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.

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here, we report its unique compact structure on the basis of four intramolecular thioether bridges introduced post-translationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.

The world is on the verge of a major antibiotic crisis as the emergence of resistant bacteria is increasing, and very few novel molecules have been discovered since the 1960s. In this context, scientists have been exploring alternatives to conventional antibiotics, such as ribosomally synthesized and post-translationally modified peptides (RiPPs). Interestingly, the highly potent in vitro antibacterial activity and safety of ruminococcin C1, a recently discovered RiPP belonging to the sactipeptide subclass, has been demonstrated. The present results show that ruminococcin C1 is efficient at curing infection and at protecting challenged mice from Clostridium perfringens with a lower dose than the conventional antibiotic vancomycin. Moreover, antimicrobial peptide (AMP) is also effective against this pathogen in the complex microbial community of the gut environment, with a selective impact on a few bacterial genera, while maintaining a global homeostasis of the microbiome. In addition, ruminococcin C1 exhibits other biological activities that could be beneficial for human health, as well as other fields of applications. Overall, this study, by using an in vivo infection approach, confirms the antimicrobial clinical potential and highlights the multiple functional properties of ruminococcin C1, thus extending its therapeutic interest.

Cystic fibrosis (CF) is associated with repeated lung bacterial infection, mainly by Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium abscessus, all known to be or becoming resistant to several antibiotics, often leading to therapeutic failure and death. In this context, antimicrobial peptides and antimicrobial polymers active against resistant strains and less prompt to cause resistance, appear as a good alternative to conventional antibiotics. In the present study, methacrylate-based copolymers obtained by radical chemistry were evaluated against CF-associated bacterial strains. Results showed that the type (Random versus Diblock) and the size of the copolymers affected their antibacterial activity and toxicity. Among the different copolymers tested, four (i.e., Random₁₀₂₀₀, Random₁₅₀₀₀, Random₂₃₉₀₀, and Diblock₉₅₀₀) were identified as the most active and the safest molecules and were further investigated. Data showed that they inserted into bacterial lipids, leading to a rapid membranolytic effect and killing of the bacterial. In relation with their fast bactericidal action and conversely to conventional antibiotics, those copolymers did not induce a resistance and remained active against antibiotic-resistant strains. Finally, the selected copolymers possessed a preventive effect on biofilm formation, although not exhibiting disruptive activity. Overall, the present study demonstrates that methacrylate-based copolymers are an interesting alternative to conventional antibiotics in the treatment of CF-associated bacterial infection.

RumC1 is a structurally unique bacteriocin with broad-spectrum efficacy, including against multidrug-resistant pathogens, yet acting by an undefined mechanism. By integrating genetics, biochemistry, computational modeling and single-cell fluorescence microscopy, we demonstrate that RumC1 is a distinct cell-wall-targeting toxin. First, all RumC1-resistant mutants isolated through a high-rate, genome-wide mutagenic screening exhibited specific impairments in peptidoglycan homeostasis regulation, pinpointing this pathway as critical for RumC1 activity. Second, RumC1 selectively accumulates within neosynthesized peptidoglycan, leading to cell growth arrest and death in a dose-dependent manner. Third, we characterize the RumIc1 immunity protein of the RumC1 biosynthetic cluster as a peptidase acting at the cell surface to protect the cells by trimming the stem peptide crucial for cell-wall assembly. As such, RumIc1 provides cross-protection against vancomycin, while RumC1 is demonstrated to act differently from this glycopeptide antibiotic. Collectively, these findings establish RumC1 as a toxin targeting a key peptidoglycan intermediate of cell wall maturation.

Les Ruminococcines C, une nouvelle famille de sactipeptides comme alternatives aux antibiotiques conventionnels: La résistance aux antibiotiques est considérée par l’Organisation Mondiale de la Santé comme une des plus sérieuses menaces de santé publique. Dans ce contexte, des alternatives aux antibiotiques sont activement étudiées, telles que les peptides synthétisés par voie ribosomale et modifiés post-traductionnellement (RiPPs). Mon projet de thèse a porté sur les Ruminococcines C (RumC), une famille de RiPPs produite par la souche E1 de Ruminococcus gnavus, un des membres proéminents du microbiote intestinal humain. Ces RiPPs sont produits uniquement dans un contexte symbiotique avec un hôte animal. Un protocole d’extraction de RumC a donc été établi à partir de contenus caecaux de rats mono-colonisés par R. gnavus E1. La caractérisation des modifications post-traductionnelles des 5 isoformes de RumC et la résolution de la structure tridimensionnelle de l’isoforme RumC1 a permis de classifier RumC comme une nouvelle famille de sactipeptides. D’autre part, la caractérisation biologique de RumC1 a démontré une forte activité antibiotique in vitro contre des pathogènes à Gram-positif. Le traitement avec RumC1 sur un modèle murin de péritonite létale, causée par Clostridium perfringens, a montré son efficacité antibactérienne in vivo. Enfin, RumC1 présente des caractéristiques propres et indispensables aux développements de médicaments comme la résistance à des traitements protéolytique ou physico-chimique ; une absence de toxicité sur tissus humains ; une faible propension à générer de la résistance; et une affinité supérieure pour les cellules procaryotes qu’eucaryotes. Par conséquent, RumC1 semble être un bon candidat pour le développement d’alternatives aux antibiotiques