Ruminococcin C, a promising antibiotic produced by a human gut symbiont

Abstract

A major public health challenge today is the resurgence of microbial infections caused by multidrug-resistant strains. Consequently, novel antimicrobial molecules are actively sought for development. In this context, the human gut microbiome is an under-explored potential trove of valuable natural molecules, such as the ribosomally-synthesized and post-translationally modified peptides (RiPPs). The biological activity of the sactipeptide subclass of RiPPs remains under-characterized. Here, we characterize an antimicrobial sactipeptide, Ruminococcin C1, purified from the caecal contents of rats mono-associated with Ruminococcus gnavus E1, a human symbiont. Its heterologous expression and post-translational maturation involving a specific sactisynthase establish a thioether network, which creates a double-hairpin folding. This original structure confers activity against pathogenic Clostridia and multidrug-resistant strains but no toxicity towards eukaryotic cells. Therefore, the Ruminococcin C1 should be considered as a valuable candidate for drug development and its producer strain R. gnavus E1 as a relevant probiotic for gut health enhancement.

Fig. 1. Biosynthesis of sactipeptides.

(A) Thioether network in previously described sactipeptides. Thioether bridges, disulfide bridges, and head-to-tail cyclization are indicated by purple, yellow, and black lines, respectively. (B) Gene regulon encoding ruminococcins C, subtilosin A, thuricin CD, and thurincin H. Purple, radical SAM enzymes; light blue, precursor peptides; dark blue, transporter systems; green, signal peptidases; yellow, response regulators; pink, immunity systems; and gray, genes of unknown function. (C) Alignment of the five RumC peptide isoforms. (D) Thioether bond formation in sactipeptides catalyzed by radical SAM enzymes.

Fig. 2. Purification and characterization of the five RumC isoforms produced in vivo.

(A) Protocol for extraction from cecal contents to obtain a purified mixture of RumCs. Fractions were selected on the basis of their anti-Cp activity throughout the purification steps. PBS, phosphate-buffered saline. (B and C) LC-MS analyses of the two fractions containing the different RumC (* and † indicate succinimide and deamidated forms of C5 and C3, respectively). RumC5 was present in two consecutive C18 reversed-phase fractions. (D) Deconvoluted mass spectrum of RumC1 eluted at 21.6 min in nano–LC-MS analyses. (E) Deconvoluted mass spectrum of RumC1 after dithiothreitol (DTT)/iodoacetamide treatment. (F) Deconvoluted mass spectrum of synthetic unmodified RumC1.

Fig. 3. Tandem mass spectra of mature RumC1 peptide from in vivo and in vitro preparations and thioether network (see table S1 for theoretical and observed masses of interest).

(A) Deconvoluted MS/MS spectrum of in vivo–matured RumC1 (1 to 44, bold sequence) showing prominent y/b and c/z fragments induced by breaking of the amide bonds preceding the residues bound to cysteines in thioether bridges. The very structured peptide produced high-intensity and unusual internal fragments (blue italics), particularly ANSH (A12-H15) and RNANANVA (R34-A41), corresponding to fragments located between two linked residues. (B) Deconvoluted MS/MS spectrum of the heterologously matured mRumC1 [containing leader peptide (italics) and four additional GAMD amino acids for cloning purposes (gray italics)], revealing the same characteristic fragmentation pattern. Peaks below 500 Da (identical for the y series of in vivo RumC1) are not shown to improve overall visibility. All masses considered are monoisotopic masses. M (last peak in each spectrum) corresponds to the nonfragmented peptide. (C) Deconvoluted MS spectrum of mRumC1 after DTT/iodoalkylation showing no mass increment. Mass of 6694.09 (ammonia loss) corresponds to a succinimide (*) form produced as a by-product of high-temperature reduction of RumC1hm before iodoalkylation. (D) Identification of bridging partners. (E) Double-hairpin–like structure of mRumC1. Cysteine residues bridged via thioether bonds are shown in purple, and their amino acid partners are indicated in orange.

Fig. 4. RumC1 antimicrobial activity.

(A) Activity spectrum of RumC1 against selected Gram-positive strains. MIC and MBC were >100 μM for the following Gram-negative strains tested: Salmonella enterica (CIP 80.39), E. coli (ATCC 8739), E. coli MR4 (DSMZ 22314), Pseudomonas aeruginosa (ATCC 9027), P. aeruginosa fluoroquinolone resistant (CIP 107398), Acinetobacter baumanii (CIP 103572), A. baumanii multiresistant (CIP 110431), and Klebsiella pneumoniae MR4 (DSMZ 26371). (B and C) Membrane permeabilization assay on Cp cells treated with RumC1 or nisin based on measurement of PI incorporation (B) or SYTOX Green staining (C). (B) Cells incubated with cetyltrimethylammonium bromide (CTAB) were used as a positive lysis control, and untreated cells were used as a negative control. (C) Cells were treated for 15 min before staining. Scale bar, 10 μm. (D) Confocal imaging of control Cp cells or Cp cells treated with RumC1 or metronidazole. Membranes were stained with FM4-64FX, and DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). RumC1 treatment leads to three morphotypes identical to the ones induced by metronidazole (fig. S9). This figure shows one of these three morphotypes, i.e., one regular cell associated with a cell three to four times longer and with uncondensed DNA throughout the cells with a few spots of highly condensed DNA. Scale bar, 2 μm.

Fig. 5. Proposed mechanism of maturation and activation of RumC1 in the human gut.

After induction of the two-component system, conventional transcription, and translation of the gene regulon Ruminococcins C, the intracellular RumC1 maturation process involves (i) an in situ posttranslational modification of the core peptide by RumMc1, leading to the inactive mRumC1; (ii) a partial cleavage of the leader peptide by RumPc, leading to the still inactive mRumC1c; (iii) an export in the intestinal lumen by RumTc; and (iv) an ex situ cleavage of the five remaining N-terminal amino acids of the leader peptide by pancreatic trypsin, leading to an active mRumC1cc (i.e., RumC1).