Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine

BACKGROUND: A seventh order of methanogens, the Methanomassiliicoccales, has been identified in diverse anaerobic environments including the gastrointestinal tracts (GIT) of humans and other animals and may contribute significantly to methane emission and global warming. Methanomassiliicoccales are phylogenetically distant from all other orders of methanogens and belong to a large evolutionary branch composed by lineages of non-methanogenic archaea such as Thermoplasmatales, the Deep Hydrothermal Vent Euryarchaeota-2 (DHVE-2, Aciduliprofundum boonei) and the Marine Group-II (MG-II). To better understand this new order and its relationship to other archaea, we manually curated and extensively compared the genome sequences of three Methanomassiliicoccales representatives derived from human GIT microbiota, “Candidatus Methanomethylophilus alvus", “Candidatus Methanomassiliicoccus intestinalis” and Methanomassiliicoccus luminyensis. RESULTS: Comparative analyses revealed atypical features, such as the scattering of the ribosomal RNA genes in the genome and the absence of eukaryotic-like histone gene otherwise present in most of Euryarchaeota genomes. Previously identified in Thermoplasmatales genomes, these features are presently extended to several completely sequenced genomes of this large evolutionary branch, including MG-II and DHVE2. The three Methanomassiliicoccales genomes share a unique composition of genes involved in energy conservation suggesting an original combination of two main energy conservation processes previously described in other methanogens. They also display substantial differences with each other, such as their codon usage, the nature and origin of their CRISPRs systems and the genes possibly involved in particular environmental adaptations. The genome of M. luminyensis encodes several features to thrive in soil and sediment conditions suggesting its larger environmental distribution than GIT. Conversely, “Ca. M. alvus” and “Ca. M. intestinalis” do not present these features and could be more restricted and specialized on GIT. Prediction of the amber codon usage, either as a termination signal of translation or coding for pyrrolysine revealed contrasted patterns among the three genomes and suggests a different handling of the Pyl-encoding capacity. CONCLUSIONS: This study represents the first insights into the genomic organization and metabolic traits of the seventh order of methanogens. It suggests contrasted evolutionary history among the three analyzed Methanomassiliicoccales representatives and provides information on conserved characteristics among the overall methanogens and among Thermoplasmat

Approaches to sequence the HTT CAG repeat expansion and quantify repeat length variation

Background: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of the HTT CAG repeat. Affected individuals inherit ≥36 repeats and longer alleles cause earlier onset, greater disease severity and faster disease progression. The HTT CAG repeat is genetically unstable in the soma in a process that preferentially generates somatic expansions, the proportion of which is associated with disease onset, severity and progression. Somatic mosaicism of the HTT CAG repeat has traditionally been assessed by semi-quantitative PCR-electrophoresis approaches that have limitations (e.g., no information about sequence variants). Genotyping-by-sequencing could allow for some of these limitations to be overcome. Objective: To investigate the utility of PCR sequencing to genotype large (>50 CAGs) HD alleles and to quantify the associated somatic mosaicism. Methods: We have applied MiSeq and PacBio sequencing to PCR products of the HTT CAG repeat in transgenic R6/2 mice carrying ∼55, ∼110, ∼255 and ∼470 CAGs. For each of these alleles, we compared the repeat length distributions generated for different tissues at two ages. Results: We were able to sequence the CAG repeat full length in all samples. However, the repeat length distributions for samples with ∼470 CAGs were biased towards shorter repeat lengths. Conclusion: PCR sequencing can be used to sequence all the HD alleles considered, but this approach cannot be used to estimate modal allele size or quantify somatic expansions for alleles ⪢250 CAGs. We review the limitations of PCR sequencing and alternative approaches that may allow the quantification of somatic contractions and very large somatic expansions

Cryptosporidium parvum can subvert the host immune response through manipulation of CRAMP expression during neonatal infection

Due to the immaturity of their immune system, neonates are highly sensitive to intestinal infections. During the neonatal period, antimicrobial peptide (AMP) composition differs substantially from that of adults. This is the case in the small intestine for the cathelicidin-related antimicrobial peptide (CRAMP) expressed preferentially in the neonatal period while conversely other AMPs such as Reg3γ are expressed later in life. Among enteric neonatal diseases, Cryptosporidiosis is a zoonotic disease and is highly prevalent in children less than 5 years old in developing countries and in neonatal ruminants worldwide. Cryptosporidium parvum is the etiological agent of this diarrheal disease and infects exclusively epithelial cells. Innate immunity is important to control the acute phase of infection in neonates with dendritic cells and IFNγ playing a major role. Antimicrobial peptides are important contributors of innate immunity, but the role of CRAMP, which is elevated in the intestine of neonates has never been investigated during Cryptosporidiosis so far. In this work, we observed in the neonatal murine model of cryptosporidiosis that unlike other antimicrobial molecules such as Reg3 and Lysozyme, CRAMP expression was significantly reduced in the intestine during infection. By using different genetically modified mouse models, we demonstrated that the reduced CRAMP expression was independent of IFN , a pro-inflammatory cytokine strongly produced during infection, but also of Myd88, an adaptor molecule involved in innate immune signalling. We also excluded the role of gut flora in this response. When C. parvum infected neonatal mice orally received exogenous CRAMP to compensate the reduced expression of this AMP, the parasitic load of neonates was significantly decreased. In addition, when free parasites were in direct contact with CRAMP, this AMP affected the viability of sporozoites. All together, these data suggest that C. parvum induces the reduction of CRAMP expression to escape the anti-parasiticidal effect of CRAMP

Functional amplification and preservation of human gut microbiota

Background: The availability of fresh stool samples is a prerequisite in most gut microbiota functional studies. Objective: Strategies for amplification and long-term gut microbiota preservation from fecal samples would favor sample sharing, help comparisons and reproducibility over time and between laboratories, and improve the safety and ethical issues surrounding fecal microbiota transplantations. Design: Taking advantage of in vitro gut-simulating systems, we amplified the microbial repertoire of a fresh fecal sample and assessed the viability and resuscitation of microbes after preservation with some common intracellular and extracellular acting cryoprotective agents (CPAs), alone and in different combinations. Preservation efficiencies were determined after 3 and 6 months and compared with the fresh initial microbiota diversity and metabolic activity, using the chemostat-based Environmental Control System for Intestinal Microbiota (ECSIM) in vitro model of the gut environment. Microbial populations were tested for fermentation gas, short-chain fatty acids, and composition of amplified and resuscitated microbiota, encompassing methanogenic archaea. Results: Amplification of the microbial repertoire from a fresh fecal sample was achieved with high fidelity. Dimethylsulfoxide, alone or mixed with other CPAs, showed the best efficiency for functional preservation, and the duration of preservation had little effect. Conclusions: The amplification and resuscitation of fecal microbiota can be performed using specialized in vitro gut models. Correct amplification of the initial microbes should ease the sharing of clinical samples and improve the safety of fecal microbiota transplantation. Abbreviations: CDI, Clostridium difficile infection; CPA, cryoprotective agent; D, DMSO, dimethylsulfoxide; FMT, fecal microbiota transplantation; G, glycerol; IBD, inflammatory bowel disease; P, PEG-4000, polyethylene glycol 4000 g.mol−1; SCFA, short-chain fatty acid; SNR, signal-to-noise rati

<em>Cryptosporidium parvum</em> can subvert the host immune response through manipulation of CRAMP expression during neonatal infection

National audienceDue to the immaturity of their immune system, neonates are highly sensitive to intestinal infections. During the neonatal period, antimicrobial peptide (AMP) composition differs substantially from that of adults. This is the case in the small intestine for the cathelicidin-related antimicrobial peptide (CRAMP) expressed preferentially in the neonatal period while conversely other AMPs such as Reg3γ are expressed later in life. Among enteric neonatal diseases, Cryptosporidiosis is a zoonotic disease and is highly prevalent in children less than 5 years old in developing countries and in neonatal ruminants worldwide. Cryptosporidium parvum is the etiological agent of this diarrheal disease and infects exclusively epithelial cells. Innate immunity is important to control the acute phase of infection in neonates with dendritic cells and IFNγ playing a major role. Antimicrobial peptides are important contributors of innate immunity, but the role of CRAMP, which is elevated in the intestine of neonates has never been investigated during Cryptosporidiosis so far. In this work, we observed in the neonatal murine model of cryptosporidiosis that unlike other antimicrobial molecules such as Reg3 and Lysozyme, CRAMP expression was significantly reduced in the intestine during infection. By using different genetically modified mouse models, we demonstrated that the reduced CRAMP expression was independent of IFN , a pro-inflammatory cytokine strongly produced during infection, but also of Myd88, an adaptor molecule involved in innate immune signalling. We also excluded the role of gut flora in this response. When C. parvum infected neonatal mice orally received exogenous CRAMP to compensate the reduced expression of this AMP, the parasitic load of neonates was significantly decreased. In addition, when free parasites were in direct contact with CRAMP, this AMP affected the viability of sporozoites. All together, these data suggest that C. parvum induces the reduction of CRAMP expression to escape the anti-parasiticidal effect of CRAMP

Le concept "Archaebiotics", vers de nouveaux probiotiques préventifs des maladies cardiovasculaires ?

Communication orale: Brugère J.F.D'origine intestinale, la trimethylamine (TMA) provient du métabolisme de divers nutriments par le microbiote intestinal (phosphatidylcholine, choline, L-carnitine, oxyde de trimethylamine TMAO). Absorbée, elle est oxydée par la flavine-monoxygénase 3 hépatique (FMO3) en TMAO qui rejoint la circulation sanguine. Très récemment, le TMAO (d'origine alimentaire via le métabolisme microbien de l'intestin) est apparu comme biomarqueur du risque cardiovasculaire et un rôle athérogène est fortement suspecté. Nous avons mis en évidence un nouvel ordre d'Archaea méthanogènes (7e ordre), présent notamment dans le tractus digestif (insectes, ruminants, homme) avec une plus grande prévalence chez les personnes âgées de 65 ans et plus (de l'ordre de 20% de la population). A partir de 3 génomes de souches digestives, des caractéristiques métaboliques et énergétiques uniques au sein du vivant ont pu être mises en évidence, telle une méthanogenèse semblant strictement limitée aux composés méthylés (méthanol, méthylamines, diméthylsulfide), sous la dépendance d'H2: le métabolisme anaérobie de la TMA est ainsi possible, et fait intervenir notamment une méthyltransférase particulière (MttB) incorporant au moment de sa synthèse, la pyrrolysine (Pyl, 22e acide aminé protéogène) par suppression traductionnelle d'un codon non-sens ambre. Cette conversion métabolique en méthane est montrée en culture pure de la seule souche isolée à ce jour (Methanomassiliicoccus luminyensis). Ceci fait de ce 7e ordre, en particulier des souches digestives, des candidats privilégiés pour une utilisation probiotique. Certaines souches montrent une meilleure adaptation à l'environnement digestif humain, avec des gènes de résistance aux sels biliaires. De plus, le méthane produit est considéré comme inerte pour l'homme (exhalé par environ 50 % de la population humaine productrice de ce gaz par son microbiote). Pour ces raisons, il serait possible de réduire la production de TMA par le microbiote intestinal au niveau intestinal, et par cela, la production hépatique de TMAO délétère, directement in situ au moment de sa synthèse intestinale, via des archaebiotiques

Shifts in the microbial community structure explain the response of soil respiration to land-use change but not to climate warming

Soil stores more carbon (C) than plants and atmosphere combined and it is vulnerable to increased microbial respiration under projected global changes including land-use change and future climate scenarios (mainly elevated temperature). Land-use change is known to have a direct impact on soil organic C and soil respiration (Rs) but the mechanisms that drive these changes remain debatable. Similarly, recent studies and simulation models predict that Rs will respond positively to projected climate warming. However, there are significant uncertainties in the magnitude and mechanisms of this feedback response of Rs to global change. To identify the mechanisms of Rs response to land-use change and climate warming, we first investigated Rs from different land use types. Soil respiration was estimated seasonally from four different Scottish land uses: moorland, birch woodland, grassland and pine forest (n = 24). Our results demonstrated that despite a dramatic loss of total C and nitrogen (N) in the soils under birch trees, the Rs in the birch woodland was similar to that of the moorland and pine forest, with Rs in the grassland being significantly higher. The microbial community structure, estimated by Multiplex Terminal-Restriction Fragment Length Polymorphism (MT-RFLP) and 454 pyrosequencing, was significantly different under each land use type. A strong correlation of Rs with soil properties (pH, inorganic N, C:N ratio and moisture) and with microbial community structure was identified. To test the impact of elevated temperature on Rs and to identify potential microbial mechanisms, we performed laboratory incubation studies. Soils from different land uses were incubated at 7 °C (mean annual temperature (MAT) in Scotland) and 10 °C (MAT + 3 °C) with and without the presence of a labile (13C-glucose) and recalcitrant (13C-lignin) form of C to identify the active groups of microbes and to determine the role of substrate availability on feedback response. The warming treatment induced an increase in Rs rates in all soils. The magnitude of the Rs response to warming was modulated by the land use types, and the Rs was more prominent in soils with high C contents. The addition of glucose substantially increased both total and rate of Rs compared to no substrate- and lignin-amended soils, providing evidence of labile C depletion as a mechanism for the thermal response of Rs. The warming treatment did not impact the composition of the active or total microbial community as revealed by phospholipid fatty acid-stable isotope probing (PLFA-SIP), MT-RFLP and 454 pyrosequencing. Our results showed that the microbial metabolic activity was higher under warming treatment suggesting that a positive feedback of Rs to increased temperature is mediated by changes in substrate availability and microbial metabolic rates