Rethinking Legionella pneumophila type IV pili and their roles in intracellular infection

项目摘要/摘要 嗜肺军团菌(LP)是军团病的病原体，军团病是一种经常致命的肺炎形式，是 发病率增加的。在天然和人造水生生境中，LP作为一种寄生在细胞内的寄生虫而蓬勃发展 阿米巴虫，通常存在于生物膜中。吸入受LP污染的水滴后，病原体生长 主要见于肺泡巨噬细胞，其次见于肺上皮细胞。关于LP菌毛或Lp菌毛 低密度脂蛋白附着于人体细胞和其他表面的基座。早期的电子显微镜显示不同 从LP表面发出的细丝，表明LP表达“长”和“短”菌毛。基因组学 其他人和我们的分析逐渐发现，LP具有典型地编码IV型菌毛的所有基因 (T4P)设备，确认至少一些LP菌毛是T4P。最近，我们发现了PilA2，一个以前的 预测编码一种与主要构件相似的蛋白质的未知基因(IVA型主要菌毛蛋白) 其他表达T4P的细菌。免疫荧光显微镜显示PilA2显著存在 在整个可视化的LP菌毛长度上，确认其作为主要菌毛的作用。有趣的是，我们发现 与PilA2相邻的是第二型IVA型主要毛蛋白基因，PilA1。从我们的RNA-Seq工作来看，pilA1和pilA2都是 表现为在液体和固体介质中生长的Lp。此外，基于pilA1、pilA2 Double的行为 突变体PilA1和PilA2各自促进抽动，这是一种表面相关的运动形式。然而，鉴于 PilA2突变体对卡氏棘阿米巴的DNA摄取和感染均有一定的抑制作用，但对DNA的摄取和感染均有抑制作用。 在聚集性方面，一个pilA1突变体的自我聚集能力受损，但DNA摄取和Ac感染正常。 因此，我们假设i)PilA2主要由促进能力、抽动和感染的T4aP组成 Ac，但对Lp聚集有抑制作用；ii)PilA1形成独特的T4aP，促进聚集为 以及抽搐运动，但不是DNA摄取或AC感染所必需的。因此，推翻长期存在的 观点来看，我们的工作提供了第一个遗传证据，证明LP阐述了不止一种类型的功能 T4P，与显示LP表面有两种大小菌毛的“旧”EM数据兼容。这项提议旨在 I)确认PilA1和PilA2是否作为主要粘连蛋白组装成不同的LP T4aP和ii)识别PilA1-T4aP和 PilA2-T4aP在人巨噬细胞的黏附和感染方面功能不同 上皮细胞，帮助人类传播LP的各种阿米巴虫的感染，以及生物膜的形成，这是另一个 体内和环境中LP的关键属性。这项工作将i)增加我们对LP的知识，一个不断增长的 临床问题，二)定义新的T4P形式和感染途径，三)对其他病原体有影响 使用T4P或是细胞内寄生虫，以及iv)为疾病治疗或预防提供了新的靶点。

PROJECT SUMMARY / ABSTRACT Legionella pneumophila (Lp) is the agent of Legionnaires disease, an oft-fatal form of pneumonia that is increasing in incidence. In natural and man-made aquatic habitats, Lp flourishes as an intracellular parasite of amoebae, often in biofilms. Following the inhalation of Lp-contaminated water droplets, the pathogen grows primarily in alveolar macrophages and secondarily in lung epithelia. Relatively little is known about Lp pili or the bases of Lp attachment to human cells and other surfaces. Early electron microscopy revealed different filaments emanating from the Lp surface, suggesting that Lp expresses “long” and “short” pili. Genomic analysis done by others and us gradually found that Lp has all the genes that typically encode a type IV pilus (T4P) apparatus affirming that at least some Lp pili are T4P. Recently, we uncovered pilA2, a previously unrecognized gene predicted to encode a protein with similarity to major building blocks (type IVa major pilins) of other T4P-expressing bacteria. Immunofluorescence microscopy has revealed that PilA2 exists prominently across the entire length of visualized Lp pili, affirming its role as a major pilin. Intriguingly, we discovered adjacent to pilA2 a second type IVa major pilin gene, pilA1. From our RNA-Seq work, both pilA1 and pilA2 are expressed by Lp growing in liquid and solid media. Moreover, based on the behavior of a pilA1 pilA2 double mutant, pilA1 and pilA2 each promote twitching, a form of surface associated motility. However, whereas a pilA2 mutant was impaired for DNA uptake and infection of Acanthamoeba castellanii (Ac) but hyper- aggregative, a pilA1 mutant was impaired for auto-aggregation but normal for DNA uptake and Ac infection. Thus, we hypothesize that i) PilA2 majorly comprises T4aP that promote competence, twitching, and infection of Ac, but are inhibitory to Lp aggregation and ii) that PilA1 forms distinct T4aP that promote aggregation as well as twitching motility but are not needed for DNA uptake or Ac infection. Thus, overturning long-standing perspectives, our work provides the first genetic evidence that Lp elaborates more than one type of functional T4P, compatible with the “old” EM data that had shown two sizes of pili on the Lp surface. This proposal aims to i) confirm if PilA1 and PilA2 assemble, as major pilins, into distinct Lp T4aP and ii) discern if PilA1-T4aP and PilA2-T4aP are functionally different in the context of adherence to and infection of human macrophages and epithelia, infection of various amoebae that help transmit Lp to humans, and biofilm formation, which is another key attribute of Lp in vivo and in the environment. This work will i) increase our knowledge of Lp, a growing clinical problem, ii) define new forms of T4P and infection pathways, iii) have implications for other pathogens that use T4P or are intracellular parasites, and iv) suggest new targets for disease treatment or prevention.