Ruminococcin C, an anti-clostridial sactipeptide produced by a prominent member of the human microbiota Ruminococcus gnavus

Abstract

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus. This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens. By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to–α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

Protective effects of Δ9‐tetrahydrocannabinol against enterotoxin‐induced acute respiratory distress syndrome are mediated by modulation of microbiota

“…Besides, R. gnavus produced peptides such as ruminococcin A (RumA) and RumC, have high activity against pathogenic clostridia and exhibit other antimicrobial activities (Balty et al, 2019;Ongey et al, 2018). In contrast, A. muciniphila is widely present in the intestines of humans and animals and known to induce IgG1 antibodies and antigenspecific T cell responses, thus enhancing immune responses (Ansaldo et al, 2019).…”

The manifold roles of microbial ribosomal peptide–based natural products in physiology and ecology

“…They have been found recently in Ruminococcus (ruminococcins from Ruminococcus gnavus isolated from the human microbiota)(32,33) and presumably in Staphylococcus (hyicin from Staphylococcus hyicus 4244). The structure of ruminococcin C was recently established, showing that this sactipeptide is stabilized by four thioether bonds that generate a double hairpin fold(32,33).…”

“…, thurincin H from Bacillus thuringiensis SF361, the twopeptide thuricin CD from the human fecal isolate B. thuringiensis DPC 6431, and huazacin or thuricin Z from B. thuringiensis serovar Huazhongensis)(25,32,93,94). They have been found recently in Ruminococcus (ruminococcins from Ruminococcus gnavus isolated from the human microbiota)(32,33) and presumably in Staphylococcus (hyicin from Staphylococcus hyicus 4244). The structure of ruminococcin C was recently established, showing that this sactipeptide is stabilized by four thioether bonds that generate a double hairpin fold(32,33).…”

The Epipeptide YydF Intrinsically Triggers the Cell Envelope Stress Response of Bacillus subtilis and Causes Severe Membrane Perturbations

“…In this work, we presented a comprehensive MOA analysis of the epipeptide (YydF * ), a novel ribosomally-synthesized and posttranslationally modified peptide (RiPP) (Benjdia et al, 2017a,b;Balty et al, 2019), produced by B. subtilis. We demonstrate that extrinsically added YydF * targets the cell envelope and dissipates membrane potential via membrane permeabilization.…”

“…These processes are accompanied by rapid loss of membrane fluidity, substantiated by lipid domain formation based in altered lipid packing. Disturbance of the lipid matrix causes drastic disorder of membrane associated proteins, thereby disrupting membrane function and homeostasis (Benjdia et al, 2017a,b;Balty et al, 2019). The global response of YydF * upon B. subtilis treatment revealed a narrow and highly specific transcriptome profile.…”