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

项目概要/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， 发病率增加。在自然和人造水生栖息地中，Lp 作为细胞内寄生虫而繁衍生息。 阿米巴原虫，通常存在于生物膜中。吸入 Lp 污染的水滴后，病原体就会生长 主要存在于肺泡巨噬细胞中，其次存在于肺上皮细胞中。人们对 Lp pili 或 Lp pili 知之甚少。 Lp 附着于人体细胞和其他表面的基础。早期的电子显微镜揭示了不同的 从 Lp 表面发出的细丝，表明 Lp 表达“长”和“短”菌毛。基因组 我们和其他人所做的分析逐渐发现 Lp 具有通常编码 IV 型菌毛的所有基因 (T4P)装置确认至少一些Lp菌毛是T4P。最近，我们发现了 pilA2，一个先前的 未识别的基因预测编码与主要构建模块（IVa型主要菌毛蛋白）相似的蛋白质 其他表达 T4P 的细菌。免疫荧光显微镜显示 PilA2 明显存在 横跨可视化 Lp pili 的整个长度，确认其作为主要 Pili 的作用。有趣的是，我们发现 与 pilA2 相邻的是第二种 IVa 型主要菌毛蛋白基因 pilA1。从我们的 RNA-Seq 工作来看，pilA1 和 pilA2 都是 用在液体和固体培养基中生长的 Lp 表示。此外，基于 pilA1 pilA2 double 的行为 突变体 pilA1 和 pilA2 各自促进抽搐，这是一种表面相关运动的形式。然而，尽管 pilA2 突变体对 DNA 摄取和卡氏棘阿米巴 (Ac) 感染的影响受到损害，但超 聚集性方面，pilA1 突变体的自动聚集受损，但 DNA 摄取和 Ac 感染正常。 因此，我们假设 i) PilA2 主要包含 T4aP，可促进能力、抽搐和感染 Ac，但抑制 Lp 聚集，并且 ii) PilA1 形成独特的 T4aP，促进聚集： 以及抽搐运动，但 DNA 摄取或 Ac 感染不需要。从而颠覆了长期以来的 从角度来看，我们的工作提供了第一个遗传证据，表明 Lp 阐述了不止一种类型的功能性 T4P，与“旧”EM 数据兼容，该数据显示 Lp 表面上有两种尺寸的菌毛。该提案旨在 i) 确认 PilA1 和 PilA2 是否作为主要菌毛蛋白组装成不同的 Lp T4aP 和 ii) 辨别 PilA1-T4aP 和 PilA2-T4aP 在粘附和感染人类巨噬细胞和 上皮细胞、帮助将 Lp 传播给人类的各种变形虫的感染以及生物膜的形成，这是另一个 Lp 在体内和环境中的关键属性。这项工作将 i) 增加我们对 Lp 的了解，Lp 是一个不断增长的 临床问题，ii) 定义新形式的 T4P 和感染途径，iii) 对其他病原体有影响 使用 T4P 或细胞内寄生虫，并且 iv) 提出了疾病治疗或预防的新靶标。

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.