The molecular basis of ferrosome organelle biogenesis and its impact on host-microbe interactions

SUMMARY Clostridioides difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of nosocomial and antibiotic-associated intestinal infections in the United States. Over the past two decades, there has been a significant rise in the incidence, severity, and economic burden of C. difficile infection (CDI). This increase can

be attributed to the limited efficacy of antibiotics, a growing recurrence rate of CDI, and the emergence of highly virulent strains. These trends underscore the pressing need for alternative strategies in the treatment of CDI. To colonize the gastrointestinal tract, C. difficile must compete with both the host and the gut microbiota for essential

nutrient iron. However, it is unclear how C. difficile adapts to nutrient iron stress in the gut during CDI. Thus, I set out to interrogate the iron homeostasis systems in C. difficile and examine their physiological function. My postdoctoral work has demonstrated that C. difficile undergoes an intracellular iron biomineralization process

and produces membrane-bound ferrosome organelles containing iron phosphate biominerals. The ferrosome organelles serve as an iron storage mechanism, protecting cells against iron intoxication upon transient iron overload. The ferrosome system is activated in the inflamed gut to combat host-mediated iron sequestration and

is important for bacterial colonization and persistence during CDI. A manuscript describing this work was recently accepted for publication in Nature. However, the molecular basis of ferrosome biogenesis is largely unknown and the implications of ferrosome formation within the context of CDI remain unclear. This project aims to

elucidate the underlying mechanisms of ferrosome formation and define its influence on host-microbe interactions. In this application, we hypothesize that (i) the ferrosome membrane derives from the cytoplasmic membrane but exhibits distinct lipid composition, (ii) iron is transported to the FezB transporter through the iron

importer FeoA3B3, aided by an iron chaperone, (iii) many other factors play roles in various stages of ferrosome formation including iron oxidation, nucleation, and biomineralization, (iv) the ferrosome system facilitates C. difficile adaptation to nutrient iron stress mediated by both the host and gut commensals, and (v) nutrient iron

exhibits profound effects on CDI outcomes, gut microbiome resilience, and host immune responses. The experiments described in this proposal will test these hypotheses, elucidate the underlying mechanisms of ferrosome biogenesis, determine the function of ferrosome organelles within the gut community, and define the

impact of nutrient iron on host-microbe interactions. Furthermore, the findings of this proposal will uncover novel factors critical for C. difficile infection and create a framework for developing effective antimicrobial therapeutics to combat this important infection.