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

重新思考嗜肺军团菌 IV 型菌毛及其在细胞内感染中的作用

• 批准号: 10738431
• 负责人: NICHOLAS P CIANCIOTTO
• 金额: $ 23.06万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-13 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10738431
• 关键词: Acanthameba infection; Acanthamoeba; Acanthamoeba castellanii; Adherence; Adhesions; Alveolar Macrophages; Amoeba genus; Area; Bacteria; Behavior; Cell Line; Cells; Clinical; Competence; DNA; Data; Development; Devices; Disease; Electron Microscopy; Environment; Epithelial Cells; Epithelium; Filament; Fimbriae Proteins; Genes; Genetic; Genomics; Habitats; Human; Immunofluorescence Microscopy; Impairment; Incidence; Infection; Inhalation; Knowledge; Legionella pneumophila; Legionnaires' Disease; Length; Link; Liquid substance; Lung; Macrophage; Mediating; Methods; Microbial Biofilms; Microscopic; Minor; Movement; Names; Organism; Parasites; Pathogenesis; Pathway interactions; Pilum; Pneumonia; Prevention; Protein Secretion; Proteins; Reporting; Research Project Grants; Risk; Role; Solid; Surface; System; Thinking; United States National Institutes of Health; Variant; Virulence; Visualization; Work; aerosolized; appendage; base; cell motility; contaminated water; disease diagnosis; disorder prevention; in vivo; man; mutant; pathogen; programs; transcriptome sequencing; transmission process; uptake; virulence gene

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.

项目总结/摘要 嗜肺军团菌（Lp）是军团菌病的病原体，军团菌病是一种致命的肺炎， 发病率上升。在自然和人造水生生境中，Lp作为细胞内寄生虫寄生于 变形虫，通常在生物膜中。在吸入被Lp污染的水滴后，病原体生长 主要在肺泡巨噬细胞中，其次在肺上皮细胞中。关于Lp皮利或其受体的了解相对较少。 Lp附着于人体细胞和其他表面的碱基。早期的电子显微镜显示 纤维从Lp表面发出，表明Lp表达“长”和“短”皮利。基因组 我们和其他人所做的分析逐渐发现，Lp具有典型编码IV型菌毛的所有基因 (T4P)确认至少一些Lp皮利是T4 P的装置。最近，我们发现了pilA 2， 预测编码与主要结构单元（IVa型主要菌毛蛋白）相似的蛋白质的未识别基因 其他表达T4 P的细菌。免疫荧光显微镜观察显示，PilA 2在细胞内的表达量显著增加， 在整个长度的可视化Lp皮利，肯定其作为一个主要的菌毛蛋白的作用。有趣的是，我们发现 与pilA 2相邻的是第二种IVa型主要菌毛蛋白基因pilA 1。根据我们的RNA-Seq工作，pilA 1和pilA 2都是 由在液体和固体培养基中生长的Lp表达。此外，基于pilA 1 pilA 2双链的行为， 突变体pilA 1和pilA 2各自促进抽搐，这是一种表面相关运动形式。然而，尽管A pilA 2突变体对卡氏阿米巴（Ac）的DNA摄取和感染有损害，但对pilA 2突变体的DNA摄取和感染无影响，而对pilA 2突变体的DNA摄取和感染无影响。 聚集性，pilA 1突变体受损的自动聚集，但正常的DNA摄取和Ac感染。 因此，我们假设i）PilA 2主要包含促进感受态、抽搐和感染的T4 aP ii）PilA 1形成不同的T4 aP，其促进聚集， 以及抽搐运动，但不需要DNA摄取或Ac感染。因此，推翻长期以来 我们的工作提供了第一个遗传学证据，表明Lp阐述了一种以上的功能性 T4 P，与“旧”EM数据兼容，该数据显示Lp表面上有两种尺寸的皮利。这项建议旨在 i）确认PilA 1和PilA 2是否作为主要菌毛组装成不同的Lp T4 aP，以及ii）辨别PilA 1-T4 aP和 PilA 2-T4 aP在粘附和感染人巨噬细胞的情况下功能不同， 上皮细胞，感染各种阿米巴原虫，帮助将Lp传播给人类，以及生物膜形成，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的认识， 临床问题，ii）定义新形式的T4 P和感染途径，iii）对其他病原体有影响 使用T4 P或为细胞内寄生虫的细胞，和iv）提出用于疾病治疗或预防的新靶点。