Functional analysis of Legionella Dot/Icm T4SS component DotA.

蛋白质分泌在细菌发病机制中起核心作用。嗜肺军团菌是军团病的病原体，通过Dot/Icm IV型分泌系统（T4SS）使大量效应蛋白易位。Dot/Icm T4SS由bbb20蛋白组成，是军团菌感染所必需的。然而，IV型分泌的分子基础在很大程度上仍然未知。我们已经证明，绝大多数DotA （Dot/Icm T4SS成分）通过Dot/Icm T4SS分泌到细胞外环境。在细菌中，DotA被整合到具有7个跨膜区域的内膜中。为了研究这种完整的内膜蛋白是如何由T4SS分泌的，我们分离了DotA分泌缺陷或DotA功能缺陷的DotA点突变体。通过军团菌在环境模型宿主castellanacanthamoeba castellanii内的生长能力来评估其DotA功能。这些突变体在DotA分泌和DotA功能上都存在缺陷，表明DotA分泌是DotA功能不可缺少的。这些突变体的精细图谱揭示了22个独立的点突变。没有发现特异的DotA区域富含突变。值得注意的是，这些点突变在DotA的细胞质和质周区域都被定位，这意味着DotA的分泌涉及多步骤过程。此外，我们发现DotF是Dot/Icm T4SS的核心成分，对于DotA的分泌是必不可少的。DotF KO菌株的DotA分泌量减少了10倍。由于DotF被认为是Dot/Icm T4SS的核心成分，我们研究了DotF KO菌株的效应物易位。数据表明，Dot F对于DotA分泌和效应体转运都是不可缺少的。

Protein secretion plays a central role in bacterial pathogenesis. Legionella pneumophlia, the causative agent of Legionnaires' disease, translocated a large array of effector proteins via the Dot/Icm type IV secretion system (T4SS). Dot/Icm T4SS is composed of>20 proteins and essential for Legionella infection. However, molecular basis of type IV secretion remains largely unknown. We had demonstrated that vast majority of DotA, a Dot/Icm T4SS component, was secreted into extracellular milieu via the Dot/Icm T4SS. In bacteria, DotA is integrated into inner membrane with seven trans-membrane regions. To address how such integral inner membrane protein is secreted by T4SS, we isolated DotA point mutants defective either in DotA secretion or in DotA function. DotA function was assessed by the ability of Legionella to grow within environmental model host Acanthamoeba castellanii We established a high-throughput screening system and isolated 57 DotA mutants. All of these mutants are defective both in DotA secretion and in DotA function, suggesting that DotA secretion is indispensable for DotA function. Fine mapping of these mutants unveiled 22 independent point mutations. No specific region of DotA rich of mutations was found. Notably, these point mutations were mapped both at cytoplasmic and periplasmic regions of DotA, implying that DotA secretion involve multi-step processes. Additonally, we found that DotF, a putative core component of Dot/Icm T4SS, is dispensable for DotA secretion. DotA secretion from DotF KO strain was reduced by 10-fold. Because DotF is thought to be a core component of Dot/Icm T4SS, we examined effector translocation from DotF KO strain. The data indicated that Dot F is dispensable both for DotA secretion and for effector translocation.