Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each
Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each
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DOI：
10.1128/jb.00635-18
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发表时间：
2019-01-01
影响因子：
3.2
通讯作者：
Ouellette, Scot P.
中科院分区：
文献类型：
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作者：
Wood, Nicholas A.;Chung, Krystal Y.;Ouellette, Scot P.
衣原体的成员是专性细胞内细菌，在其发育周期中分为两种不同的功能和形态形式，初级体（EBs）和网状体（RBs）。EBs是一种不分裂的小电子密度形式，可以感染宿主细胞。RBs是较大的非传染性复制形式，在膜结合的囊泡内发育，称为包涵体。鉴于这种细菌的每种发育形式的独特性质，我们假设Clp蛋白酶系统通过降解来自一种或另一种发育形式的特定蛋白质，在蛋白质组转换中起着不可或缺的作用。衣原体有5个未被鉴定的clp基因，clpX、clpC、两个clpP类似物和clpB。在其他细菌中，ClpC和ClpX是atp酶，它们展开并将蛋白质提供给被降解的ClpP蛋白酶，而ClpB是一种去聚集酶。在这里，我们重点描述了ClpP的相似之处。转录分析和免疫印迹法确定这些基因在周期中期表达。这些蛋白的生物信息学分析确定了对活性重要的关键残基。非活性cipP突变体在衣原体中的过表达表明每个ClpP平行体具有独立的功能。为了进一步探索这些差异，我们使用细菌双杂交实验和重组蛋白的天然凝胶分析来确定ClpP蛋白之间的相互作用。检测到ClpP蛋白的同型相互作用，但未检测到ClpP相似物之间的异型相互作用。有趣的是，在体外检测到ClpP2的蛋白酶活性，而不是ClpP1。这种活性是由已知能激活ClpP的抗生素刺激的，ClpP也能阻断衣原体的生长。我们的数据表明，衣原体ClpP相似物可能在这种重要病原体中起着独特而关键的作用。沙眼衣原体是世界范围内可预防的感染性失明和细菌性传播感染的主要原因。衣原体是发育调节的专性细胞内病原体，在两种功能和形态形式之间交替，具有不同的蛋白质谱。我们假设蛋白质降解是发育周期的一个关键方面。Clp蛋白酶系统是细菌中参与蛋白质周转的一个关键系统。在这里，我们通过检测它们在衣原体中的表达、它们的寡聚能力和它们的蛋白水解活性来表征这两种衣原体ClpP相似物。这项工作将有助于了解细胞内生物背景下进化多样性的Clp蛋白酶，这可能有助于研究其他临床相关的细胞内细菌。
Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive cipP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCE Chlarnydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.