Nicolas Parisot

The Human Gut Chip "HuGChip'', an explorative phylogenetic microarray for determining gut microbiome diversity at family level

Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearsons correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays.

Genome Sequence of "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1, a Third Thermoplasmatales-Related Methanogenic Archaeon from Human Feces

ABSTRACT “Candidatus Methanomassiliicoccus intestinalis” Issoire-Mx1 is a methanogenic archaeon found in the human gut and is a representative of the novel order of methanogens related to Thermoplasmatales. Its complete genome sequence is presented here.

Detecting unknown sequences with DNA microarrays: explorative probe design strategies.

Designing environmental DNA microarrays that can be used to survey the extreme diversity of microorganisms existing in nature, represents a stimulating challenge in the field of molecular ecology. Indeed, recent efforts in metagenomics have produced a substantial amount of sequence information from various ecosystems, and will continue to accumulate large amounts of sequence data given the qualitative and quantitative improvements in the next-generation sequencing methods. It is now possible to take advantage of these data to develop comprehensive microarrays by using explorative probe design strategies. Such strategies anticipate genetic variations and thus are able to detect known and unknown sequences in environmental samples. In this review, we provide a detailed overview of the probe design strategies currently available to construct both phylogenetic and functional DNA microarrays, with emphasis on those permitting the selection of such explorative probes. Furthermore, exploration of complex environments requires particular attention on probe sensitivity and specificity criteria. Finally, these innovative probe design approaches require exploiting newly available high-density microarray formats.

Gene Capture Coupled to High-Throughput Sequencing as a Strategy for Targeted Metagenome Exploration

Next-generation sequencing (NGS) allows faster acquisition of metagenomic data, but complete exploration of complex ecosystems is hindered by the extraordinary diversity of microorganisms. To reduce the environmental complexity, we created an innovative solution hybrid selection (SHS) method that is combined with NGS to characterize large DNA fragments harbouring biomarkers of interest. The quality of enrichment was evaluated after fragments containing the methyl coenzyme M reductase subunit A gene (mcrA), the biomarker of methanogenesis, were captured from a Methanosarcina strain and a metagenomic sample from a meromictic lake. The methanogen diversity was compared with direct metagenome and mcrA-based amplicon pyrosequencing strategies. The SHS approach resulted in the capture of DNA fragments up to 2.5 kb with an enrichment efficiency between 41 and 100%, depending on the sample complexity. Compared with direct metagenome and amplicons sequencing, SHS detected broader mcrA diversity, and it allowed efficient sampling of the rare biosphere and unknown sequences. In contrast to amplicon-based strategies, SHS is less biased and GC independent, and it recovered complete biomarker sequences in addition to conserved regions. Because this method can also isolate the regions flanking the target sequences, it could facilitate operon reconstructions.

Annotation of microsporidian genomes using transcriptional signals

High-quality annotation of microsporidian genomes is essential for understanding the biological processes that govern the development of these parasites. Here we present an improved structural annotation method using transcriptional DNA signals. We apply this method to re-annotate four previously annotated genomes, which allow us to detect annotation errors and identify a significant number of unpredicted genes. We then annotate the newly sequenced genome of Anncaliia algerae. A comparative genomic analysis of A. algerae permits the identification of not only microsporidian core genes, but also potentially highly expressed genes encoding membrane-associated proteins, which represent good candidates involved in the spore architecture, the invasion process and the microsporidian-host relationships. Furthermore, we find that the ten-fold variation in microsporidian genome sizes is not due to gene number, size or complexity, but instead stems from the presence of transposable elements. Such elements, along with kinase regulatory pathways and specific transporters, appear to be key factors in microsporidian adaptive processes.

Impact of clay mineral, wood sawdust or root organic matter on the bacterial and fungal community structures in two aged PAH-contaminated soils.

The high organic pollutant concentration of aged polycyclic aromatic hydrocarbon (PAH)-contaminated wasteland soils is highly recalcitrant to biodegradation due to its very low bioavailability. In such soils, the microbial community is well adapted to the pollution, but the microbial activity is limited by nutrient availability. Management strategies could be applied to modify the soil microbial functioning as well as the PAH contamination through various amendment types. The impact of amendment with clay minerals (montmorillonite), wood sawdust and organic matter plant roots on microbial community structure was investigated on two aged PAH-contaminated soils both in laboratory and 1-year on-site pot experiments. Total PAH content (sum of 16 PAHs of the US-EPA list) and polar polycyclic aromatic compounds (pPAC) were monitored as well as the available PAH fraction using the Tenax method. The bacterial and fungal community structures were monitored using fingerprinting thermal gradient gel electrophoresis (TTGE) method. The abundance of bacteria (16S rRNA genes), fungi (18S rRNA genes) and PAH degraders (PAH-ring hydroxylating dioxygenase and catechol dioxygenase genes) was followed through qPCR assays. Although the treatments did not modify the total and available PAH content, the microbial community density, structure and the PAH degradation potential changed when fresh organic matter was provided as sawdust and under rhizosphere influence, while the clay mineral only increased the percentage of catechol-1,2-dioxygenase genes. The abundance of bacteria and fungi and the percentage of fungi relative to bacteria were enhanced in soil samples supplemented with wood sawdust and in the plant rhizospheric soils. Two distinct fungal populations developed in the two soils supplemented with sawdust, i.e. fungi related to Chaetomium and Neurospora genera and Brachyconidiellopsis and Pseudallescheria genera, in H and NM soils respectively. Wood sawdust amendment favoured the development of PAH-degrading bacteria holding Gram-negative PAH-ring hydroxylating dioxygenase, catechol-1,2-dioxygenase and catechol-2,3-dioxygenase genes. Regarding the total community structure, bacteria closely related to Thiobacillus (β-Proteobacteria) and Steroidobacter (γ-Proteobacteria) genera were favoured by wood sawdust amendment. In both soils, plant rhizospheres induced the development of fungi belonging to Ascomycota and related to Alternaria and Fusarium genera. Bacteria closely related to Luteolibacter (Verrucomicrobia) and Microbacterium (Actinobacteria) were favoured in alfalfa and ryegrass rhizosphere.

KASpOD—a web service for highly specific and explorative oligonucleotide design

SUMMARY KASpOD is a web service dedicated to the design of signature sequences using a k-mer-based algorithm. Such highly specific and explorative oligonucleotides are then suitable for various goals, including Phylogenetic Oligonucleotide Arrays. AVAILABILITY http://g2im.u-clermont1.fr/kaspod. CONTACT eric.peyretaillade@udamail.fr SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Oral exposure to environmental pollutant benzo[a]pyrene impacts the intestinal epithelium and induces gut microbial shifts in murine model.

Gut microbiota dysbiosis are associated with a wide range of human diseases, including inflammatory bowel diseases. The physiopathology of these diseases has multifactorial aetiology in which environmental factors, particularly pollution could play a crucial role. Among the different pollutants listed, Polycyclic Aromatic Hydrocarbons (PAHs) are subject to increased monitoring due to their wide distribution and high toxicity on Humans. Here, we used 16S rRNA gene sequencing to investigate the impact of benzo[a]pyrene (BaP, most toxic PAH) oral exposure on the faecal and intestinal mucosa-associated bacteria in C57BL/6 mice. Intestinal inflammation was also evaluated by histological observations. BaP oral exposure significantly altered the composition and the abundance of the gut microbiota and led to moderate inflammation in ileal and colonic mucosa. More severe lesions were observed in ileal segment. Shifts in gut microbiota associated with moderate inflammatory signs in intestinal mucosa would suggest the establishment of a pro-inflammatory intestinal environment following BaP oral exposure. Therefore, under conditions of genetic susceptibility and in association with other environmental factors, exposure to this pollutant could trigger and/or accelerate the development of inflammatory pathologies.

Metatranscriptomics Reveals the Active Bacterial and Eukaryotic Fibrolytic Communities in the Rumen of Dairy Cow Fed a Mixed Diet

Ruminants have a unique ability to derive energy from the degradation of plant polysaccharides through the activity of the rumen microbiota. Although this process is well studied in vitro, knowledge gaps remain regarding the relative contribution of the microbiota members and enzymes in vivo. The present study used RNA-sequencing to reveal both the expression of genes encoding carbohydrate-active enzymes (CAZymes) by the rumen microbiota of a lactating dairy cow and the microorganisms forming the fiber-degrading community. Functional analysis identified 12,237 CAZymes, accounting for 1% of the transcripts. The CAZyme profile was dominated by families GH94 (cellobiose-phosphorylase), GH13 (amylase), GH43 and GH10 (hemicellulases), GH9 and GH48 (cellulases), PL11 (pectinase) as well as GH2 and GH3 (oligosaccharidases). Our data support the pivotal role of the most characterized fibrolytic bacteria (Prevotella, Ruminocccus and Fibrobacter), and highlight a substantial, although most probably underestimated, contribution of fungi and ciliate protozoa to polysaccharide degradation. Particularly these results may motivate further exploration of the role and the functions of protozoa in the rumen. Moreover, an important part of the fibrolytic bacterial community remains to be characterized since one third of the CAZyme transcripts originated from distantly related strains. These findings are used to highlight limitations of current metatranscriptomics approaches to understand the functional rumen microbial community and opportunities to circumvent them.

Hijacking of host cellular functions by an intracellular parasite, the microsporidian Anncaliia algerae.

Intracellular pathogens including bacteria, viruses and protozoa hijack host cell functions to access nutrients and to bypass cellular defenses and immune responses. These strategies have been acquired through selective pressure and allowed pathogens to reach an appropriate cellular niche for their survival and growth. To get new insights on how parasites hijack host cellular functions, we developed a SILAC (Stable Isotope Labeling by Amino Acids in Cell culture) quantitative proteomics workflow. Our study focused on deciphering the cross-talk in a host-parasite association, involving human foreskin fibroblasts (HFF) and the microsporidia Anncaliia algerae, a fungus related parasite with an obligate intracellular lifestyle and a strong host dependency. The host-parasite cross-talk was analyzed at five post-infection times 1, 6, 12 and 24 hours post-infection (hpi) and 8 days post-infection (dpi). A significant up-regulation of four interferon-induced proteins with tetratricopeptide repeats IFIT1, IFIT2, IFIT3 and MX1 was observed at 8 dpi suggesting a type 1 interferon (IFN) host response. Quantitative alteration of host proteins involved in biological functions such as signaling (STAT1, Ras) and reduction of the translation activity (EIF3) confirmed a host type 1 IFN response. Interestingly, the SILAC approach also allowed the detection of 148 A. algerae proteins during the kinetics of infection. Among these proteins many are involved in parasite proliferation, and an over-representation of putative secreted effectors proteins was observed. Finally our survey also suggests that A. algerae could use a transposable element as a lure strategy to escape the host innate immune system.