72 h post-symptom onset revealed that P1 failed at developing neutralizing antibodies and a potent CD4 memory response (lack of SARS-CoV-2 specific CD4+IL-2+ cells) and CD8 effector response (CD8+IFNγ+ cells). The second participant (P2) developed post-vaccination SARS-CoV-2 infection by a β variant, associated with a short period of viral excretion and symptoms. Despite displaying initially high levels and polyfunctional T cell responses, P2 lacked initial β-directed neutralizing antibodies. Both participants developed and/or increased their neutralization activity and cellular responses against all variants, namely, β and δ variants that lasts up to 3 months after breakthrough infection.ConclusionsAn analysis of cellular and humoral response suggests two possible mechanisms of breakthrough infection: a poor immune response to vaccine and viral evasion to neutralizing antibodies.

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa1-3. It has since spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of around 32 mutations in spike-located mostly in the N-terminal domain and the receptor-binding domain-that may enhance viral fitness and enable antibody evasion. Here we isolated an infectious Omicron virus in Belgium from a traveller returning from Egypt. We examined its sensitivity to nine monoclonal antibodies that have been clinically approved or are in development4, and to antibodies present in 115 serum samples from COVID-19 vaccine recipients or individuals who have recovered from COVID-19. Omicron was completely or partially resistant to neutralization by all monoclonal antibodies tested. Sera from recipients of the Pfizer or AstraZeneca vaccine, sampled five months after complete vaccination, barely inhibited Omicron. Sera from COVID-19-convalescent patients collected 6 or 12 months after symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titres 6-fold to 23-fold lower against Omicron compared with those against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and, to a large extent, vaccine-elicited antibodies. However, Omicron is neutralized by antibodies generated by a booster vaccine dose. ispartof: NATURE vol:602 issue:7898 pages:671-+ ispartof: location:England status: published

The largest gene families in eukaryotes are subject to allelic exclusion, but mechanisms underpinning single allele selection and inheritance remain unclear. Here, we describe a protein complex sustaining variant surface glycoprotein (VSG) allelic exclusion and antigenic variation in Trypanosoma brucei parasites. The VSG-exclusion-1 (VEX1) protein binds both telomeric VSG-associated chromatin and VEX2, an ortholog of nonsense-mediated-decay helicase, UPF1. VEX1 and VEX2 assemble in an RNA polymerase-I transcription-dependent manner and sustain the active, subtelomeric VSG-associated transcription compartment. VSG transcripts and VSG coats become highly heterogeneous when VEX proteins are depleted. Further, the DNA replication-associated chromatin assembly factor, CAF-1, binds to and specifically maintains VEX1 compartmentalisation following DNA replication. Thus, the VEX-complex controls VSG-exclusion, while CAF-1 sustains VEX-complex inheritance in association with the active-VSG. Notably, the VEX2-orthologue and CAF-1 in mammals are also implicated in exclusion and inheritance functions. In trypanosomes, these factors sustain a highly effective and paradigmatic immune evasion strategy., Monoallelic expression of variant surface glycoprotein genes (VSGs) is essential for immune evasion by Trypanosoma brucei. Here, Faria et al. show that the VEX protein complex controls VSG allelic exclusion, and that CAF‐1 sustains inheritance of the VEX‐complex in association with the active VSG.

Staphylococcus aureus is the most prevalent pathogen isolated from diabetic foot infections (DFIs). The purpose of this study was to evaluate its behavior in an in vitro model mimicking the conditions encountered in DFI. Four clinical S. aureus strains were cultivated for 16 weeks in a specific environment based on the wound-like medium biofilm model. The adaptation of isolates was evaluated as follows: by Caenorhabditis elegans model (to evaluate virulence), by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) (to evaluate expression of the main virulence genes), and by Biofilm Ring test® (to assess the biofilm formation). After 16 weeks, the four S. aureus had adapted their metabolism, with the development of small colony variants and the loss of b-hemolysin expression. The in vivo nematode model suggested a decrease of virulence, confirmed by qRT-PCRs, showing a significant decrease of expression of the main staphylococcal virulence genes tested, notably the toxin-encoding genes. An increased expression of genes involved in adhesion and biofilm was noted. Our data based on an in vitro model confirm the impact of environment on the adaptation switch of S. aureus to prolonged stress environmental conditions. These results contribute to explore and characterize the virulence of S. aureus in chronic wounds.

International audience; Macrophages participate to the first line of defense against infectious agents. Microbial pathogens evolved sophisticated mechanisms to escape macrophage killing. Here, we review recent discoveries and emerging concepts on bacterial molecular strategies to subvert macrophage immune responses. We focus on the expanding number of fascinating subversive tools developed by Listeria monocytogenes, Staphylococcus aureus, and pathogenic Yersinia spp., illustrating diversity and commonality in mechanisms used by microorganisms with different pathogenic lifestyles.

International audience; Haemophilus influenzae harbours a complex array of factors to resist human complement attack. As non-typeable H. influenzae (NTHi) strains do not possess a capsule, their serum resistance mainly depends on other mechanisms including LOS decoration. In this report, we describe the identification of a highly serum resistant, nasopharyngeal isolate (NTHi23) by screening a collection of 77 clinical isolates. For NTHi23, we defined the MLST sequence type 1133, which matches the profile of a previously published invasive NTHi isolate. A detailed genetic analysis revealed that NTHi23 shares several complement evading mechanisms with invasive disease isolates. These mechanisms include the functional expression of a retrograde phospholipid trafficking system and the presumable decoration of the LOS structure with sialic acid. By screening the NTHi23 population for spontaneous decreased serum resistance, we identified a clone, which was about 103-fold more sensitive to complement-mediated killing. Genome-wide analysis of this isolate revealed a phase variation in the N'-terminal region of lpsA, leading to a truncated version of the glycosyltransferase (LpsA). We further showed that a NTHi23 lpsA mutant exhibits a decreased invasion rate into human alveolar basal epithelial cells. Since only a small proportion of the NTHi23 population expressed the serum sensitive phenotype, resulting from lpsA phase-off, we conclude that the nasopharyngeal environment selected for a population expressing the intact and functional glycosyltransferase.

International audience; Leishmania (Viannia) braziliensis is responsible for the largest number of American tegumentary leishmaniasis (ATL) in Brazil. ATL can present several clinical forms including typical (TL) and atypical (AL) cutaneous and mucocutaneous (ML) lesions. To identify parasite and host factors potentially associated with these diverse clinical manifestations, we first surveyed the expression of two virulence-associated glycoconjugates, lipophosphoglycan (LPG) and the metalloprotease GP63 by a panel of promastigotes of Leishmania braziliensis (L. braziliensis) strains isolated from patients with different clinical manifestations of ATL and from the sand fly vector. We observed a diversity of expression patterns for both LPG and GP63, which may be related to strain-specific polymorphisms. Interestingly, we noted that GP63 activity varies from strain to strain, including the ability to cleave host cell molecules. We next evaluated the ability of promastigotes from these L. braziliensis strains to modulate phagolysosome biogenesis in bone marrow-derived macrophages (BMM), by assessing phagosomal recruitment of the lysosome-associated membrane protein 1 (LAMP-1) and intraphagosomal acidification. Whereas, three out of six L. braziliensis strains impaired the phagosomal recruitment of LAMP-1, only the ML strain inhibited phagosome acidification to the same extent as the L. donovani strain that was used as a positive control. While decreased phagosomal recruitment of LAMP-1 correlated with higher LPG levels, decreased phagosomal acidification correlated with higher GP63 levels. Finally, we observed that the ability to infect and replicate within host cells did not fully correlate with the inhibition of phagosome maturation. Collectively, our results revealed a diversity of strain-specific phenotypes among L. braziliensis isolates, consistent with the high genetic diversity within Leishmania populations.

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International audience; Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus, one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores (i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host.

International audience; What are the new approaches and emerging ideas to prevent leptospirosis, a neglected bacterial re-emerging zoonotic disease? How do Leptospira interrogans escape the host defenses? We aim here to review and discuss the most recent literature that provides some answers to these questions, in particular data related to a better understanding of adaptive and innate immunity towards leptospires, and design of vaccines. This is an opinion paper, not a comprehensive review. We will try to highlight the new strategies and technologies boosting the search for drugs and vaccines. We will also address the bottlenecks and difficulties impairing the search for efficient vaccines and the many gaps in our knowledge of immunity against leptospirosis. Finally, we aim to delineate how Leptospira spp. escape the innate immune responses of Toll-Like receptors (TLR) and Nod-Like receptors (NLR). The rational use of TLR and NLR agonists as adjuvants could be key to design future vaccines against pathogenic leptospires.

International audience; An emerging realization of infectious disease is that pathogens can cause a high incidence of genetic instability within the host as a result of infection-induced DNA lesions. These often lead to classical hallmarks of cancer, one of which is the ability to evade apoptosis despite the presence of numerous genetic mutations that should be otherwise lethal. The Human Immunodeficiency Virus type 1 (HIV-1) is one such pathogen as it induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages. As a consequence there is long-term dissemination of the pathogen specifically by these infected yet surviving host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses like HIV-1, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21. As is typical for a long noncoding RNA, lincRNA-p21 mediates its activities in a complex with one of its two protein binding partners, namely HuR and hnRNP-K. In this work, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering the lincRNA-p21 partner HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering lincRNA-p21's other protein partner hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Of particular note, this MAP2K1/ERK2 pro-survival cascade is switched off during T cell maturation and is thus unavailable for similar viral manipulation in mature CD4+ T cells. We show that the introduction of MAP2K1, ERK2, or HDM2 inhibitors in HIV-infected macrophages results in apoptosis, providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and its apoptosis protein partner hnRNP-K. Together, these results reveal a unique example of pathogenic control over mammalian apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages.

International audience; B cells have long been regarded as simple antibody production units, but are now becoming known as key players in both adaptive and innate immune responses. However, several bacteria, viruses and parasites have evolved the ability to manipulate B cell functions to modulate immune responses. Pathogens can affect B cells indirectly, by attacking innate immune cells and altering the cytokine environment, and can also target B cells directly, impairing B cell-mediated immune responses. In this Review, we provide a summary of recent advances in elucidating direct B cell-pathogen interactions and highlight how targeting this specific cell population benefits different pathogens.

International audience; The era of antiretroviral therapy has made HIV-1 infection a manageable chronic disease for those with access to treatment. Despite treatment, virus persists in tissue reservoirs seeded with long-lived infected cells that are resistant to cell death and immune recognition. Which cells contribute to this reservoir and which factors determine their persistence are central questions that need to be answered to achieve viral eradication. In this chapter, we describe how cell susceptibility to infection, resistance to cell death, and immune-mediated killing as well as natural cell life span and turnover potential are central components that allow persistence of different lymphoid and myeloid cell subsets that were recently identified as key players in harboring latent and actively replicating virus. The relative contribution of these subsets to persistence of viral reservoir is described, and the open questions are highlighted.

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.

Background: Despite advances toward an improved understanding of the evasive mechanisms leading to the establishment of cystic echinococcosis, the discovery of specific immunosuppressive mechanisms and related factors are of great interest in the development of an immunotherapeutic approach. Objective: To elucidate immunosuppressive effects of bioactive factors contained in chromatographic fractions from hydatid cystic fluid (HCF) of Echinococcus granulosus. Methods: Hydatid cystic fluid was fractionated by reverse phase chromatography. Non-specific Concanavalin A-driven proliferation of spleen cells was used to determine specific inhibitory fractions. Trypan blue exclusion test and flowcytometry analysis were performed to check whether highly inhibitory fractions of HCF have apoptotic effect on peritoneal macrophages. Western blot analysis was used to determine proteolytic effects of parasitic antigens on major histocompatibility complex (MHC) class II (I-a) contained in membrane proteins extract from macrophages. Results: High concentrations of HCF and few of chromatographic fractions suppressed spleen cells proliferation. Fractions 7 and 35 were the highest inhibitory fractions. Specifically fraction 35 and to a lesser extent HCF induced apoptosis in peritoneal naive macrophages. However, HCF and the fraction 7 proteolytically altered the expression of MHC class II molecules on peritoneal macrophages. The proteolytic molecule was identified to be a serine protease. Macrophages taken at the chronic and end phase from cystic echinococcosis-infected mice were able to uptake and process C-Ovalbumine-FITC. These cells expressed a drastically reduced level of (I-a) molecules. Conclusion: Our study present new aspects of immune suppression function of E. granulosus. Further molecular characterization of apoptotic and proteolytic factors might be useful to develop immunotherapeutic procedure to break down their inhibitory effects.

Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. IMPORTANCE Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual.

International audience; Subversion of host immune responses is the key infection strategy employed by most, if not all, human pathogens. Modulation of the host innate response by pathogens has been vastly documented. Yet, especially for bacterial infections, it was only recently that cells of the adaptive immune response were recognized as targets of bacterial weapons such as the type III secretion system (T3SS) and its effector proteins. In this review, we focus on the recent advances made in the understanding of how the enteroinvasive bacterium Shigella flexneri interferes with the host adaptive response by targeting T lymphocytes, especially their migration capacities.

International audience; Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

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International audience; Pseudomonas aeruginosa is responsible for persistent infections in cystic fibrosis patients, suggesting an ability to circumvent innate immune defenses. This bacterium uses the kynurenine pathway to catabolize tryptophan. Interestingly, many host cells also produce kynurenine, which is known to control immune system homeostasis. We showed that most strains of P. aeruginosa isolated from cystic fibrosis patients produce a high level of kynurenine. Moreover, a strong transcriptional activation of kynA (the first gene involved in the kynurenine pathway) was observed upon contact with immune cells and particularly with neutrophils. In addition, using coculture of human neutrophils with various strains of P. aeruginosa producing no (ΔkynA) or a high level of kynurenine (ΔkynU or ΔkynA pkynA), we demonstrated that kynurenine promotes bacterial survival. In addition, increasing the amount kynurenine inhibits reactive oxygen species production by activated neutrophils, as evaluated by chemiluminescence with luminol or isoluminol or SOD-sensitive cytochrome c reduction assay. This inhibition is due neither to a phagocytosis defect nor to direct NADPH oxidase inhibition. Indeed, kynurenine has no effect on oxygen consumption by neutrophils activated by PMA or opsonized zymosan. Using in vitro reactive oxygen species-producing systems, we showed that kynurenine scavenges hydrogen peroxide and, to a lesser extent, superoxide. Kynurenine׳s scavenging effect occurs mainly intracellularly after bacterial stimulation, probably in the phagosome. In conclusion, the kynurenine pathway allows P. aeruginosa to circumvent the innate immune response by scavenging neutrophil reactive oxygen species production.

International audience; LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material., Author Summary Natural genetic transformation, first discovered in Streptococcus pneumoniae by Griffith in 1928, is observed in many Gram-negative and Gram-positive bacteria. This process promotes genome plasticity and adaptability. In particular, it enables many human pathogens such as Streptococcus pneumoniae, Staphylococcus aureus or Neisseria gonorrhoeae to acquire resistance to antibiotics and/or to escape vaccines through the binding and incorporation of new genetic material. While it is well established that this process requires the binding and internalization of external DNA, the molecular details of these steps are unknown. In this study, we discovered a new appendage at the surface of S. pneumoniae cells. We show that this appendage is similar in morphology and composition to appendages called Type IV pili commonly found in Gram-negative bacteria. We demonstrate that this new pneumococcal pilus is essential for transformation and that it directly binds DNA. We propose that the transformation pilus is an essential piece of the transformation apparatus by capturing exogenous DNA at the bacterial cell surface.

The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.

Background For an efficient immune response against viral infection, dendritic cells (DCs) must express a coordinate repertoire of receptors that allow their recruitment to the sites of inflammation and subsequently to the secondary lymphoid organs in response to chemokine gradients. Several pathogens are able to subvert the chemokine receptor expression and alter the migration properties of DCs as strategy to escape from the immune control. Findings Here we report the inhibitory effect of Human Herpesvirus 8 (HHV-8) on the migratory behavior of immature and mature DCs. We found that the virus altered the DC chemokine receptor expression and chemokine induced migration. Moreover HHV-8 was also able to interfere with basal motility of DCs by inducing cytoskeleton modifications. Conclusion Based on our findings, we suggest that HHV-8 is able to subvert the DC migration capacity and this represents an additional mechanism which interferes with their immune-functions.

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective., Author Summary Mycobacterium tuberculosis is one of the most life-threatening pathogens of all time. Despite the development of vaccines and antibiotics, this pathogen is still a major public health problem. Also the HIV epidemic has an important impact on the rise of M. tuberculosis infections since immunodeficient people are highly susceptible. Commonly, M. tuberculosis has been thought to reside in a membrane-bound compartment within its host cells during the entire infection cycle from invasion to cell death. Using a fluorescence-based method, we provide evidence that M. tuberculosis is able to rupture its membrane-bound compartment and gain access to the host cytosol, where it can elicit cell death. Furthermore, we show that this effect is dependent on a functional type VII secretion system named ESX-1. Most importantly, we were able to track the dynamics of infection to understand the consequences of M. tuberculosis phagosomal rupture. This revealed that phagosomal rupture results in cell toxicity and host cell death involving necrosis. Together, our data provide a new angle in the worldwide fight against M. tuberculosis and could lead to new approaches in the development of innovative treatments.

High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25-27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16-24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1-13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.

Human group IIA-secreted phospholipase A2 (sPLA2-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA2-IIA. Analysis of a set of clinical isolates demonstrated that an ∼500-fold higher concentration of sPLA2-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA2-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA2-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (∼30-fold) to sPLA2-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA2-IIA. Importantly, using transgenic mice expressing human sPLA2-IIA, we showed that the sortase A-mediated sPLA2-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA2-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA2-IIA and we showed that sPLA2-IIA contributes to the host defense against S. pyogenes infection.

International audience; This review gives an update of current knowledge on the clinical pleiomorphism of Leishmania, with a special emphasis on the case of asymptomatic carriage. The first part describes the numerous unusual expressions of the disease that occur besides the classic (visceral, cutaneous, and mucocutaneous) forms of leishmaniases. The second part deals with progress in the understanding of disease outcome in humans, and the possible future approaches to improve our knowledge in the field. The third part highlights the role of the too often neglected asymptomatic carrier compartment. This group could be key to understanding infraspecific differences in virulence and pathogenicity of the parasite, as well as identifying the genetic determinants involved in the expression of the disease.

Zaire Ebola virus (ZEBOV) is the most lethal Ebola virus species and one of the most virulent human pathogens, causing a severe hemorrhagic fever syndrome that ends in the death of 90% of symptomatic patients within a few days (11). ZEBOV caused numerous human outbreaks in Central Africa between 1976 and 2008 and also devastated wild populations of nonhuman primates (2, 10, 13). Recent studies show that ZEBOV infection induces profound suppression of adaptive immunity, characterized by massive B- and T-lymphocyte apoptosis both in humans and in experimentally infected animals (1, 3, 5–6, 14). Lymphocytes are not infected by ZEBOV, and the mechanisms leading to this massive lymphocyte apoptosis are unclear. As the final shock-like syndrome associated with fatal ZEBOV infection resembles the toxic shock syndrome observed during infections with Gram-positive bacteria with superantigen (SAg) activity, we wondered if ZEBOV might also exhibit SAg activity. SAgs are microbial proteins that bind simultaneously to major histocompatibility complex class II molecules and to the T-cell receptor (TCR) Vβ region. This “bridge” skews the T-cell repertoire by amplifying specific T-cell Vβ subsets, which then are either rapidly deleted by activated cell death or become anergic (4, 7). Thus, we examined the TCR Vβ repertoire of peripheral blood mononuclear cells isolated from acutely infected patients by using PCR-based methods. Surprisingly, TCR Vβ12, Vβ13.2, and Vβ17 mRNA expression was undetectable throughout the symptomatic phase in ZEBOV victims, in survivors but also in three of seven asymptomatic individuals identified during the 1996 Booue outbreak in Gabon (9) (Fig. 1). Furthermore, expression of the other TCR-Vβ chains in deceased patients diminished as the disease progressed, reaching very low levels the day before death. In contrast, mRNA expression of the other Vβ chains was normally upregulated to the same level as β-actin mRNA in asymptomatic individuals but also at the beginning and end of the symptomatic phase and after recovery in survivors. Fig. 1. Reverse transcription (RT)-PCR analysis of the TCR Vβ repertoire during human Ebola virus infection. Agarose gel electrophoresis of PCR products obtained with various (n = 20) combinations of Vβ gene family-specific and Cβ oligonucleotide ... Together, these data show that, whatever the clinical outcome, human ZEBOV infection is associated with mRNA downregulation of three TCR Vβ subsets, indicating either anergy or deletion of the three corresponding T-lymphocyte populations. These results are consistent with ZEBOV SAg activity, the first time that it is suggested in the Filoviridae family. Whereas a large number of bacterial SAgs have been identified, only two families of viruses have SAg activity in humans, namely, Herpesviridae and Rhabdoviridae (8, 12). These findings may represent the “missing link” in our understanding of ZEBOV pathogenicity. The SAg activity of ZEBOV might contribute to the extraordinarily rapid and profound T-lymphocyte depletion observed during fatal infection. The high viral load observed during fatal ZEBOV infection, together with the simultaneous targeting of three Vβ T-cell subsets observed here, would elicit massive lymphocyte activation, rapidly resulting in the deletion of large populations of Vβ12-, Vβ13.2-, and Vβ17-bearing T cells. The loss of mRNA expression of the other Vβ T cells would be due to the proportionally small decline in these subsets.

International audience; NK cells present in the peripheral blood respond rapidly to pathogens or pathogen-infected cells by various means including cytotoxicity and production of cytokines. Whether decidual NK (dNK) cells are able to play a similar role when the pregnant uterus is infected by viruses is still largely unknown. Decidual NK cells are generally considered as poorly cytotoxic when compared to their peripheral blood counterparts. However, we have recently demonstrated that freshly isolated dNK cells from healthy early pregnant uterus do have a cytotoxic potential mediated by the specific engagement of NKp46 activating receptor. We further found that the co-engagement of CD94/NKG2A inhibiting receptor drastically inhibits the cytolytic function of dNK. This latter observation suggests that in situ the CD94/NKG2A receptor interaction with its HLA-E specific ligand is a dominant negative regulatory mechanism that prevents unwanted dNK cell cytotoxicity in non-infected pregnant uterus. How do dNK cells behave when they are activated by virus-infected cells present at the maternal-fetal interface? Largely based on data obtained from circulating NK cells, this review briefly discusses the following questions: Does uterine viral infection promote decidual NK cell proliferative capacity in situ? Are dNK cells able to kill virus-infected autologous decidual target cells and thus limit the virus spreading to the fetus? Which viral-mediated signal(s) and molecular interactions may subvert inhibition of dNK cytotoxic potential? Does uterine viral infection promote decidual NK cell secretion of cytokines and chemokines that boost the anti-viral immune response?

International audience; Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Preventive modalities are absent and the current antiviral treatment is limited by resistance, toxicity, and high costs. Viral entry is required for initiation, spread, and maintenance of infection, and thus is a promising target for antiviral therapy. HCV entry is a highly orchestrated process involving viral and host cell factors. These include the viral envelope glycoproteins E1 and E2, CD81, scavenger receptor BI, and tight junction proteins claudin-1 and occludin. Recent studies in preclinical models and HCV-infected patients have demonstrated that the virus has developed multiple strategies to escape host immune responses during viral entry. These include evasion from neutralizing antibodies and viral spread by cell-cell transmission. These challenges have to be taken into account for the design of efficient antiviral strategies. Thus, a detailed understanding of the mechanisms of viral entry and escape is a prerequisite to define viral and cellular targets and develop novel preventive and therapeutic antivirals. This review summarizes the current knowledge about the molecular mechanisms of HCV entry into hepatocytes, highlights novel targets and reviews the current preclinical and clinical development of compounds targeting entry. Proof-of-concept studies suggest that HCV entry inhibitors are a novel and promising class of antivirals widening the preventive and therapeutic arsenal against HCV infection.

International audience; More than 20 years ago, immunologists discovered that resistance and susceptibility to experimental infection with the intracellular protozoan Leishmania major was associated with the development of T-helper 1 (Th1)- and Th2-dominated immune responses, respectively. This infectious disease model was later used to identify and assess the role of key factors, such as interleukin-12 (IL-12) and IL-4, in Th1 and Th2 maturation. While infection by Leishmania remains a popular model for immunologists who wish to assess the role of their favorite molecule in T-cell differentiation, other investigators have tried to better understand how Leishmania interact with its insect and mammalian hosts. In this review, we discuss some of these new data with an emphasis on the early events that shape the immune response to Leishmania and on the immune evasion mechanisms that allow this parasite to avoid the development of sterilizing immunity and to secure its transmission to a new host.

International audience; The study of infectious disease concerns the interaction between the host species and a pathogen organism. The analysis of such complex systems is improving with the evolution of high-throughput technologies and advanced computational resources. This article reviews integrative, systems-oriented approaches to understanding mechanisms underlying infection, immune response and inflammation to find biomarkers of disease and design new drugs. We focus on the systems biology process, especially the data gathering and analysis techniques rather than the experimental technologies or latest computational resources.

Staphylococcus aureus, a pathogen responsible for hospital and community-acquired infections, expresses many virulence factors under the control of numerous regulatory systems. Here we show that one of the small pathogenicity island RNAs, named SprD, contributes significantly to causing disease in an animal model of infection. We have identified one of the targets of SprD and our in vivo data demonstrate that SprD negatively regulates the expression of the Sbi immune-evasion molecule, impairing both the adaptive and innate host immune responses. SprD interacts with the 5′ part of the sbi mRNA and structural mapping of SprD, its mRNA target, and the ‘SprD-mRNA’ duplex, in combination with mutational analysis, reveals the molecular details of the regulation. It demonstrates that the accessible SprD central region interacts with the sbi mRNA translational start site. We show by toeprint experiments that SprD prevents translation initiation of sbi mRNA by an antisense mechanism. SprD is a small regulatory RNA required for S. aureus pathogenicity with an identified function, although the mechanism of virulence control by the RNA is yet to be elucidated., Author Summary Bacteria possess numerous and diverse means of gene regulation using RNA molecules, including small RNAs (sRNAs). Here we show that one sRNA is essential for a major human bacterial pathogen, Staphylococcus aureus, to cause a disease in an animal model of infection. Our study provides evidence that this RNA regulates the expression of an immune evasion molecule secreted by the bacterium to impair the host immune responses, and we have solved the mechanism of the RNA-based regulation at molecular level. So far, the mechanism of bacterial virulence controlled by SprD is unrevealed, but that small RNA has a huge impact in the course of a bacterial infection. It implies possible new strategies in fighting against that major human and animal bacterial pathogen in preventing the expression of this regulatory RNA.

International audience; During phagocytosis, microorganisms are taken up by immune cells into phagosomes. Through membrane-trafficking events mediated by SNARE proteins, phagosomes fuse with lysosomes, generating degradative phagolysosomes. Phagolysosomes contribute to host immunity by linking microbial killing within these organelles with antigen processing for presentation on MHC class I or II molecules to T cells. We show that the intracellular parasite Leishmania evades immune recognition by inhibiting phagolysosome biogenesis. The Leishmania cell surface metalloprotease GP63 cleaves a subset of SNAREs, including VAMP8. GP63-mediated VAMP8 inactivation or Vamp8 disruption prevents the NADPH oxidase complex from assembling on phagosomes, thus altering their pH and degradative properties. Consequently, the presentation of exogenous Leishmania antigens on MHC class I molecules, also known as cross-presentation, is inhibited, resulting in reduced T cell activation. These findings indicate that Leishmania subverts immune recognition by altering phagosome function and highlight the importance of VAMP8 in phagosome biogenesis and antigen cross-presentation.

Variations in disease enhancement Secondary Dengue virus (DENV) infections can be dangerous if levels of antibodies from prior infection are inadequate to clear the virus. This RNA flavivirus exploits the presence of lower levels of heterotypic antibodies to infect immunoglobulin Fcγ receptor–bearing cells. Many RNA viruses also exhibit antigenic variation, which classically allows evasion of immune responses. Katzelnick et al . investigated whether antigenic variation in DENV has a biological function in a virus that courts immune responses to enhance replication (see the Perspective by Rohani and Drake). Using antigenic cartography on a panel of more than 400 DENV1-4 subtype samples isolated in Bangkok, Thailand, the authors found that antigenic variation in virus populations oscillated between similarity and dissimilarity across subtypes over time, with outbreaks correlating with periods of antigenic dissimilarity within serotypes. This pattern may be at least in part a result of the conflicting evolutionary pressures of immune evasion and immune enhancement. —CA