# The role of a Clostridioides difficile P-type ATPase in ferrosome formation and its impact on cellular physiology and pathogenesis > **NIH NIH R00** · YALE UNIVERSITY · 2023 · $249,000 ## Abstract SUMMARY Clostridioides difficile is a Gram-positive, spore-forming anaerobic pathogen, and the leading cause of nosocomial and antibiotic-associated intestinal infections. Susceptibility to C. difficile infection (CDI) often follows antibiotic treatment and subsequent disruption of the resident intestinal microbiota, however the rise of infections in healthy young adults suggests that additional bacterial and host factors make important contributions to CDI. To colonize the gastrointestinal tract, C. difficile must compete with host metal sequestrating proteins that withhold nutrient metals to restrict microbial growth in a process termed nutritional immunity. Iron is the most well studied metal in the host-pathogen interface and is withheld by host iron-sequestering proteins such as calprotectin and lactoferrin. However, it is unknown how calprotectin and lactoferrin affect metal availability in the gastrointestinal tract and impact the outcome of CDI. It is also unclear how C. difficile adapts to metal limitation mediated by these two proteins and circumvents nutritional immunity during CDI. Thus, we set out to interrogate the iron homeostatic systems in C. difficile and examine their physiological function. Our preliminary data demonstrate that C. difficile undergoes an intracellular iron biomineralization process and produces faceted iron oxide granules (ferrosomes) to maintain iron balance during transient iron overload. We also discovered that a P1B6-ATPase transporter (which we have named FezB), regulated by both iron and the ferric uptake regulator Fur, is required for ferrosome formation. Additionally, ferrosomes isolated from C. difficile cells exhibit a highly ordered crystalline lattice structure that is distinct from any known iron oxide minerals. In this application, we hypothesize that (i) FezB transports iron into ferrosomes and interacts with other factors during ferrosome formation, (ii) ferrosomes serve as an important iron storage strategy and alleviate iron overload and oxidative stress, (iii) stored iron in ferrosomes is released through a specific mechanism to support growth under iron limitation, (iv) ferrosomes are produced within the vertebrate host to combat host iron sequestration, and (v) this ferrosome system is activated in the inflamed gut and required for bacterial colonization and survival during CDI. Experiments described in this proposal will test these hypotheses, elucidate the underlying mechanism of FezB- dependent ferrosome formation, define the structural features of the ferrosome, and determine its impact on cellular physiology and C. difficile pathogenesis. Furthermore, the findings from this application will determine the significance of host-mediated iron sequestration during CDI and create a framework for developing effective antimicrobial therapeutics to combat this important infection. ## Key facts - **NIH application ID:** 10894965 - **Project number:** 4R00AI168483-03 - **Recipient organization:** YALE UNIVERSITY - **Principal Investigator:** Hualiang Pi - **Activity code:** R00 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2023 - **Award amount:** $249,000 - **Award type:** 4N - **Project period:** 2022-08-15 → 2024-07-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10894965 ## Citation > US National Institutes of Health, RePORTER application 10894965, The role of a Clostridioides difficile P-type ATPase in ferrosome formation and its impact on cellular physiology and pathogenesis (4R00AI168483-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10894965. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*