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

艰难梭菌 P 型 ATP 酶在铁体形成中的作用及其对细胞生理学和发病机制的影响

基本信息

批准号：
10428260
负责人：
Hualiang Pi
金额：
$ 10.71万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-15 至 2024-07-31
项目状态：
已结题

项目摘要

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.

概括梭状芽胞杆菌艰难梭菌是一种革兰氏阳性，形成孢子的厌氧病原体，是导致的主要原因医院和抗生素相关的肠道感染。经常遵循对艰难梭菌感染（CDI）的敏感性抗生素治疗以及随后的居民肠道菌群的破坏，但是感染的兴起在健康的年轻人中，成年人表明，其他细菌和宿主因素为CDI做出了重要贡献。要定居胃肠道，艰难梭菌必须与宿主金属隔离蛋白质竞争扣留营养金属以限制称为营养免疫的过程中的微生物生长。铁是最大的在宿主 - 病原体界面中进行了良好研究的金属，并通过宿主铁序列蛋白（例如钙骨和乳铁蛋白。但是，尚不清楚钙染色素和乳铁蛋白如何影响金属的可用性胃肠道并影响CDI的结果。还不清楚艰难梭菌如何适应金属这两种蛋白质介导的限制和CDI期间的营养免疫。因此，我们着手询问艰难梭菌中的铁稳态系统并检查其生理功能。我们的初步数据表明，艰难梭菌经历了细胞内铁生物矿化过程并产生方面铁氧化铁颗粒（铁质体）在瞬态铁超负荷过程中保持铁平衡。我们还发现 P1B6-ATPase转运蛋白（我们命名为FEZB），由铁和铁摄取调节剂调节毛皮是铁体形成所必需的。此外，从艰难梭菌细胞中分离出的铁质体表现出高度有序的结晶晶格结构，与任何已知的氧化铁矿物质不同。在此应用程序中，我们假设（i）FEZB将铁运输到铁体体中，并在铁质体期间与其他因素相互作用形成，（ii）铁质体是一种重要的铁储存策略，并减轻铁超载和氧化压力，（iii）铁质体中储存的铁通过特定机制释放，以支持铁下的生长限制，（iv）在脊椎动物宿主中产生铁体体以对抗宿主铁固存，（v）在发炎的肠道中激活了铁染色体系统，并且在CDI期间细菌定殖和存活所必需。该提案中描述的实验将检验这些假设，阐明了FEZB-的潜在机制依赖的铁体形成，定义铁染色体的结构特征，并确定其对细胞生理和艰难梭菌发病机理。此外，此应用程序的发现将确定 CDI期间宿主介导的铁隔离的重要性，并创建了一个有效的框架抗菌治疗剂可抵抗这种重要的感染。