Lactic acid bacteria (LAB)-infecting bacteriophages (phages, or bacterial viruses) use diverse host-binding mechanisms, yet the overall picture of the interactions between LAB phages and their host remains incomplete. Unraveling the molecular details of phage-LAB interactions is essential not only for decoding phage biology, but also for industrial and public health purposes since LAB are important micro-organisms in food fermentations and in the human gut microbiota. Phages infecting the LAB species Lactococcus lactis and Streptococcus thermophilus have enjoyed extensive scientific scrutiny since they may disrupt LAB-dependent processes in dairy plants with serious concomitant economic losses. In contrast, there is a significant knowledge gap on the interactions between plant-associated LAB and their phage, even though they may also significantly impact fermentation processes. This is true for fermented beverages as exemplified by the emblematic field of winemaking that heavily relies on the LAB species Oenococcus oeni. Recently, we have shown that representative phages that infect O. oeni possess host-binding devices of distinct composition and morphology, and being different from those of lactococcal and streptococcal phages, that likely employ novel host-binding mechanisms. Moreover, we have observed that wine polyphenolic compounds (PCs), which are abundant in the O. oeni ecological niche, can interfere with the phage infection process. These organic compounds, being sterically similar to cell surface saccharides recognized by phages, may occupy phage receptor-binding sites, thereby preventing host binding. Moreover, PCs could also induce modifications in the cell wall saccharide composition, which would also prevent phages from binding to their host. In this context, our overall aim is to unravel molecular interactions between the wine LAB O. oeni, their viral predators, and PCs. We will work on three representative oenophages, all of which infect the same strain but use different host-binding devices differently affected by wine PCs. We will leverage complementary approaches covering the fields of structural biology, biochemistry, and microbiology, to meet our stated aim. We will 1) determine structure-function relationships of distinct host-binding devices combining cryo-electron microscopy, X-ray crystallography, biophysical characterization of protein-ligand interactions, and host cell-binding assays, 2) explore host-binding capabilities of these phages and the impact of PCs combining phenotypic analyses (generation of bacteriophage-insensitive mutants, phage plaque assays, adsorption tests) and comparative genomics, and 3) map phage-specific host cell saccharide receptors and examine the potential effects of PCs on the synthesis of these receptors through the analysis of gene expression, cell wall biochemical composition, and chemical structure of surface polysaccharides. Investigating molecular interactions between the wine LAB O. oeni and its phages, as a model system of the interactions between plant-related LABs and their phages, will significantly advance current knowledge of phage biology and structure, while simultaneously defining the role and potential inhibitory action of plant PCs on phage infection. We will produce important knowledge of LAB-phage interactions with expected high gains for the wine industry, as well as other plant-fermented products. Of note, plant-based fermented products are currently one of the most innovative and dynamic food categories, in response to the increasing popularity of vegetarian and vegan diets in western countries. Lastly, addressing the role of plant PCs on phage-host interactions may also lead to a better understanding of gut microbiota dynamics and to the rational development of ‘green’ phage-based biocontrol strategies, thereby opening perspectives in the socio-economically important fields of human health and agriculture.

Listeria monocytogenes is a food-borne bacterial contaminant causing a dangerous zoonosis, listeriosis. The virulence mechanisms of this pathogen have been extensively studied, but its asymptomatic carriage in the host is poorly understood. We have discovered a phase of intracellular persistence of Listeria that could explain this portage. This phase is accompanied by a phenotypic switch of Listeria, which enters a state of dormancy in a vacuolar niche. The objectives of the PERMALI project are to develop tools and methods for the detection of persistent intracellular Listeria, and to use them on ex vivo models of listeriosis and clinical (human/animal) samples. The results will allow a better knowledge of the adaptive strategies developed by L. monocytogenes to nest, undetectably, in hosts. They will generate diagnostic and risk management methods to better track Listeria.

Mycotoxins in the trichothecenes family are natural contaminants found in cereals. Resistant to heating, they contaminate the food chain and reach the consumer's plate. On the other hand, an increased number of Europeans harbor in their intestinal microbiota E. coli strains producing a genotoxin, colibactin, suspected of promoting colorectal cancer. We have recently demonstrated that the genotoxicity of colibactin is strongly exacerbated by the presence of a tricothecene in the diet. The objective of this project is to evaluate how this association could contribute to the development of neoplastic colorectal lesions. The project combines three research teams recognized internationally for their expertise on bacterial genotoxins, food mycotoxins, and the health effects of the microbiota. This project will appreciate for the first time the combined risk of a food contaminant and microbiota in colorectal cancer.

Polymorphisms in autophagy-related genes, which lead to dysregulated autophagy, Western diet and an abnormal colonization of the ileal mucosa with adherent-invasive E. coli (AIEC) have been revealed as risk factors for Crohn’s disease (CD). This project aims at (i) investigating the effect of Western diet on autophagy, (ii) examining the impact of the combination of Western diet and host genetic susceptibility (with dysregulated autophagy) on intestinal homeostasis, gut microbiota, restriction of CD-associated AIEC, and on inflammatory responses, and (iii) identifying the bacterial species and their metabolites which could exert beneficial effects on intestinal functions via modulating autophagy. Thus, this project will contribute to a better understanding of the etiology of MC and, in the future, to the development of personalized therapies based on the modulation of autophagy.

The peptidoglycan is an essential and specific component of the bacterial cell wall. It consists of long glycan chains crosslinked by short peptide motifs. This semi-rigid polymer surrounds the cytoplasmic membrane, allowing the cell to resist internal osmotic pressure and providing each bacterium a specific shape. Peptidoglycan is a real Achilles' heel for bacteria, as evidenced by the existence of a very large number of antibacterial agents that degrade it or interfere with its biosynthesis. The majority of peptidoglycan biosynthesis enzymes are gathered within multiprotein complexes that coordinate and regulate their activities both spatially and temporally. These complexes, called elongasome and divisome, present a precise organization and dynamics during the cell cycle. Their formation and progression are mainly controlled from inside the cell by filamentous proteins forming the bacterial cytoskeleton. The constitutive elements of peptidoglycan are synthesised in the cytoplasm and then linked to the undecaprenyl-phosphate, a specific lipid that allows the translocation of the disaccharide-peptide subunits across the membrane with the aid of a flippase. The polymerisation of the peptidoglycan is then ensured by different enzymes, which catalyse transglycosylase and transpeptidase reactions and release the lipid carrier in the undecaprenyl-pyrophosphate form on the outer side of the membrane. The recycling of undecaprenyl-phosphate is essential for peptidoglycan biosynthesis because this lipid is present in a low copy number per cell and is used for the synthesis of other cell wall polymers. It is recycled by the action of phosphatases of BacA or PAP2 type, which act immediately after the action of peptidoglycan polymerases and whose plurality in the same bacterium raises the question of their respective roles. The lipid is then translocated to the inner side of the membrane by an unknown mechanism in order to enter a new cycle of biosynthesis. The recycling of the lipid carrier represents a critical point in the control of the biogenesis of the cell wall, which has only been partially elucidated. In particular, the interplay between lipid recycling enzymes and the elongasome and divisome peptidoglycan biosynthesis machineries is not yet established. The BacWall project aims to understand the overall recycling of undecaprenyl-phosphate in the model bacterium Escherichia coli. The role of BacA and PAP2 proteins in cell wall biogenesis will be addressed by complementary and multidisciplinary approaches (genetics, biophysical, biochemical and microscopic). We will study their potential flippase activity, analyse their localisation and dynamics during the cell cycle and identify their protein partners. Due to its essential role, this metabolic pathway opens the way to new antibacterial strategies. Consequently, the recycling of undecaprenyl-phosphate will also be studied in the multidrug resistant pathogen Enterococcus faecalis. We will measure the impact of an impairment of this metabolism, by antibacterial agents or by gene inactivation, on the fitness and virulence of this pathogen using mouse models of infection and colonisation. We will also elucidate the mechanism of action of a recently identified bacteriocin that targets the BacA protein of E. faecalis. This study will pave the way for the exploitation of this bacteriocin as an antibacterial agent directed against enterococci, as well as the development of engineering experiments aiming at extending or specifying its spectrum of action against other pathogenic species.