Functional analysis of Legionella Dot/Icm T4SS component DotA.

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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.