Sulfated fucans, frequently referred to simply as fucans, constitute a class of polysaccharides first isolated in 1913. For many years fucans were regarded only as a potential source of l-fucose, although their anticoagulant activity was known. Even as the potent effects of fucans on physiological systems have become better characterized, structural studies have lagged behind. Recently the search for new drugs has raised increased interest in sulfated fucans. In the past few years, several structures of algal and invertebrate fucans have been solved, and many aspects of their biological activity have been elucidated. From this work emerges a more interesting picture of this class of polysaccharides than was previously suspected. The availability of purified fucans and fucan fractions with simple, but varied structures, in conjunction with the development of new enzymatic tools, demonstrate that the biological properties of sulfated fucans are not only a simple function of their charge density but also are determined by detailed structural features.

Faecalibacterium prausnitzii is a major member of the Firmicutes phylum and one of the most abundant bacteria in the healthy human microbiota. F. prausnitzii depletion has been reported in several intestinal disorders, and more consistently in Crohn’s disease (CD) patients. Despite its importance in human health, only few microbiological studies have been performed to isolate novel F. prausnitzii strains in order to better understand the biodiversity and physiological diversity of this beneficial commensal species. In this study, we described a protocol to isolate novel F. prausnitzii strains from feces of healthy volunteers as well as a deep molecular and metabolic characterization of these isolated strains. These F. prausnitzii strains were classified in two phylogroups and 3 clusters according to 16S rRNA sequences and results support that they would belong to two different genomospecies or genomovars as no genome sequencing has been performed in this work. Differences in enzymes production, antibiotic resistance and immunomodulatory properties were found to be strain-dependent. So far, all F. prausnitzii isolates share some characteristic such as i) the lack of epithelial cells adhesion, plasmids, anti-microbial and hemolytic activity and ii) the presence of DNAse activity. Furthermore, Short Chain Fatty Acids (SCFA) production was assessed for the novel isolates as these products influence intestinal homeostasis. Indeed, the butyrate production has been correlated to the capacity to induce IL-10, an anti-inflammatory cytokine, in peripheral blood mononuclear cells (PBMC) but not to the ability to block IL-8 secretion in TNF-α-stimulated HT-29 cells, reinforcing the hypothesis of a complex anti-inflammatory pathway driven by F. prausnitzii. Altogether, our results suggest that some F. prausnitzii strains could represent good candidates as next-generation probiotic.

In the human gastrointestinal tract, bacterial β-D-glucuronidases (BG; E.C. 3.2.1.31) are involved both in xenobiotic metabolism and in some of the beneficial effects of dietary compounds. Despite their biological significance, investigations are hampered by the fact that only a few BGs have so far been studied. A functional metagenomic approach was therefore performed on intestinal metagenomic libraries using chromogenic glucuronides as probes. Using this strategy, 19 positive metagenomic clones were identified but only one exhibited strong β-D-glucuronidase activity when subcloned into an expression vector. The cloned gene encoded a β-D-glucuronidase (called H11G11-BG) that had distant amino acid sequence homologies and an additional C terminus domain compared with known β-D-glucuronidases. Fifteen homologs were identified in public bacterial genome databases (38–57% identity with H11G11-BG) in the Firmicutes phylum. The genomes identified derived from strains from Ruminococcaceae, Lachnospiraceae, and Clostridiaceae. The genetic context diversity, with closely related symporters and gene duplication, argued for functional diversity and contribution to adaptive mechanisms. In contrast to the previously known β-D-glucuronidases, this previously undescribed type was present in the published microbiome of each healthy adult/child investigated (n= 11) and was specific to the human gut ecosystem. In conclusion, our functional metagenomic approach revealed a class of BGs that may be part of a functional core specifically evolved to adapt to the human gut environment with major health implications. We propose consensus motifs for this unique Firmicutes β-D-glucuronidase subfamily and for the glycosyl hydrolase family 2.

The large-scale application of genomic and metagenomic sequencing technologies has yielded a number of insights about the metabolic potential of symbiotic human gut microbes. Nevertheless, the molecular basis of the interactions between commensal bacteria and their host remained to be investigated. Bacteria colonizing the mucosal layer that overlies the gut epithelium are exposed to highly sulfated glycans (i.e. mucin and glycosaminoglycans). These polymers can serve as potential nutrient sources, but their high sulfate content usually prevents their degradation. Commensal bacteria such as Bacteroides thetaiotaomicron possess more predicted sulfatase genes than in the human genome, the physiological functions of which are largely unknown. To be active, sulfatases must undergo a critical post-translational modification catalyzed in anaerobic bacteria by the radical AdoMet enzyme anaerobic sulfatase-maturating enzyme (anSME). In the present study, we have tested the role of this pathway in Bacteroides thetaiotaomicron which, in addition to 28 predicted sulfatases, possesses a single predicted anSME. In vitro studies revealed that deletion of the gene encoding its anSME (BT0238) results in loss of sulfatase activity and impaired ability to use sulfated polysaccharides as carbon sources. Co-colonization of formerly germ-free mice with both isogenic strains (i.e. wild-type or ΔanSME), or invasion experiments involving introduction of one followed by the other strain established that anSME activity and the sulfatases activated via this pathway, are important fitness factors for B. thetaiotaomicron, especially when mice are fed a simple sugar diet that requires this saccharolytic bacterium to adaptively forage on host glycans as nutrients. Whole genome transcriptional profiling of wild-type and the anSME mutant in vivo revealed that loss of this enzyme alters expression of genes involved in mucin utilization and that this disrupted ability to access mucosal glycans likely underlies the observed pronounced colonization defect. Comparative genomic analysis reveals that 100% of 46 fully sequenced human gut Bacteroidetes contain homologs of BT0238 and genes encoding sulfatases, suggesting that this is an important and evolutionarily conserved feature for bacterial adaptation to life in this habitat.

Sulfatases are a highly conserved family of enzymes found in all three domains of life. To be active, sulfatases undergo a unique post-translational modification leading to the conversion of either a critical cysteine ("Cys-type" sulfatases) or a serine ("Ser-type" sulfatases) into a Calpha-formylglycine (FGly). This conversion depends on a strictly conserved sequence called "sulfatase signature" (C/S)XPXR. In a search for new enzymes from the human microbiota, we identified the first sulfatase from Firmicutes. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that this enzyme undergoes conversion of its critical cysteine residue into FGly, even though it has a modified (C/S)XAXR sulfatase signature. Examination of the bacterial and archaeal genomes sequenced to date has identified many genes bearing this new motif, suggesting that the definition of the sulfatase signature should be expanded. Furthermore, we have also identified a new Cys-type sulfatase-maturating enzyme that catalyzes the conversion of cysteine into FGly, in anaerobic conditions, whereas the only enzyme reported so far to be able to catalyze this reaction is oxygen-dependent. The new enzyme belongs to the radical S-adenosyl-l-methionine enzyme superfamily and is related to the Ser-type sulfatase-maturating enzymes. This finding leads to the definition of a new enzyme family of sulfatase-maturating enzymes that we have named anSME (anaerobic sulfatase-maturating enzyme). This family includes enzymes able to maturate Cys-type as well as Ser-type sulfatases in anaerobic conditions. In conclusion, our results lead to a new scheme for the biochemistry of sulfatases maturation and suggest that the number of genes and bacterial species encoding sulfatase enzymes is currently underestimated.