Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX 77843, USA.
Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21755-60. doi: 10.1073/pnas.1010485107. Epub 2010 Nov 23.
Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based β-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila. Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and (ii) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila. Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii.
贝氏考克斯体是一种专性细胞内细菌病原体,可引起急性和慢性 Q 热。这种细菌拥有一种与嗜肺军团菌的 Dot/Icm 高度相似的 IV 型分泌系统(T4SS),被认为对其感染力至关重要。考克斯氏体 T4SS 的蛋白质底物被预测有助于形成有利于其细胞内生长的吞噬体。然而,由于缺乏遗传系统,考克斯氏菌 Dot/Icm 的蛋白质转移尚未得到证实。在这项研究中,我们使用荧光β-内酰胺酶(TEM1)易位测定法以及替代宿主嗜肺军团菌中的钙调蛋白依赖性环化酶(CyaA)测定法,报告了鉴定出的 32 种考克斯氏菌 Dot/Icm 系统的底物。值得注意的是,鉴定出的 26 种 T4SS 底物是没有预测功能的假设蛋白。候选分泌底物是通过(i)遗传筛选鉴定与 T4SS 组件 DotF 相互作用的考克斯氏菌蛋白,以及(ii)生物信息学方法从考克斯氏菌和嗜肺军团菌中检索预测与以前报道的 Dot/Icm 系统底物相关的特征的候选基因获得的。此外,我们开发了一种穿梭质粒,允许在考克斯氏菌中表达重组蛋白作为 TEM 融合产物。使用该系统,我们证明了用嗜肺军团菌鉴定的 Dot/Icm 底物也被考克斯氏菌易位,该过程需要其 C 末端,为考克斯氏菌中的功能性 T4SS 提供了直接的遗传证据。