MicroAnnot: A Dedicated Workflow for Accurate Microsporidian Genome Annotation.

With nearly 1700 species, Microsporidia represent a group of obligate intracellular eukaryotes with veterinary, economic and medical impacts. To help understand the biological functions of these microorganisms, complete genome sequencing is routinely used. Nevertheless, the proper prediction of their gene catalogue is challenging due to their taxon-specific evolutionary features. As innovative genome annotation strategies are needed to obtain a representative snapshot of the overall lifestyle of these parasites, the MicroAnnot tool, a dedicated workflow for microsporidian sequence annotation using data from curated databases of accurately annotated microsporidian genes, has been developed. Furthermore, specific modules have been implemented to perform small gene (<300 bp) and transposable element identification. Finally, functional annotation was performed using the signature-based InterProScan software. MicroAnnot's accuracy has been verified by the re-annotation of four microsporidian genomes for which structural annotation had previously been validated. With its comparative approach and transcriptional signal identification method, MicroAnnot provides an accurate prediction of translation initiation sites, an efficient identification of transposable elements, as well as high specificity and sensitivity for microsporidian genes, including those under 300 bp.

The intracellular parasite Anncaliia algerae induces a massive miRNA down-regulation in human cells.

Anncaliia algerae belongs to microsporidia, a group of obligate intracellular parasites related to fungi. These parasites are largely spread in water and food-webs and can infect a wide variety of hosts ranging from invertebrates to vertebrates including humans. In humans, microsporidian infections are mainly opportunistic as immunocompetent hosts can clear parasites naturally. Recent studies however have reported persistent microsporidian infections and have highlighted them as a risk factor in colon cancer. This may be a direct result of cell infection or may be an indirect effect of the infectious microenvironment and the host's response. In both cases, this raises the question of the effects of microsporidian infection at the host and host-cell levels. We aimed to address the question of human host intracellular response to microsporidian infection through a transcriptomic kinetic study of human foreskin fibroblasts (HFF) infected with A.algerae, a human infecting microsporidia with an exceptionally wide host range. We focused solely on host response studying both coding and small non-coding miRNA expression. Our study revealed a generalized down-regulation of cell miRNAs throughout infection with up to 547 different miRNAs downregulated at some timepoints and also transcriptomic dysregulations that could facilitate parasite development with immune and lipid metabolism genes modulation. We also hypothesize possible small nucleic acid expropriation explaining the miRNA downregulation. This work contributes to a better understanding of the dialogue that can occur between an intracellular parasite and its host at the cellular level, and can guide future studies on microsporidian infection biology to unravel the mode of action of these minimalist parasites at the tissue or host levels.We have also generated a kinetic and comprehensive transcriptomic data set of an infectious process that can help support comparative studies in the broader field of parasitology. Lastly, these results may warrant for caution regarding microsporidian exposure and persistent infections.

Identification and localization of polar tube proteins in the extruded polar tube of the microsporidian Anncaliia algerae.

Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae. While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae.

Genome-Wide Analysis of Antigen 43 (Ag43) Variants: New Insights in Their Diversity, Distribution and Prevalence in Bacteria.

Antigen 43 (Ag43) expression induces aggregation and biofilm formation that has consequences for bacterial colonisation and infection. Ag43 is secreted through the Type 5 subtype "a" secretion system (T5aSS) and is a prototypical member of the family of self-associating autotransporters (SAATs). As a T5aSS protein, Ag43 has a modular architecture comprised of (i) a signal peptide, (ii) a passenger domain that can be subdivided into three subdomains (SL, EJ, and BL), (iii) an autochaperone (AC) domain, and (iv) an outer membrane translocator. The cell-surface SL subdomain is directly involved in the "Velcro-handshake" mechanism resulting in bacterial autoaggregation. Ag43 is considered to have a ubiquitous distribution in E. coli genomes and many strains harbour multiple agn43 genes. However, recent phylogenetic analyses indicated the existence of four distinct Ag43 classes exhibiting different propensities for autoaggregation and interactions. Given the knowledge of the diversity and distribution of Ag43 in E. coli genomes is incomplete, we have performed a thorough in silico investigation across bacterial genomes. Our comprehensive analyses indicate that Ag43 passenger domains cluster in six phylogenetic classes associated with different SL subdomains. The diversity of Ag43 passenger domains is a result of the association of the SL subtypes with two different EJ-BL-AC modules. We reveal that agn43 is almost exclusively present among bacterial species of the Enterobacteriaceae family and essentially in the Escherichia genus (99.6%) but that it is not ubiquitous in E. coli. The gene is typically present as a single copy but up to five copies of agn43 with different combinations of classes can be observed. The presence of agn43 as well as its different classes appeared to differ between Escherichia phylogroups. Strikingly, agn43 is present in 90% of E. coli from E phylogroup. Our results shed light on Ag43 diversity and provide a rational framework for investigating its role in E. coli ecophysiology and physiopathology.

Draft Genome Sequence of Tubulinosema ratisbonensis, a Microsporidian Species Infecting the Model Organism Drosophila melanogaster.

We present the draft genome sequence of Tubulinosema ratisbonensis, a microsporidium species infecting Drosophila melanogaster A total of 3,013 protein-encoding genes and an array of transposable elements were identified. This work represents a necessary step to develop a novel model of host-parasite relationships using the highly tractable genetic model D. melanogaster.

ASaiM: a Galaxy-based framework to analyze microbiota data.

Background: New generations of sequencing platforms coupled to numerous bioinformatics tools have led to rapid technological progress in metagenomics and metatranscriptomics to investigate complex microorganism communities. Nevertheless, a combination of different bioinformatic tools remains necessary to draw conclusions out of microbiota studies. Modular and user-friendly tools would greatly improve such studies. Findings: We therefore developed ASaiM, an Open-Source Galaxy-based framework dedicated to microbiota data analyses. ASaiM provides an extensive collection of tools to assemble, extract, explore, and visualize microbiota information from raw metataxonomic, metagenomic, or metatranscriptomic sequences. To guide the analyses, several customizable workflows are included and are supported by tutorials and Galaxy interactive tours, which guide users through the analyses step by step. ASaiM is implemented as a Galaxy Docker flavour. It is scalable to thousands of datasets but also can be used on a normal PC. The associated source code is available under Apache 2 license at https://github.com/ASaiM/framework and documentation can be found online (http://asaim.readthedocs.io). Conclusions: Based on the Galaxy framework, ASaiM offers a sophisticated environment with a variety of tools, workflows, documentation, and training to scientists working on complex microorganism communities. It makes analysis and exploration analyses of microbiota data easy, quick, transparent, reproducible, and shareable.

Characterization of an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC-MS system: application to Arthrospira platensis.

We developed an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC-MS system run in SIM mode. The profiling approach was optimized for the derivatization protocol of some 60 targeted metabolites. The performance of two silylation reagents (MSTFA and BSTFA) that allowed the comprehensive derivatization of 42 key intermediary metabolites of the 60 initially targeted (organic acids, phosphate derivatives, monosaccharides and amino acids) was measured. The experimental results unequivocally showed that the MSTFA reagent met mandatory criteria including ease of handling (a very simple one-step protocol was developed), comprehensiveness of derivatization (the 42 compounds covered the extended metabolic pathways of the central carbon metabolism, with a coverage percentage ranging from 17% for the worst to 90% for the best result), optimized response coefficient of the whole derivatives (median value greater than the others by one order of magnitude) and repeatability of the protocol (RSD value below 25% for the whole procedure). When tested in real conditions (cyanobacteria polar extract), the experimental results showed that the profiling methodology was adequately repeatable (RSD = 35%) to ensure quantification results comparable with much more sensitive analytical techniques (capillary electrophoresis/mass spectrometry and liquid chromatography/triple quadrupole mass spectrometry system), while needing only about twice the quantity of biomass. Graphical abstract Schematic overview of an easy-to-use profiling method for the routine analysis of the central metabolism using a low-resolution GC-MS system.

Environmental Pollutant Benzo[a]Pyrene Impacts the Volatile Metabolome and Transcriptome of the Human Gut Microbiota.

Benzo[a]pyrene (B[a]P) is a ubiquitous, persistent, and carcinogenic pollutant that belongs to the large family of polycyclic aromatic hydrocarbons. Population exposure primarily occurs via contaminated food products, which introduces the pollutant to the digestive tract. Although the metabolism of B[a]P by host cells is well known, its impacts on the human gut microbiota, which plays a key role in health and disease, remain unexplored. We performed an in vitro assay using 16S barcoding, metatranscriptomics and volatile metabolomics to study the impact of B[a]P on two distinct human fecal microbiota. B[a]P exposure did not induce a significant change in the microbial structure; however, it altered the microbial volatolome in a dose-dependent manner. The transcript levels related to several metabolic pathways, such as vitamin and cofactor metabolism, cell wall compound metabolism, DNA repair and replication systems, and aromatic compound metabolism, were upregulated, whereas the transcript levels related to the glycolysis-gluconeogenesis pathway and bacterial chemotaxis toward simple carbohydrates were downregulated. These primary findings show that food pollutants, such as B[a]P, alter human gut microbiota activity. The observed shift in the volatolome demonstrates that B[a]P induces a specific deviation in the microbial metabolism.

Comparative genomics of microsporidian genomes reveals a minimal non-coding RNA set and new insights for transcription in minimal eukaryotic genomes.

Microsporidia are ubiquitous intracellular pathogens whose opportunistic nature led to their increased recognition with the rise of the AIDS pandemic. As the RNA world was largely unexplored in this parasitic lineage, we developed a dedicated in silico methodology to carry out exhaustive identification of ncRNAs across the Encephalitozoon and Nosema genera. Thus, the previously missing U1 small nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs) targeting only the LSU rRNA were highlighted and were further validated using 5' and 3'RACE-PCR experiments. Overall, the 15 ncRNAs that were found shared between Encephalitozoon and Nosema spp. may represent the minimal core set required for parasitic life. Interestingly, the systematic presence of a CCC- or GGG-like motif in 5' of all ncRNA and mRNA gene transcripts regardless of the RNA polymerase involved suggests that the RNA polymerase machineries in microsporidia species could use common factors. Our data provide additional insights in accordance with the simplification processes observed in these reduce genomes and underline the usefulness of sequencing closely related species to help identify highly divergent ncRNAs in these parasites.

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.

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.

Sequence capture by hybridization to explore modern and ancient genomic diversity in model and nonmodel organisms.

The recent expansion of next-generation sequencing has significantly improved biological research. Nevertheless, deep exploration of genomes or metagenomic samples remains difficult because of the sequencing depth and the associated costs required. Therefore, different partitioning strategies have been developed to sequence informative subsets of studied genomes. Among these strategies, hybridization capture has proven to be an innovative and efficient tool for targeting and enriching specific biomarkers in complex DNA mixtures. It has been successfully applied in numerous areas of biology, such as exome resequencing for the identification of mutations underlying Mendelian or complex diseases and cancers, and its usefulness has been demonstrated in the agronomic field through the linking of genetic variants to agricultural phenotypic traits of interest. Moreover, hybridization capture has provided access to underexplored, but relevant fractions of genomes through its ability to enrich defined targets and their flanking regions. Finally, on the basis of restricted genomic information, this method has also allowed the expansion of knowledge of nonreference species and ancient genomes and provided a better understanding of metagenomic samples. In this review, we present the major advances and discoveries permitted by hybridization capture and highlight the potency of this approach in all areas of biology.

Targeted Gene Capture by Hybridization to Illuminate Ecosystem Functioning.

Microbial communities are extremely abundant and diverse on earth surface and play key role in the ecosystem functioning. Thus, although next-generation sequencing (NGS) technologies have greatly improved knowledge on microbial diversity, it is necessary to reduce the biological complexity to better understand the microorganism functions. To achieve this goal, we describe a promising approach, based on the solution hybrid selection (SHS) method for the selective enrichment in a target-specific biomarker from metagenomic and metatranscriptomic samples. The success of this method strongly depends on the determination of sensitive, specific, and explorative probes to assess the complete targeted gene repertoire. Indeed, in this method, RNA probes were used to capture large DNA or RNA fragments harboring biomarkers of interest that potentially allow to link structure and function of communities of interest.

Distribution of Dehalococcoidia in the Anaerobic Deep Water of a Remote Meromictic Crater Lake and Detection of Dehalococcoidia-Derived Reductive Dehalogenase Homologous Genes.

Here we describe the natural occurrence of bacteria of the class Dehalococcoidia (DEH) and their diversity at different depths in anoxic waters of a remote meromictic lake (Lake Pavin) using 16S rRNA gene amplicon sequencing and quantitative PCR. Detected DEH are phylogenetically diverse and the majority of 16S rRNA sequences have less than 91% similarity to previously isolated DEH 16S rRNA sequences. To predict the metabolic potential of detected DEH subgroups and to assess if they encode genes to transform halogenated compounds, we enriched DEH-affiliated genomic DNA by using a specific-gene capture method and probes against DEH-derived 16S rRNA genes, reductive dehalogenase genes and known insertion sequences. Two reductive dehalogenase homologous sequences were identified from DEH-enriched genomic DNA, and marker genes in the direct vicinity confirm that gene fragments were derived from DEH. The low sequence similarity with known reductive dehalogenase genes suggests yet-unknown catabolic potential in the anoxic zone of Lake Pavin.

Probe Design Strategies for Oligonucleotide Microarrays.

Oligonucleotide microarrays have been widely used for gene detection and/or quantification of gene expression in various samples ranging from a single organism to a complex microbial assemblage. The success of a microarray experiment, however, strongly relies on the quality of designed probes. Consequently, probe design is of critical importance and therefore multiple parameters should be considered for each probe in order to ensure high specificity, sensitivity, and uniformity as well as potentially quantitative power. Moreover, to assess the complete gene repertoire of complex biological samples such as those studied in the field of microbial ecology, exploratory probe design strategies must be also implemented to target not-yet-described sequences. To design such probes, two algorithms, KASpOD and HiSpOD, have been developed and they are available via two user-friendly web services. Here, we describe the use of this software necessary for the design of highly effective probes especially in the context of microbial oligonucleotide microarrays by taking into account all the crucial parameters.

The Prediction and Validation of Small CDSs Expand the Gene Repertoire of the Smallest Known Eukaryotic Genomes.

The proper prediction of the gene catalogue of an organism is essential to obtain a representative snapshot of its overall lifestyle, especially when it is not amenable to culturing. Microsporidia are obligate intracellular, sometimes hard to culture, eukaryotic parasites known to infect members of every animal phylum. To date, sequencing and annotation of microsporidian genomes have revealed a poor gene complement with highly reduced gene sizes. In the present paper, we investigated whether such gene sizes may have induced biases for the methodologies used for genome annotation, with an emphasis on small coding sequence (CDS) gene prediction. Using better delineated intergenic regions from four Encephalitozoon genomes, we predicted de novo new small CDSs with sizes ranging from 78 to 255 bp (median 168) and corroborated these predictions by RACE-PCR experiments in Encephalitozoon cuniculi. Most of the newly found genes are present in other distantly related microsporidian species, suggesting their biological relevance. The present study provides a better framework for annotating microsporidian genomes and to train and evaluate new computational methods dedicated at detecting ultra-small genes in various organisms.

Capturing prokaryotic dark matter genomes.

Prokaryotes are the most diverse and abundant cellular life forms on Earth. Most of them, identified by indirect molecular approaches, belong to microbial dark matter. The advent of metagenomic and single-cell genomic approaches has highlighted the metabolic capabilities of numerous members of this dark matter through genome reconstruction. Thus, linking functions back to the species has revolutionized our understanding of how ecosystem function is sustained by the microbial world. This review will present discoveries acquired through the illumination of prokaryotic dark matter genomes by these innovative approaches.

Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment.

Draft Genomes of Three Strains Representative of the Bacillus anthracis Diversity Found in France.

We report here the draft genomes of three Bacillus anthracis strains isolated in France: 08-8_20 (A.Br.001/002), 99-100 (A.Br.011/009), and 00-82 (B.Br CNEVA). The total lengths of assemblies are 5,440,708 bp, 5,446,472 bp, and 5,436,014 bp for 08-8_20, 99-100, and 00-82, respectively.

Microsporidian genomes harbor a diverse array of transposable elements that demonstrate an ancestry of horizontal exchange with metazoans.

Microsporidian genomes are the leading models to understand the streamlining in response to a pathogenic lifestyle; they are gene-poor and often possess small genomes. In this study, we show a feature of microsporidian genomes that contrasts this pattern of genome reduction. Specifically, genome investigations targeted at Anncaliia algerae, a human pathogen with a genome size of 23 Mb, revealed the presence of a hitherto undetected diversity in transposable elements (TEs). A total of 240 TE families per genome were identified, exceeding that found in many free-living fungi, and searches of microsporidian species revealed that these mobile elements represent a significant portion of their coding repertoire. Their phylogenetic analysis revealed that many cases of ancestry involve recent and bidirectional horizontal transfers with metazoans. The abundance and horizontal transfer origin of microsporidian TEs highlight a novel dimension of genome evolution in these intracellular pathogens, demonstrating that factors beyond reduction are at play in their diversification.