Bacterial membrane fusion sustains commensal resilience during enteric pathogen infection

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Title: Bacterial membrane fusion sustains commensal resilience during enteric pathogen infection Project Summary: The trillions of commensal microbes in the human gut, collectively known as the microbiota, play a pivotal role in host health by regulating immunity and defense against pathogens. Perturbations such as enteric infections can trigger severe intestinal inflammation, which destabilizes the microbial ecosystem and drives it towards dysbiosis, a state linked to disease exacerbation. Commensal resilience, or the ability of beneficial microbes to withstand or recover from such perturbations, is therefore essential for sustaining gut homeostasis and overall health. Yet, the molecular mechanisms underlying this resilience remain poorly understood. One critical challenge that arises during infection is the activation of a host process known as nutritional immunity, which restricts microbial access to essential metals like iron and poses a significant threat to commensal survival. While the enteric pathogen Salmonella Typhimurium (S. Tm) overcomes iron limitation by producing high-affinity iron chelators, or siderophores, how commensals such as Bacteroides vulgatus adapt to iron limitation remains largely unknown. Our preliminary findings reveal that B. vulgatus does not utilize free or protein-bound siderophores; instead, it depends on outer membrane vesicle (OMV) fusion to access siderophores produced by S. Tm, a mechanism distinct from classical siderophore uptake. Using genome-wide forward genetics, we identified key determinants of OMV fusion, notably enzymes involved in peptidoglycan remodeling. Our data show that cell wall remodeling is essential for OMV-bacterial membrane fusion, and that mutants defective in this process display impaired iron acquisition. Building on these strong preliminary data, this proposal aims to define how OMV fusion orchestrates commensal iron acquisition and resilience in the inflamed gut. Specific Aim 1 will determine how OMV fusion facilitates iron uptake and supports B. vulgatus fitness during gut inflammation, using reductionist in vitro systems and murine models of infectious colitis. Specific Aim 2 will elucidate the molecular and structural basis of OMV fusion, focusing on the contribution of peptidoglycan remodeling. This work is innovative because it leverages a unique bacterial behavior and integrates high-throughput genetic screens, live-cell imaging, and high-resolution cryo-electron tomography to provide a mechanistic understanding of OMV-mediated nutrient acquisition in commensal bacteria. While membrane fusion is a well-established process in eukaryotes, its mechanisms and physiological significance in prokaryotes remain almost entirely unexplored. The impact of this research lies in uncovering peptidoglycan remodeling as a critical component of membrane fusion and commensal resilience during gut inflammation, presenting a new frontier in microbial physiology. Ultimately, this work has the potential to transform our understanding of how beneficial microbes survive and adapt within the inflamed intestine, laying the groundwork for rational interventions to bolster the gut microbiota in patients with inflammatory disease.