Marsh James W, Wee Bryan A, Tyndall Joel D A, Lott William B, Bastidas Robert J, Caldwell Harlan D, Valdivia Raphael H, Kari L, Huston Wilhelmina M
Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), 60 Musk Avenue, Kelvin Grove, QLD, 4059, Australia.
National School of Pharmacy, University of Otago, PO Box 56, Dunedin, 9054, New Zealand.
BMC Microbiol. 2015 Sep 30;15:194. doi: 10.1186/s12866-015-0533-2.
Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism's intracellular developmental cycle. HtrA is a critical stress response serine protease and chaperone for many bacteria and in C. trachomatis has been previously shown to be important for heat stress and the replicative phase of development using a chemical inhibitor of the CtHtrA activity. In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized.
SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis. The C. trachomatis strains containing the SNVs with marked reductions in proteolysis were investigated in cell culture to identify phenotypes that could be linked to CtHtrA function.
The strain harboring the SNV with the most marked impact on proteolysis (cthtrA P370L) was detected to have a significant reduction in the production of infectious elementary bodies.
This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.
沙眼衣原体是全球最常见的细菌性性传播感染病原体，也是可预防性失明的主要原因。由于该生物体的细胞内发育周期，用于研究沙眼衣原体的遗传方法直到最近才得以开发。HtrA是许多细菌关键的应激反应丝氨酸蛋白酶和伴侣蛋白，在沙眼衣原体中，先前已证明使用CtHtrA活性的化学抑制剂时，其对热应激和发育的复制阶段很重要。在本研究中，鉴定并表征了cthtrA基因中化学诱导的单核苷酸变异（SNV），这些变异导致了氨基酸替代（A240V、G475E和P370L）。
最初使用重组蛋白技术在体外对SNV进行生化表征，以确认其对蛋白水解的功能影响。在细胞培养中研究含有蛋白水解显著减少的SNV的沙眼衣原体菌株，以鉴定可能与CtHtrA功能相关的表型。
检测到对蛋白水解影响最显著的SNV（cthtrA P370L）所在的菌株，其感染性原体的产生显著减少。
这提供了遗传学证据，表明CtHtrA对沙眼衣原体的发育周期至关重要。
