Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA.
School of Biological Sciences, Southern Illinois University Carbondale, Carbondale, Illinois, USA.
mBio. 2020 Sep 1;11(5):e02016-20. doi: 10.1128/mBio.02016-20.
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 , ClpX is encoded within an operon 3' to 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 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 transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in by a plasmid copy of Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation. 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 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.
是一种专性细胞内细菌，在其复杂的发育周期中，细菌在两种功能和形态上明显不同的形式之间分化，即原体（EB）和网状体（RB），每种形式都表达自己特有的专门蛋白质组。初级（EB 到 RB）和次级（RB 到 EB）分化都需要蛋白质周转，我们假设蛋白酶对于介导分化至关重要。Clp 蛋白酶系统在细菌中得到很好的保守，并且对蛋白质周转很重要。该系统至少依赖于丝氨酸蛋白酶亚基 ClpP 和一个 AAA+ATP 酶，如 ClpX，该酶识别并展开 ClpP 降解的底物。在 中，ClpX 编码在一个操纵子 3'到 我们提供的证据表明，衣原体的 ClpX 和 ClpP2 同源物对于生物体的存活和发育是必不可少的。我们在这里证明，衣原体的 ClpX 是一种功能性 ATP 酶，并形成预期的同六聚体 过表达缺乏 ATP 酶活性的 ClpX 突变体对 DNA 复制或次级分化的影响有限，但仍然降低了 EB 的活力，并观察到 EB 形态的明显缺陷。相反，过表达催化失活的 ClpP2 突变体显著影响发育周期的进展，降低了生物体的总数。使用 CRISPR 干扰阻断 转录导致细菌生长减少，而在 中，通过质粒拷贝的 互补了这一效应。总的来说，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具有不同的功能。 是全球传染性失明的主要原因，也是国内外报告最多的细菌性性传播感染。鉴于经济负担、缺乏批准的疫苗以及广谱抗生素治疗感染的使用，了解衣原体的生长和发育对于开发新型靶向抗生素至关重要。Clp 蛋白是细菌中重要且保守的蛋白酶系统。我们的工作强调了衣原体 Clp 蛋白对这种具有临床重要性的细菌的重要性。此外，我们的研究表明 Clp 系统在衣原体发育周期的进展中起着不可或缺的作用，这可能有助于建立衣原体属和其他细菌各自发育周期进展的模型。我们的工作还为 Clp 特异性研究的不断发展做出了贡献，这进一步强调了该系统在细菌进化过程中的重要性和多功能性，并进一步验证了 Clp 蛋白作为药物靶点的合理性。
