Siderophores of Legionella pneumophila

项目总结 嗜肺军团菌(LP)是军团病的病原体，军团病是一种日益常见的 肺炎。在肺部，LP主要生长在巨噬细胞中，受损的宿主细胞产生的细胞因子会触发 严重的炎症。铁对LP在胞外和胞内的生长至关重要，也对LP引起 疾病。20年来，我的实验室一直在研究LP Iron收购方面发挥领导作用。我们之前 发现LP在延期的化学定义介质(CDM)中生长时会分泌一个铁载体 这种铁载体是肺中LP生长所必需的。最近，我们确定了分子结构， 发现它是一种多羧酸盐，与根铁蛋白相同，根铁蛋白是一种铁载体，最先在真菌中发现。 除了证明纯化的根铁蛋白能够介导LP铁的吸收外，我们还表征了 参与其生物合成、输出、输入和调控的基因和蛋白质。有趣的是，过去的研究使用 来自真菌的根铁蛋白表明，铁载体能够与其他金属结合，包括锌、铜和锰。 提示根铁蛋白在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，如果根铁蛋白介导其他金属的吸收，以及它是否也有结合的能力 (消耗)宿主细胞中的金属，从而引发受感染的巨噬细胞和上皮细胞释放细胞因子。 有趣的是，Lp根铁蛋白的外膜受体LbtU在结构上与已知的铁载体不同 受体，包括没有与能量转导的TonB结合的周质N末端尾巴。这个，再加上 由于LP不编码TonB或合作伙伴ExbB和ExbD，这意味着LbtU是一种新的类型 受体，中介摄取，不同于目前的范例。因此，我们的目标是确定 与LbtU相互作用的蛋白质(S)，包括TonB-ExbBD的Lp相关，从而建立了一个新的 铁载体跨细菌膜运输的机制。在研究根铁蛋白时，我们发现 Lp分泌两个额外的铁载体的证据，其中一个也是在Lp生长期间产生的 延迟的CDM和另一种是在复杂的缓冲酵母浸膏肉汤中生长产生的 铁含量中等偏低。后者依赖于FrgA，一种铁调节的LbtA样蛋白，它 我们之前已经证明，巨噬细胞的LP感染需要。因此，对这些的结构性确定 铁载体和进一步的突变分析，如在人源化的肺炎小鼠模型中，将导致 增加了对LP铁获得和发病机制的了解。这一努力也为 发现了一种新型的铁载体，并了解到“新的”铁载体具有临床应用的潜力 用途，如铁超载的治疗或作为抗生素载体的衍生物的生成。

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.