Free-living nematodes as vectors and reservoirs for Legionella pneumophila

嗜肺军团菌是军团病的病原体，其几种毒力机制对人类肺组织的感染表现出高度特异性。这些机制，这似乎是主要相关的多细胞背景下，不能很容易地推断出环境军团菌原生动物的关系。最近，我们证实了从温泉和冷却塔中分离的几种线虫类群中含有L。嗜肺菌这些观察结果，结合模式生物秀丽隐杆线虫的感染研究，以及土壤生活线虫可以作为其他人类病原体的载体的证据，表明水生栖息地的分类群也可以作为L.嗜肺菌在后续的全面实地采样活动，在自然（23个游泳湖）和技术（1个淋浴，7个冷却塔）的栖息地，我们确定冷却塔作为主要的环境水体，军团菌和线虫共存。各自的优势L. pneumophila菌株和线虫分类群（例如Acrobeloides sp.，双壳藻属，Monhystrellasp.，（Plectus sp.）分离培养。拟议的项目现在将确定L. pneumophila-nematode相互作用，第一次考虑到自由生活的线虫类群发生在体内与军团菌。作为初始步骤，L.在潜在的载体/储库宿主上的嗜肺线虫的数量，即细菌生长以及线虫的生活周期，生物量和食物选择。除冷却塔中的优势自由生活类群外，C。elegans和L. pneumophila Corby作为“基线模型”。其次，通过线虫的摄食、定殖和脱落试验研究了线虫对细菌的转运。pneumophila Corby突变体在宿主粘附性方面的差异。另一个焦点是L.嗜肺菌和线虫。在此，主要的问题，是什么决定了生物相互作用的结果，是解决：是线虫喂养军团菌或军团菌使用线虫作为资源？脂肪酸被用作标记物，以解开运输从喂养，和代谢的碳通量进行定量的稳定同位素（13 C）分析。最后，在半自然条件下，用接种mCherry标记的L.嗜肺菌该工作计划提供了一个深入了解的持久性，传播和后生动物致病性的L。嗜肺菌它将为改善技术供水系统的卫生翻新工程铺平道路。

Several virulence mechanisms of Legionella pneumophila, the causative agent of Legionnaires´ disease, exhibit a high specificity for the infection of human lung tissue. These mechanisms, which seem to be mainly relevant in a multicellular context, cannot be deduced easily from the environmental Legionella-protozoa relationship. Recently, we demonstrated that several nematode taxa, which were isolated from a thermal spring and a cooling tower, harbored L. pneumophila. These observations, taken together with infection studies in the model organism Caenorhabditis elegans, and the evidence that soil-living nematodes can act as vectors for other human pathogens, suggest that taxa from aquatic habitats may also serve as environmental reservoirs or vectors for L. pneumophila. In a follow up comprehensive field sampling campaign across natural (23 swimming lakes) and technical (1 shower, 7 cooling towers) habitats, we identified cooling towers as major environmental water bodies, where Legionella and nematodes co-occur. The respective dominant L. pneumophila strains and nematode taxa (e.g. Acrobeloides sp., Diploscapter sp., Monhystrella sp., Plectus sp.) were isolated and cultured. The proposed project will now determine the mechanisms behind the L. pneumophila-nematode interactions, for the first time taking into account free-living nematode taxa occurring in vivo with Legionella. As initial step the impact of L. pneumophila on the potential vectors/reservoir hosts is assigned, i.e. bacterial growth as well as nematode´s life cycle, biomass and food choice. Besides dominant free-living taxa from the cooling towers, C. elegans and L. pneumophila Corby are employed as “baseline models”. Next, the bacterial transport by nematodes is investigated by ingestion, colonization and shedding assays with L. pneumophila Corby mutants differing in host adherence. Another focus is on the carbon flow between L. pneumophila and nematodes. Herewith, the main question, what determines the outcome in the biotic interaction, is addressed: Is the nematode feeding on Legionella or is Legionella using nematodes as resource? Fatty acids are used as marker to disentangle transport from feeding, and the metabolic carbon fluxes are quantified by stable isotope (13C) profiling. Finally, nematode grazing and vector function is modelled under semi-natural conditions with nematode-cured biofilms inoculated with mCherry-labeled L. pneumophila. This working program offers a deep insight into the persistence, dissemination and metazoan pathogenicity of L. pneumophila. It will pave the way for improved hygienic refurbishment works in technical water systems.