Nicolas Terrapon

A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility

Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.

Molecular traces of alternative social organization in a termite genome

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

Glycan complexity dictates microbial resource allocation in the large intestine

The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

Complex pectin metabolism by gut bacteria reveals novel catalytic functions

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.

Automatic prediction of polysaccharide utilization loci in Bacteroidetes species

MOTIVATIONnA bacterial polysaccharide utilization locus (PUL) is a set of physically linked genes that orchestrate the breakdown of a specific glycan. PULs are prevalent in the Bacteroidetes phylum and are key to the digestion of complex carbohydrates, notably by the human gut microbiota. A given Bacteroidetes genome can encode dozens of different PULs whose boundaries and precise gene content are difficult to predict.nnnRESULTSnHere, we present a fully automated approach for PUL prediction using genomic context and domain annotation alone. By combining the detection of a pair of marker genes with operon prediction using intergenic distances, and queries to the carbohydrate-active enzymes database (www.cazy.org), our predictor achieved above 86% accuracy in two Bacteroides species with extensive experimental PUL characterization.nnnAVAILABILITY AND IMPLEMENTATIONnPUL predictions in 67 Bacteroidetes genomes from the human gut microbiota and two additional species, from the canine oral sphere and from the environment, are presented in our database accessible at www.cazy.org/PULDB/index.php.

Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides

Diet shapes host and gut microbe fitness The human gut microbiota is hugely diverse, with many strain variants having a multiplicity of effects on host metabolism and immunity. To define some of these functions, Wu et al. made libraries of mutants of Bacteroides species known for their capacity to process otherwise intractable dietary fiber. Germ-free mice colonized with defined gut microbiota communities containing the mutants were fed specific diets containing different ratios of fat and fiber. Genes, strains, and species were identified that were associated with specific metabolic pathways. The community responses to dietary shifts were manipulated in an attempt to characterize species for their probiotic or therapeutic potential. Science, this issue 10.1126/science.aac5992> To design probiotics, gut microbe fitness determinants and niches were characterized and responses to dietary changes monitored. INTRODUCTION Relatively little is known about the genetic factors that allow members of the human gut microbiota to occupy their niches. Identification of these factors is important for understanding mechanisms that determine microbiota assembly and perturbation through diet, disease, and clinical treatments. Discovery of these factors should enable new approaches for intervening therapeutically in the functional properties of the human gut microbiota. We present a generalizable approach by which to identify fitness determinants for multiple bacterial strains simultaneously in a model human gut microbiota, obtain gene-level characterization of responses to diet change, and design prebiotics for precision microbiota manipulation. RATIONALE We developed a method—multi-taxon INsertion Sequencing (INSeq)—for monitoring the behavior of tens of thousands of transposon (Tn) mutants of multiple bacterial species and strains simultaneously in the guts of gnotobiotic mice. We focused on four prominent human gut Bacteroides: one strain of B. cellulosilyticus, one strain of B. ovatus, and two strains of B. thetaiotaomicron. INSeq libraries, each composed of 87,000 to 167,000 isogenic Tn mutant strains, were produced (single site of Tn insertion per mutant strain; a total of 11 to 26 Tn insertions represented in the library per gene; and 82 to 92% genes covered per genome). The four mutant libraries were introduced into germ-free mice together with 11 wild-type species commonly present in the human gut microbiota. Animals were given a diet rich in fat and simple sugars but devoid of complex polysaccharides [diet 1 (D1)] or one rich in plant polysaccharides and low in fat (D2), either monotonously or in the sequence D1-D2-D1 or D2-D1-D2. Wecalculated a “fitness index” for each gene on the basis of the relative abundance of its INSeq reads in the fecal or cecal microbiota compared with the input library. In vivo INSeq data were correlated with INSeq data generated from organisms cultured under defined in vitro conditions; microbial RNA-seq profiling of the community’s metatranscriptome; and reconstructions of metabolic pathways, regulons, and polysaccharide utilization loci. On the basis of the results, we designed a prebiotic intervention. RESULTS Multi-taxon INSeq (i) provided a digital readout of the remarkably consistent pattern of community assembly; (ii) identified shared as well as species-, strain-, and diet-specific fitness determinants associated with a variety of metabolic or nutrient processing pathways, including those involving amino acids, carbohydrates, and vitamins/cofactors; (iii) enabled quantitative gene-level measurement of the resilience of the responses to diet perturbations; (iv) revealed that arabinoxylan, the most common hemicellulose in cereals, could be used to deliberately manipulate the representation of Bacteroides cellulosilyticus; and (v) defined the niche adjustments of this and the other Bacteroides to arabinoxylan supplementation of the high-fat diet. CONCLUSION In principle, the approach described can be used to obtain a more comprehensive understanding of how host genotype, diet, physiologic, metabolic, and immune factors, as well as pathologic states, affect niches in gut and nongut habitats, as well as to facilitate development of therapeutic interventions for modifying community structure/function. Identification of a prebiotic that increases the abundance of B. cellulosilyticus. (Left) The four mutant libraries were pooled together with 11 other phylogenetically diverse wild-type strains, and this consortium, representing an artificial human gut microbiota, was introduced into germ-free mice. Community assembly, the effects of diet, and recovery from diet oscillations were characterized at a community, strain, and gene level in these gnotobiotic animals by use of multi-taxon INSeq. (Middle) Multi-taxon INSeq revealed an arabinoxylan utilization locus in B. cellulosilyticus that is critical for the organism’s fitness in the high-fat/simple-sugar diet (D1) context but not in the D2 context. A homologous arabinoxylan utilization locus in B. ovatus was not a fitness determinant with either diet. (Right) Consistent with this finding, supplementation of drinking water with arabinoxylan in mice consuming D1 selectively increased the abundance of B. cellulosilyticus but not B. ovatus. Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in different ordered sequences. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability, and resilience and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness.

Rapid similarity search of proteins using alignments of domain arrangements

MOTIVATIONnHomology search methods are dominated by the central paradigm that sequence similarity is a proxy for common ancestry and, by extension, functional similarity. For determining sequence similarity in proteins, most widely used methods use models of sequence evolution and compare amino-acid strings in search for conserved linear stretches. Probabilistic models or sequence profiles capture the position-specific variation in an alignment of homologous sequences and can identify conserved motifs or domains. While profile-based search methods are generally more accurate than simple sequence comparison methods, they tend to be computationally more demanding. In recent years, several methods have emerged that perform protein similarity searches based on domain composition. However, few methods have considered the linear arrangements of domains when conducting similarity searches, despite strong evidence that domain order can harbour considerable functional and evolutionary signal.nnnRESULTSnHere, we introduce an alignment scheme that uses a classical dynamic programming approach to the global alignment of domains. We illustrate that representing proteins as strings of domains (domain arrangements) and comparing these strings globally allows for a both fast and sensitive homology search. Further, we demonstrate that the presented methods complement existing methods by finding similar proteins missed by popular amino-acid-based comparison methods.nnnAVAILABILITYnAn implementation of the presented algorithms, a web-based interface as well as a command-line program for batch searching against the UniProt database can be found at http://rads.uni-muenster.de. Furthermore, we provide a JAVA API for programmatic access to domain-string–based search methods.

DoMosaics: software for domain arrangement visualization and domain-centric analysis of proteins

UNLABELLEDnDoMosaics is an application that unifies protein domain annotation, domain arrangement analysis and visualization in a single tool. It simplifies the analysis of protein families by consolidating disjunct procedures based on often inconvenient command-line applications and complex analysis tools. It provides a simple user interface with access to domain annotation services such as InterProScan or a local HMMER installation, and can be used to compare, analyze and visualize the evolution of domain architectures.nnnAVAILABILITY AND IMPLEMENTATIONnDoMosaics is licensed under theApache License, Version 2.0, and binaries can be freely obtained from www.domosaics.net.

Ninety-nine de novo assembled genomes from the moose ( Alces alces ) rumen microbiome provide new insights into microbial plant biomass degradation

The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

Unraveling the pectinolytic function of Bacteroides xylanisolvens using a RNA-seq approach and mutagenesis

BackgroundDiet and particularly dietary fibres have an impact on the gut microbiome and play an important role in human health and disease. Pectin is a highly consumed dietary fibre found in fruits and vegetables and is also a widely used additive in the food industry. Yet there is no information on the effect of pectin on the human gut microbiome. Likewise, little is known on gut pectinolytic bacteria and their enzyme systems. This study was undertaken to investigate the mechanisms of pectin degradation by the prominent human gut symbiont Bacteroides xylanisolvens.ResultsTranscriptomic analyses of B. xylanisolvens XB1A grown on citrus and apple pectins at mid- and late-log phases highlighted six polysaccharide utilization loci (PUL) that were overexpressed on pectin relative to glucose. The PUL numbers used in this report are those given by Terrapon et al. (Bioinformatics 31(5):647-55, 2015) and found in the PUL database: http://www.cazy.org/PULDB/. Based on their CAZyme composition, we propose that PUL 49 and 50, the most overexpressed PULs on both pectins and at both growth phases, are involved in homogalacturonan (HG) and type I rhamnogalacturonan (RGI) degradation, respectively. PUL 13 and PUL 2 could be involved in the degradation of arabinose-containing side chains and of type II rhamnogalacturonan (RGII), respectively. Considering that HG is the most abundant moiety (>70 %) within pectin, the importance of PUL 49 was further investigated by insertion mutagenesis into the susC-like gene. The insertion blocked transcription of the susC-like and the two downstream genes (susD-like/FnIII). The mutant showed strong growth reduction, thus confirming that PUL 49 plays a major role in pectin degradation.ConclusionThis study shows the existence of six PULs devoted to pectin degradation by B. xylanisolvens, one of them being particularly important in this function. Hence, this species deploys a very complex enzymatic machinery that probably reflects the structural complexity of pectin. Our findings also highlight the metabolic plasticity of B. xylanisolvens towards dietary fibres that contributes to its competitive fitness within the human gut ecosystem. Wider functional and ecological studies are needed to understand how dietary fibers and especially plant cell wall polysaccharides drive the composition and metabolism of the fibrolytic and non-fibrolytic community within the gut microbial ecosystem.