Sylvie Kieffer-Jaquinod

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.

Viruses hijack host ribosomes to synthesize their proteins and have evolved various strategies to manipulate translation, including the association of numerous non-ribosomal proteins, known as ribosome-associated proteins (RAPs), with the core ribosome. However, current methods of capturing RAPs that associate transiently and/or in small amounts with ribosomes are suboptimal. To address this issue, we developed an enhanced ribosome affinity purification (eRAP) method that enables the capture of over 500 previously unreported RAPs. Using eRAP, we investigated whether the composition of ribosomes changes during infection by two very different viruses: herpes simplex virus 1 (HSV-1) and Sindbis virus (SINV). Our results revealed that viral infection does not substantially alter the composition of core ribosomes but does drive extensive remodeling of RAPs in a virus-specific manner. Functional analyses with SINV showed that several of these RAPs are critical for viral propagation. Additionally, we discovered that the RAP ASCC3 plays a novel role in promoting the expression of secretory proteins, including viral glycoproteins. This occurs independently of ASCC3’s canonical function of rescuing ribosome collisions. Together, our findings establish the ribosome as a dynamic regulatory hub that undergoes remodeling to achieve virus-specific translational output, highlighting the functional significance of ribosomal heterogeneity.

Candida albicans has been recently added to the WHO critical priority group of fungal pathogens based on its impact on global public health. C. albicans is a mucosal commensal yeast in humans that can cause severe gastrointestinal-borne disseminated candidiasis in immunocompromised patients. C. albicans interaction with enterocytes is thus a key step in the pathophysiology of disseminated candidiasis. Here, we show that, during the first steps of the infection process, C. albicans releases a peptide from the Repressed by Tup1 protein 1 (Rbt1), which disorganizes the cell‒cell adhesion junctions in Caco-2 intestinal epithelial cells by notably downregulating constitutive proteins of the tight junction complex (i.e. ZO-1). This is the first report to show that a peptide of a human pathogenic fungus promotes fungal invasion into the gut epithelium by disorganizing the protective epithelial barrier.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a valuable class of natural products, often featuring macrocyclization, which enhances stability and rigidity to achieve specific conformations, frequently underlying antibiotic activity. ChlB is a metalloenzyme with two catalytic domains─a radical S-adenosyl-l-methionine (SAM) domain and an α-ketoglutarate-dependent oxygenase─that work in tandem to sequentially form three cyclophanes and introduce three hydroxyl groups into its substrate peptide, ChlA. Here, we present the crystal structure of the radical SAM domain of ChlB in complex with ChlA, revealing the mechanism underlying cyclophane formation. These structures also elucidate how the leader sequence of ChlA interacts with ChlB. By combining structural, in vitro, and in vivo approaches, we determined the precise sequence of the three cyclophane formations, interspersed with hydroxylation events. Our findings demonstrate a back-and-forth movement of the core peptide between the radical SAM domain and the oxygenase domain, which drives the stepwise modification process, leading to the fully modified peptide.

Cytokines of the interleukin (IL)-1 family are widely expressed in epithelial surfaces, including the epidermis, where they play a key role in the maintenance of barrier integrity and host defense. A recent report associated the IL-1 family member IL-33 with stress granules (SGs) in epithelial cells. Formation of SGs is promoted by the aggregation of proteins harboring low complexity regions (LCRs). In this study, using computational analyses, we predicted the presence of LCRs in six of the eleven IL-1 family members. Among these, IL-38 contained a long LCR and localized to Ras GTPase-activating protein binding protein 1 (G3BP1) positive SGs, as well as to G3BP1 negative intracellular protein condensates in keratinocytes exposed to oxidative stress (OS). In addition, we identified two highly aggregation-prone amyloid core (AC) motifs in the IL-38 LCR and detected the formation of amyloid IL-38 aggregates in response to OS in cells and in vitro . Disulfide bond mapping, in silico modelling and the analysis of specific cysteine mutants supported a model in which specific oxidation-sensitive cysteines act as redox switches to modify the conformation of IL-38 and thus the surface exposure of its ACs, shuttling it from a soluble state into biomolecular condensates. Finally, the presence of IL-38 granules in human epidermal layers highly exposed to environmental OS suggests that oxidation-induced formation of amyloid aggregates, as a previously unrecognized intrinsic biological property of IL-38, may be physiologically relevant at this epithelial barrier.

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.

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.