Siderophores of Legionella pneumophila

嗜肺军团菌的铁载体

• 批准号: 10172838
• 负责人: NICHOLAS P CIANCIOTTO
• 金额: $ 44.79万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-23 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10172838
• 关键词: Aerosols; Anabolism; Antibiotics; Assimilations; Bacteria; Binding; Buffers; Cells; Chemicals; Clinical; Co-Immunoprecipitations; Complex; Coupled; Data; Devices; Disease; Epithelial; Epithelial Cells; Gene Proteins; Generations; Genes; Gram-Negative Bacteria; Growth; Human; IL8 gene; Impairment; Infection; Inflammation; Inhalation; Iron; Iron Overload; Knowledge; LCN2 gene; Leadership; Legionella pneumophila; Legionnaires' Disease; Libraries; Lung; Mediating; Membrane; Metals; Mus; Mutagenesis; N-terminal; Names; Outcome; Pathogenesis; Pathway interactions; Pneumonia; Production; Proteins; Regulation; Role; Siderophores; Structure; Tail; Testing; Time; Virulence; Virulence Factors; Yeasts; base; contaminated water; cytokine; disorder prevention; fungus; humanized mouse; macrophage; mutant; novel; periplasm; pneumonia model; receptor; siderophore receptors; uptake

PROJECT SUMMARY Legionella pneumophila (Lp) is the agent of Legionnaires' disease, an increasingly common form of pneumonia. In the lungs, Lp grows primarily in macrophages, and cytokines from damaged host cells trigger severe inflammation. Iron is vital to Lp growth in extra- and intracellular niches and for the ability of Lp to cause disease. For >20 years, my lab has served a leadership role in the study of Lp iron acquisition. We previously showed that Lp secretes a siderophore when grown in a deferrated chemically-defined medium (CDM) and that this siderophore is required for Lp growth in lungs. Recently, we determined the structure of the molecule, finding it to be a polycarboxylate identical to rhizoferrin, a siderophore that had been first discovered in fungi. Besides demonstrating the ability of purified rhizoferrin to mediate Lp iron uptake, we also characterized the genes and proteins involved in its biosynthesis, export, import, and regulation. Interestingly, past studies using rhizoferrin from fungi showed that the siderophore is able to bind other metals, including Zn, Cu, and Mn, suggesting that the role of rhizoferrin in Lp pathogenesis may be multi-faceted. Thus, we will determine, for the first time, if rhizoferrin mediates the uptake of other metals and whether it also has the capacity to bind (deplete) metals in host cells and thereby trigger cytokine release from infected macrophages and epithelia. Intriguingly, LbtU, the outer membrane receptor for Lp rhizoferrin, differs in structure from known siderophore receptors, including not having a periplasmic N-terminal tail that binds energy-transducing TonB. This, coupled with the fact that Lp does not encode TonB or partners ExbB and ExbD, implies that LbtU is a new type of receptor, mediating uptake that is distinct from current paradigms. Consequently, we aim to identify the protein(s) that interacts with LbtU, including the Lp correlate of TonB-ExbBD, and thereby establish a new mechanism for siderophore transport across bacterial membranes. While studying rhizoferrin, we uncovered evidence that Lp secretes two additional siderophores, with one also being made during Lp growth in deferrated CDM and the other being produced upon growth in complex, buffered yeast extract broth that is only moderately-low in iron. The latter siderophore was dependent on FrgA, an iron-regulated, LbtA-like protein that we had previously shown is needed for Lp infection of macrophages. Thus, structural determination of these siderophores and further mutant analyses, as in a humanized mouse model of pneumonia, will lead to increased understanding of both Lp iron acquisition and pathogenesis. This effort also offers the chance for discovering a new type of siderophore, with the knowledge that “new” siderophores have potential for clinical usage, such as the treatment of iron overload or generation of derivatives that act as carrier for antibiotics.

项目概要 嗜肺军团菌 (Lp) 是军团病的病原体，军团病是一种越来越常见的疾病形式 在肺部，Lp 主要在巨噬细胞中生长，受损宿主细胞的细胞因子会触发。 铁对于 Lp 在细胞外和细胞内的生长以及 Lp 引起的能力至关重要。 20 年来，我的实验室一直在 Lp 铁获取的研究中发挥着领导作用。 表明 Lp 在延迟化学成分确定培养基 (CDM) 中生长时会分泌铁载体，并且 最近，我们确定了该分子的结构， 发现它是一种与根铁蛋白相同的多羧酸盐，根铁蛋白是一种首先在真菌中发现的铁载体。 除了证明纯化的根铁蛋白介导 Lp 铁吸收的能力外，我们还表征了 过去的研究使用隐含的、参与其生物合成、输出、输入和调节的基因和蛋白质。 来自真菌的根铁蛋白表明铁载体能够结合其他金属，包括锌、铜和锰， 表明根铁蛋白在 Lp 发病机制中的作用可能是多方面的，因此，我们将确定，对于 第一次，根铁蛋白是否介导其他金属的吸收以及它是否也具有结合的能力 （耗尽）宿主细胞中的金属，从而触发受感染的巨噬细胞和上皮细胞释放细胞因子。 有趣的是，LbtU（Lp 根铁蛋白的外膜受体）在结构上与已知的铁载体不同 受体，包括不具有结合能量转导 TonB 的周质 N 末端尾部。 事实上 Lp 不编码 TonB 或伙伴 ExbB 和 ExbD，这意味着 LbtU 是一种新型 受体，介导与当前研究的范式不同的吸收，我们的目标是确定 与 LbtU 相互作用的蛋白质，包括 TonB-ExbBD 的 Lp 相关蛋白，从而建立一个新的 在研究根铁蛋白时，我们发现了铁载体跨细菌膜运输的机制。 有证据表明 Lp 分泌两种额外的铁载体，其中一种也是在 Lp 生长过程中产生的 延迟 CDM 和另一种是在复杂的缓冲酵母提取物肉汤中生长时产生的，该肉汤仅 后者铁载体依赖于 FrgA，一种铁调节的 LbtA 样蛋白， 我们之前已经表明，巨噬细胞的 Lp 感染是必需的，因此，需要确定这些细胞的结构。 铁载体和进一步的突变分析，如在肺炎的人源化小鼠模型中，将导致 增加对 Lp 铁的获取和发病机制的了解。这项工作也提供了机会。 发现一种新型铁载体，认识到“新”铁载体具有临床应用潜力 用途，例如治疗铁过载或产生作为抗生素载体的衍生物。