Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires’ disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.

嗜肺军团菌是一种革兰氏阴性细菌，能够在广泛的水生原生动物宿主中复制。嗜肺军团菌也是一种机会性人类病原体，可感染肺中的巨噬细胞和上皮细胞并导致军团病。II型分泌系统是一种关键的毒力因子嗜肺军团菌并用于促进低温下的细菌生长、调节生物膜形成、调节宿主对感染的反应、促进粘蛋白凝胶的细菌渗透，并且是感染初期细胞内生长所必需的。这嗜肺军团菌II 型分泌系统从细菌中输出至少 25 种底物，其中包括 NttA 到 NttG 在内的几种底物含有独特的氨基酸序列，这些序列通常在细菌之外观察不到。军团菌属。NttA、NttC 和 NttD 是感染几种变形虫物种所必需的，但尚不清楚其他新型底物对其宿主的影响。在这项研究中，我们表明 NttE 是最佳感染卡氏棘阿米巴和Vermamoeba vermamoeba vermiformis阿米巴原虫是典型的菌落形态所必需的嗜肺军团菌. 此外，我们报告了 NttA、NttC 和 NttE 的原子结构，并通过结合生物物理和生化假设驱动的方法，我们提出了这些底物在感染期间的新功能。这项工作为未来研究新型 II 型底物功能的机械理解以及这些功能与嗜肺军团菌生态和疾病。