Explore the vast range of titles available.

Background Comparing the composition of microbial communities among groups of interest (e.g., patients vs healthy individuals) is a central aspect in microbiome research. It typically involves sequencing, data processing, statistical analysis and graphical display. Such an analysis is normally obtained by using a set of different applications that require specific expertise for installation, data processing and in some cases, programming skills. Results Here, we present SHAMAN, an interactive web application we developed in order to facilitate the use of (i) a bioinformatic workflow for metataxonomic analysis, (ii) a reliable statistical modelling and (iii) to provide the largest panel of interactive visualizations among the applications that are currently available. SHAMAN is specifically designed for non-expert users. A strong benefit is to use an integrated version of the different analytic steps underlying a proper metagenomic analysis. The application is freely accessible at http://shaman.pasteur.fr/ , and may also work as a standalone application with a Docker container (aghozlane/shaman), conda and R. The source code is written in R and is available at https://github.com/aghozlane/shaman . Using two different datasets (a mock community sequencing and a published 16S rRNA metagenomic data), we illustrate the strengths of SHAMAN in quickly performing a complete metataxonomic analysis. Conclusions With SHAMAN, we aim at providing the scientific community with a platform that simplifies reproducible quantitative analysis of metagenomic data.

HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of β-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control. Here, Claireaux et al. show that people who naturally control HIV infection express lower levels of the viral co-receptor CCR5 in specific CD4 + T cells, and that this results from mutations or receptor internalization by CD4 + T cell-produced chemokines.

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.

Virus-specific CD8+ T cells play a central role in HIV-1 natural controllers to maintain suppressed viremia in the absence of antiretroviral therapy. These cells display a memory program that confers them stemness properties, high survival, polyfunctionality, proliferative capacity, metabolic plasticity, and antiviral potential. The development and maintenance of such qualities by memory CD8+ T cells appear crucial to achieving natural HIV-1 control. Here, we show that targeting the signaling pathways Wnt/transcription factor T cell factor 1 (Wnt/TCF-1) and mTORC through GSK3 inhibition to reprogram HIV-specific CD8+ T cells from noncontrollers promoted functional capacities associated with natural control of infection. Features of such reprogrammed cells included enrichment in TCF-1+ less-differentiated subsets, a superior response to antigen, enhanced survival, polyfunctionality, metabolic plasticity, less mTORC1 dependency, an improved response to γ-chain cytokines, and a stronger HIV-suppressive capacity. Thus, such CD8+ T cell reprogramming, combined with other available immunomodulators, might represent a promising strategy for adoptive cell therapy in the search for an HIV-1 cure.

Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective antimicrobial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. In this work, we develop a multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells during transitions between separate environmental conditions. With this platform, we implement a dynamic single-cell screening for pheno-tuning compounds, which induce a phenotypic change and decrease cell-to-cell variation, aiming to undermine the entire bacterial population and make it more vulnerable to other drugs. We apply this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our lead compound impairs Mycobacterium tuberculosis via a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that harnessing phenotypic variation represents a successful approach to tackle pathogens that are increasingly difficult to treat. Tuberculosis is a major global health threat. Here, the authors develop a single-cell drug discovery approach and identify a compound that tunes bacterial phenotypic variation. This enhances the activity of anti-tubercular drugs against the pathogen.

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

Background Host-associated microbes, collectively known as the microbiota, play an important role in the biology of multicellular organisms. In mosquito vectors of human pathogens, the gut bacterial microbiota influences vectorial capacity and has become the subject of intense study. In laboratory studies of vector biology, genetic effects are often inferred from differences between geographically and genetically diverse colonies of mosquitoes that are reared in the same insectary. It is unclear, however, to what extent genetic effects can be confounded by uncontrolled differences in the microbiota composition among mosquito colonies. To address this question, we used 16S metagenomics to compare the midgut bacterial microbiome of six laboratory colonies of Aedes aegypti recently derived from wild populations representing the geographical range and genetic diversity of the species. Results We found that the diversity, abundance, and community structure of the midgut bacterial microbiome was remarkably similar among the six different colonies of Ae. aegypti , regardless of their geographical origin. We also confirmed the relatively low complexity of bacterial communities inhabiting the mosquito midgut. Conclusions Our finding that geographically diverse colonies of Ae. aegypti reared in the same insectary harbor a similar gut bacterial microbiome supports the conclusion that the gut microbiota of adult mosquitoes is environmentally determined regardless of the host genotype. Thus, uncontrolled differences in microbiota composition are unlikely to represent a significant confounding factor in genetic studies of vector biology.

Background The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly. Methods Fecal samples from healthy adults were stored at -80'C as such or diluted in RNAlater0 and subjected to 2 extraction protocols with mechanical lysis: the Powersoil (R) MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote (R) (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (http://shaman.pasteur.fr/). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance. Results We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater (R) impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon. Conclusion Our results showed that RNAlater (R) moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater (R) remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction.

Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5′-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR −/− hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5’TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.