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: 10894965
• 负责人: Hualiang Pi
• 金额: $ 24.9万
• 依托单位: YALE UNIVERSITY
• 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 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10894965
• 关键词: ATP phosphohydrolase; Affect; Anaerobic Bacteria; Antibiotic Therapy; Antibiotics; Bacillus subtilis; Bacterial Physiology; Binding; Biochemical; Biological Assay; Biological Process; Cell physiology; Cells; Chemistry; Clostridium difficile; Co-Immunoprecipitations; Consumption; Cytoplasmic Granules; Data; Drug Metabolic Detoxication; Environment; Equilibrium; Exhibits; Fluorescent in Situ Hybridization; Fractionation; Gastrointestinal tract structure; Genes; Genetic; Gram-Positive Bacteria; Growth; Homeostasis; In Vitro; Infection; Integration Host Factors; Iron; Iron Overload; Kinetics; Lactoferrin; Leukocyte L1 Antigen Complex; Life; Life Style; Maps; Mediating; Metals; Minerals; Molecular; Names; Nutrient; Nutritional Immunity; Outcome; Oxidative Stress; Pathogenesis; Patients; Peptide Nucleic Acids; Physiological; Predisposition; Process; Property; Proteins; Proteomics; Reproduction spores; Role; Stains; Staphylococcus aureus; Structure; Substrate Specificity; System; Techniques; Testing; Therapeutic; Time; Toxic effect; antimicrobial; biomineralization; combat; 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.

概括 艰难梭菌是一种革兰氏阳性、形成孢子的厌氧病原体，是引起艰难梭菌感染的主要原因。 医院内和抗生素相关的肠道感染。通常会出现对艰难梭菌感染 (CDI) 的易感性 抗生素治疗和随后肠道微生物群的破坏，然而感染的增加 健康年轻人的研究表明，其他细菌和宿主因素对 CDI 起着重要作用。 为了在胃肠道定殖，艰难梭菌必须与宿主金属螯合蛋白竞争，这些蛋白 在称为营养免疫的过程中抑制营养金属以限制微生物生长。铁是最多的 宿主-病原体界面中的金属被充分研究，并被宿主铁螯合蛋白抑制，例如 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利用率 胃肠道并影响 CDI 的结果。目前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并规避 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，艰难梭菌经历细胞内铁生物矿化过程并产生多面体 氧化铁颗粒（铁体）可在短暂的铁过载期间维持铁平衡。我们还发现 P1B6-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 毛皮是铁体形成所必需的。此外，从艰难梭菌细胞中分离的铁体表现出高度 有序晶格结构，不同于任何已知的氧化铁矿物。在这个应用程序中，我们 假设 (i) FezB 将铁转运到铁体中并在铁体过程中与其他因素相互作用 形成，（ii）铁体作为重要的铁储存策略并减轻铁过载和氧化 应激，(iii) 铁体中储存的铁通过特定机制释放，以支持铁条件下的生长 限制，(iv) 铁体在脊椎动物宿主内产生以对抗宿主铁固存，以及 (v) 这 铁体系统在发炎的肠道中被激活，是 CDI 期间细菌定植和生存所必需的。 本提案中描述的实验将测试这些假设，阐明 FezB- 的基本机制 依赖的铁体形成，定义铁体的结构特征，并确定其对 细胞生理学和艰难梭菌发病机制。此外，该应用程序的结果将确定 宿主介导的铁螯合在 CDI 过程中的重要性，并为开发有效的 对抗这种重要感染的抗菌疗法。