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The intestinal microbiota contributes to the global wellbeing of their host by their fundamental role in the induction and maintenance of a healthy immune system. Commensal bacteria shape the mucosal immune system by influencing the proportion and the activation state of anti-inflammatory regulatory T cells (Treg) by metabolites that are still only partially unravelled. Microbiota members such as Clostridiales provide a transforming growth factor beta (TGF beta)-rich environment that promotes the accumulation of Treg cells in the gut. The intestinal epithelial cells (IECs) take a central part in this process, as they are a major source of TGF beta 1 upon bacterial colonisation. In this study, we investigated which gut commensal bacteria were able to regulate the TGFB1 human promoter in IECs using supernatants from cultured bacteria. We reported that Firmicutes and Fusobacteria supernatants were the most potent TGFB1 modulators in HT-29 cells. Furthermore, we demonstrated that butyrate was the main metabolite in bacterial supernatants accounting for TGF beta 1 increase. This butyratedriven effect was independent of the G-protein coupled receptors GPR41, GPR43 and GPR109a, the transporter MCT1 as well as the transcription factors NF-kappa B and AP-1 present on TGFB1 promoter. Interestingly, HDAC inhibitors were inducing a similar TGFB1 increase suggesting that butyrate acted through its HDAC inhibitor properties. Finally, our results showed that SP1 was the main transcription factor mediating the HDAC inhibitor effect of butyrate on TGFB1 expression. This is, to our knowledge, the first characterisation of the mechanisms underlying TGFB1 regulation in IEC by commensal bacteria derived butyrate.

The human GI tract is a complex and still poorly understood environment, inhabited by one of the densest microbial communities on earth. The gut microbiota is shaped by millennia of evolution to co-exist with the host in commensal or symbiotic relationships. Members of the gut microbiota perform specific molecular functions important in the human gut environment. This can be illustrated by the presence of a highly expanded repertoire of proteins involved in carbohydrate metabolism, in phase with the large diversity of polysaccharides originating from the diet or from the host itself that can be encountered in this environment. In order to identify other bacterial functions that are important in the human gut environment, we investigated the distribution of functional groups of proteins in a group of human gut bacteria and their close non-gut relatives. Complementary to earlier global comparisons between different ecosystems, this approach should allow a closer focus on a group of functions directly related to the gut environment while avoiding functions related to taxonomically divergent microbiota composition, which may or may not be relevant for gut homeostasis. We identified several functions that are overrepresented in the human gut bacteria which had not been recognized in a global approach. The observed under-representation of certain other functions may be equally important for gut homeostasis. Together, these analyses provide us with new information about this environment so critical to our health and well-being.

Sinorhizobium meliloti possesses three distinct catalases to cope with oxidative stress: two monofunctional catalases (KatA and KatC) and one bifunctional catalase-peroxydase (KatB). The katB gene is constitutively expressed during growth in batch culture and is not induced under oxidative stress conditions. In contrast, the expression of katA and katC genes is mainly regulated at the transcription level in these conditions. A differential expression of kat genes was observed during the development of the nodule. A high expression of katA gene was detected in bacteroids, suggesting that the nitrogen-fixation process induces a strong oxidative stress. In contrast, bacteria express katB and katC genes and not the H2O2-inducible katA gene in infection threads despite the detection of H2O2 around the bacteria. A katB katC double mutant nodulated poorly and displayed abnormal infection. After nonefficient release into plant cells, bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Our results indicate that these two catalases are essential for the establishment of the symbiosis. They also suggest that the bacteria are in a nonexponential growth phase in infection threads and corroborate previous studies on the growth rate of bacteria inside the plant.

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli-B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host–microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers. Seeking order among our gut microbes The human gut microbiota consists of a huge number of species and varies greatly between individuals. A comparative metagenomic analysis of the human gut microbiomes of 39 individuals from 6 countries shows that despite this diversity, the microbiota composition can be classified into at least 3 distinct groups, or enterotypes. The enterotypes contain functional markers that correlate with individual features such as age and body mass index, a feature that may be of use in the diagnosis of numerous human disorders such as colorectal cancer and diabetes.

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, ~150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively

The commensal bacterium Enterococcus faecalis is a common cause of nosocomial infections worldwide. The increasing prevalence of multi-Antibiotic resistant E. faecalis strains reinforces this public health concern. Despite numerous studies highlighting several pathology-related genetic traits, the molecular mechanisms of E. faecalis virulence remain poorly understood. In this work, we studied 23 bacterial proteins that could be considered as virulence factors or involved in the Enterococcus interaction with the host. We systematically tested their interactions with human proteins using the Human ORFeome library, a set of 12,212 human ORFs, in yeast. Among the thousands of tested interactions, one involving the E. faecalis virulence factor ElrA and the human protein FHL2 was evidenced by yeast two-hybrid and biochemically confirmed. Further molecular characterizations allowed defining an FHL2-interacting domain (FID) of ElrA. Deletion of the FID led to an attenuated in vivo phenotype of the mutated strain clearly indicating that this interaction is likely to contribute to the multifactorial virulence of this opportunistic pathogen. Altogether, our results show that FHL2 is the first host cellular protein directly targeted by an E. faecalis virulence factor and that this interaction is involved in Enterococcus pathogenicity. © 2017 The Author(s).

Several reactive oxygen and nitrogen species (ROS/RNS) are continuously produced in plants as by-products of aerobic metabolism or in response to stresses. Depending on the nature of the ROS and RNS, some of them are highly toxic and rapidly detoxified by various cellular enzymatic and non-enzymatic mechanisms. Whereas plants have many mechanisms with which to combat increased ROS/RNS levels produced during stress conditions, under other circumstances plants appear to generate ROS/RNS as signalling molecules to control various processes encompassing the whole lifespan of the plant such as normal growth and development stages. This review aims to summarize recent studies highlighting the involvement of ROS/RNS, as well as the low molecular weight thiols, glutathione and homoglutathione, during the symbiosis between rhizobia and leguminous plants. This compatible interaction initiated by a molecular dialogue between the plant and bacterial partners, leads to the formation of a novel root organ capable of fixing atmospheric nitrogen under nitrogen-limiting conditions. On the one hand, ROS/RNS detection during the symbiotic process highlights the similarity of the early response to infection by pathogenic and symbiotic bacteria, addressing the question as to which mechanism rhizobia use to counteract the plant defence response. Moreover, there is increasing evidence that ROS are needed to establish the symbiosis fully. On the other hand, GSH synthesis appears to be essential for proper development of the root nodules during the symbiotic interaction. Elucidating the mechanisms that control ROS/RNS signalling during symbiosis could therefore contribute in defining a powerful strategy to enhance the efficiency of the symbiotic interaction.

The commensal bacterium Enterococcus faecalis is a common cause of nosocomial infections worldwide. The increasing prevalence of multi-antibiotic resistant E. faecalis strains reinforces this public health concern. Despite numerous studies highlighting several pathology-related genetic traits, the molecular mechanisms of E. faecalis virulence remain poorly understood. In this work, we studied 23 bacterial proteins that could be considered as virulence factors or involved in the Enterococcus interaction with the host. We systematically tested their interactions with human proteins using the Human ORFeome library, a set of 12,212 human ORFs, in yeast. Among the thousands of tested interactions, one involving the E. faecalis virulence factor ElrA and the human protein FHL2 was evidenced by yeast two-hybrid and biochemically confirmed. Further molecular characterizations allowed defining an FHL2-interacting domain (FID) of ElrA. Deletion of the FID led to an attenuated in vivo phenotype of the mutated strain clearly indicating that this interaction is likely to contribute to the multifactorial virulence of this opportunistic pathogen. Altogether, our results show that FHL2 is the first host cellular protein directly targeted by an E. faecalis virulence factor and that this interaction is involved in Enterococcus pathogenicity.

In recent years, graph-processing has become an essential class of workloads with applications in a rapidly growing number of fields. Graph-processing typically uses large input sets, often in multi-gigabyte scale, and data-dependent graph traversal methods exhibiting irregular memory access patterns. Recent work demonstrates that, due to the highly irregular memory access patterns of data-dependent graph traversals, state-of-the-art graph-processing workloads spend up to 80 % of the total execution time waiting for memory accesses to be served by the DRAM. The vast disparity between the Last Level Cache (LLC) and main memory latencies is a problem that has been addressed for years in computer architecture. One of the prevailing approaches when it comes to mitigating this performance gap between modern CPUs and DRAM is cache replacement policies. In this work, we characterize the challenges drawn by graph-processing workloads and evaluate the most relevant cache replacement policies.