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.

项目摘要 军团肺炎（LP）是军团疾病的毒剂，一种日益常见的形式 肺炎。在肺中，LP主要在巨噬细胞中生长，而受损宿主细胞的细胞因子触发 严重的炎症。铁对于细胞内和细胞内壁ches的LP生长至关重要，以及LP引起的能力 疾病。 20年来，我的实验室在LP Iron获取研究中一直担任领导角色。我们以前 表明LP在递延化学定义的培养基（CDM）和 该铁载体是肺中LP生长所必需的。最近，我们确定了分子的结构， 发现它是一种与根茎的聚碳酸盐酸盐，这是一种在真菌中首次发现的铁载体。 除了证明纯化的根莫弗蛋白介导LP铁吸收的能力外，我们还表征了 基因和蛋白质参与其生物合成，出口，进口和调节。有趣的是，过去的研究使用 真菌的根莫法林表明，铁载体能够结合其他金属，包括Zn，Cu和Mn， 表明根氧铁蛋白在LP发病机理中的作用可能是多方面的。那我们将确定 第一次，如果根茎介导其他金属的摄取，以及它是否具有约束力的能力 （耗尽）宿主细胞中的金属，从而从感染的巨噬细胞和上皮引发细胞因子释放。 有趣的是，LBTU是LP根茎的外膜受体，其结构与已知的铁载体不同 受体，包括没有结合能量转移的tonb的周质N末端尾巴。这个，耦合 由于LP不编码TONB或合作伙伴EXBB和EXBD，这意味着LBTU是一种新型 受体，介导与当前范式不同的摄取。因此，我们旨在确定 与LBTU相互作用的蛋白质，包括tonb-exBBD的LP相关，从而建立了一个新的 跨细菌膜的铁载体转运的机制。在研究根莫弗蛋白时，我们发现了 LP分泌另外两个铁载体的证据，其中一个在LP增长过程中也是 递延的CDM，另一种是在复杂的缓冲酵母提取物肉汤中生长而产生的，仅是 铁中适中。后者的铁载体依赖于FRGA，这是一种铁调节的LBTA样蛋白，该蛋白 我们以前已显示出巨噬细胞的LP感染。那是这些的结构确定 与人性化的肺炎小鼠模型一样 对LP铁的获取和发病机理的了解增加了。这项努力也为 发现一种新型的铁载体，并知道“新”的铁载体具有临床的潜力 用法，例如对铁超载的处理或起源于抗生素载体的衍生物。