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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10428260
• 关键词: ATP phosphohydrolase; Affect; Anaerobic Bacteria; Antibiotic Therapy; Antibiotics; Bacillus subtilis; Bacterial Physiology; Binding; Biochemical; Biological Assay; Biological Process; Cell physiology; Cells; Clostridium difficile; Consumption; Cytoplasmic Granules; Data; Drug Metabolic Detoxication; Environment; Equilibrium; Exhibits; Fluorescent in Situ Hybridization; Fractionation; Gastrointestinal tract structure; Genes; Genetic; Gram-Positive Bacteria; Growth; In Vitro; Infection; Integration Host Factors; Iron; Iron Overload; Kinetics; Lactoferrin; Lead; Leukocyte L1 Antigen Complex; Life; Life Style; Light; Mediating; Metals; Minerals; Molecular; Names; Nutrient; Nutritional Immunity; Outcome; Oxidative Stress; Pathogenesis; Patients; Peptide Nucleic Acids; Physiological; Predisposition; Process; Property; Proteins; Proteomics; Relapse; Reproduction spores; Role; Stains; Staphylococcus aureus; Structure; Substrate Specificity; System; Techniques; Testing; Therapeutic; Time; Toxic effect; antimicrobial; biomineralization; combat; crystallinity; defined contribution; enteric infection; experimental study; ferryl iron; gut colonization; gut inflammation; gut microbiota; in vivo; insight; iron deficiency; iron oxide; microbial; microbiota; mutant; pathogen; pathogenic bacteria; prevent; transposon sequencing; trend; uptake; young adult

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.

总结 艰难梭菌是一种革兰氏阳性的、形成孢子的厌氧病原体，并且是导致腹泻的主要原因。 医院内感染和寄生虫相关的肠道感染。对C.艰难梭菌感染（CDI）通常伴随 抗生素治疗和随后的居民肠道微生物群的破坏，然而，感染的增加， 在健康的年轻人表明，额外的细菌和宿主因素作出重要贡献的CDI。 C.艰难梭菌必须与宿主金属螯合蛋白竞争， 在称为营养免疫的过程中保留营养金属以限制微生物生长。铁是最 已被充分研究的金属在宿主-病原体界面中，并被宿主铁螯合蛋白质如 钙卫蛋白和乳铁蛋白。然而，目前尚不清楚钙卫蛋白和乳铁蛋白如何影响金属的可用性， 胃肠道和影响CDI的结果。目前还不清楚C。difficile适应金属 限制由这两种蛋白质介导，并在CDI期间规避营养免疫。因此，我们开始 询问C.并检查其生理功能。我们的初步 数据表明，C. difficile经历细胞内铁生物矿化过程，并产生多面 铁氧化物颗粒（铁小体），以在瞬时铁过载期间维持铁平衡。我们还发现 一种P1 B6-ATP酶转运蛋白（我们将其命名为FezB），受铁和铁吸收调节剂的调节 毛是铁小体形成所必需的。此外，从C. difficile细胞表现出高度的 有序的晶格结构，不同于任何已知的氧化铁矿物。在本申请中，我们 假设（i）FezB将铁转运到铁小体中，并在铁小体过程中与其他因子相互作用 形成，（ii）铁小体作为一个重要的铁储存策略，并减轻铁过载和氧化 应力，（iii）铁小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）在脊椎动物宿主内产生铁小体以对抗宿主铁螯合，和（v）这 铁小体系统在发炎的肠道中被激活，并且是CDI期间细菌定植和存活所需的。 本提案中描述的实验将验证这些假设，阐明FezB的潜在机制， 依赖于铁小体的形成，定义铁小体的结构特征，并确定其对 细胞生理学和C.难治性发病机制此外，该应用程序的结果将决定 在CDI过程中宿主介导铁螯合的意义，并建立一个框架， 抗微生物疗法来对抗这种重要的感染。