The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development
The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development
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DOI：
10.1128/mbio.02016-20
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发表时间：
2020-09-01
期刊：
影响因子：
6.4
通讯作者：
Ouellette, Scot P.
中科院分区：
文献类型：
--
作者：
Wood, Nicholas A.;Blocker, Amanda M.;Ouellette, Scot P.
沙眼衣原体（Chlamydia trachomatis）是一种专性细胞内细菌，其经历复杂的发育周期，其中细菌区分两种功能和形态上不同的形式，即基本体（EB）和网状体（RB），每种形式表达其自身的专门蛋白质库。原发性（EB到RB）和继发性（RB到EB）分化都需要蛋白质周转，我们假设蛋白酶对介导分化至关重要。Clp蛋白酶系统在细菌中非常保守，并且对于蛋白质周转非常重要。最低限度地，该系统依赖于丝氨酸蛋白酶亚基ClpP和AAA+ ATP酶，如ClpX，其识别并展开ClpP降解的底物。在衣原体中，ClpX在clpP2的3'操纵子内编码。我们提出的证据表明，衣原体ClpX和ClpP2同源物是必不可少的有机体的生存能力和发展。我们在这里证明，衣原体ClpX是一个功能性的ATP酶，并在体外形成预期的homohexamer。缺乏ATP酶活性的ClpX突变体的过表达对DNA复制或二级分化的影响有限，但尽管如此，仍降低了EB的活力，并观察到EB形态的明显缺陷。相反，催化失活的ClpP2突变体的过表达通过减少生物体的总数而显著影响发育周期进程。使用CRISPR干扰阻断clpP2X转录导致细菌生长减少，并且这种效应通过clpP2的质粒拷贝反式补充。综上所述，我们的数据表明，ClpX和相关的ClpP2在衣原体发育周期的进展和分化中发挥不同的功能。重要性沙眼衣原体是全球感染性失明的主要原因，也是国内外报道最多的细菌性传播感染。鉴于经济负担，缺乏批准的疫苗，以及使用广谱抗生素治疗感染，了解衣原体的生长和发育对于新型靶向抗生素的发展至关重要。Clp蛋白在细菌中构成重要且保守的蛋白酶系统。我们的工作突出了衣原体Clp蛋白对这种临床重要细菌的重要性。此外，我们的研究暗示Clp系统在衣原体发育周期进展中发挥着不可或缺的作用，这可能有助于建立衣原体如何在体内发育的模型。和其他细菌通过它们各自的发育周期进展。我们的工作还有助于越来越多的Clp特异性研究，强调了该系统在整个细菌进化过程中的重要性和多功能性，并进一步验证了Clp蛋白作为药物靶点。
Chlamydia trachomatis is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the elementary body (EB) and reticulate body (RB), each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiations require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and an AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, ClpX is encoded within an operon 3' to clpP2. We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro. Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2. Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation.IMPORTANCE Chlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets.