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Significant Role for ladC in Initiation of Legionella pneumophila Infection

机译：ladC在引发嗜肺军团菌感染中的重要作用

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Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladCN430A/R434A, encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladCN430A/R434A resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.
机译：以前，我们在嗜肺军团菌中存在但在其他军团菌物种中不存在的一组基因中鉴定了 ladC 。在这里，我们构建了 L的 ladC 突变体。肺炎并评估其在哺乳动物细胞系和 Acanthamoeba castellanii 中的复制能力。与野生型 L相比， ladC 突变体的回收率显着降低。肺炎在早期的时间点，与 ladC 互补但被 ladC N430A / R434A 逆转后被逆转，编码一种假定的催化活性蛋白质的衍生物。实际上， ladC :: Km与 ladC N430A / R434A 的互补导致在人类和变形虫细胞感染模型中出现严重的复制缺陷，它没有遵循典型的显性阴性表型。使用差异免疫荧光染色从细胞内细菌区分粘附，我们发现 ladC 突变体显示出对THP-1巨噬细胞的粘附减少了10倍，但是THP-1细胞的摄取没有差异。当在A / J小鼠体内进行测试时， ladC 突变体的竞争指数在72小时内下降了五倍，表明与野生型 L相比，其衰减显着。肺炎。尽管LadC定位于细菌内膜表明该蛋白可能参与调节毒力基因表达的信号传导途径，但微阵列分析表明 ladC 不会影响 L的转录谱。肺炎在体外或 A期间。卡氏菌感染。尽管尚不清楚LadC调节细菌与宿主细胞之间初始相互作用的机制，但我们已经确定LadC在 L中起着重要作用。肺炎感染。

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