The role of a Clostridioides difficile P-type ATPase in ferrosome formation and its impact on cellular physiology and pathogenesis

摘要 艰难梭状芽胞杆菌是一种革兰氏阳性、形成芽胞的厌氧病原菌，是引起肺炎的主要原因。 医院内和抗生素相关的肠道感染。艰难梭菌感染(CDI)的易感性通常如下 抗生素治疗和随后扰乱常驻肠道微生物区系，然而感染的上升 在健康的年轻人中的研究表明，额外的细菌和宿主因素对CDI有重要贡献。 艰难梭菌必须与宿主金属隔离蛋白竞争才能在胃肠道内定植 在一种称为营养免疫的过程中抑制营养金属以限制微生物的生长。铁是最多的 对宿主-病原体界面上的金属进行了充分的研究，并被宿主的铁隔离蛋白所抑制，如 钙保护素和乳铁蛋白。然而，目前尚不清楚钙保护素和乳铁蛋白是如何影响体内金属利用率的。 并影响CDI的转归。艰难梭菌如何适应金属也不清楚。 在CDI过程中，这两种蛋白介导的限制和规避营养免疫。因此，我们开始着手 询问艰难梭菌中的铁稳态系统，并检查它们的生理功能。我们的预赛 数据表明艰难梭菌经历了细胞内铁的生物矿化过程，并产生多面性 在短暂的铁超载期间维持铁平衡的氧化铁颗粒(铁体)。我们还发现， 一个P1B6-ATPase转运体(我们已经命名为FezB)，受铁和铁摄取调节因子调节 皮毛，是形成铁体所必需的。此外，从艰难梭菌细胞中分离的铁体表现出高度的 有别于任何已知的氧化铁矿物的有序晶格结构。在此应用程序中，我们 假设(I)FezB转运铁进入铁体，并在铁体过程中与其他因素相互作用 形成，(二)铁体作为一种重要的铁储存策略，减轻铁过载和氧化 压力，(Iii)铁小体中储存的铁通过一种特定的机制释放，以支持铁下的生长 局限性，(Iv)在脊椎动物宿主内产生铁体以对抗宿主铁的隔离，以及(V)这 在CDI期间，发炎的肠道中激活了铁体系统，这是细菌定植和生存所必需的。 这项建议中描述的实验将检验这些假设，阐明FezB-的潜在机制- 独立的铁体形成，定义铁体的结构特征，并确定其对 艰难梭菌的细胞生理学和致病机制。此外，这项申请的结果将确定 宿主介导的铁封存在CDI中的意义并为开发有效的框架创造框架 抗菌疗法来对抗这种重要的感染。

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.