尽管基因组高度减少,沙眼衣原体经历了一个复杂的发育周期,其中细菌区分以下两种功能和形态上不同的形式:非复制基本体(EB)和非传染性,复制网状体(RB)。EB和RB之间的转换不是由重新分配细胞内蛋白质的分裂事件介导的。相反,原发性(EB到RB)和继发性(RB到EB)分化可能都需要大量蛋白质周转。靶向蛋白质降解的一个系统是核糖体拯救的转译系统,在翻译过程中停滞的多肽用杂合tRNA-mRNA编码的SsrA标签标记,tmRNA。ClpX识别SsrA标记,导致ClpXP介导的降解。我们假设ClpX通过靶向蛋白质降解在衣原体分化中起作用。我们发现,与识别SsrA标记的底物相关的ClpX中特定基序内的关键残基(R230A)的突变导致消除了二次分化,而不会减少衣原体复制或发育周期的进展,如转录物所测量的。此外,通过化学和靶向遗传方法抑制转译也阻碍了衣原体的发育。tmRNA的敲低以及随后与SsrA标签中突变的等位基因的互补紧密地表现出ClpXR230A的过表达,因此表明ClpX对SsrA标记底物的识别在次级分化中起着至关重要的作用。一起来看,这些数据提供了对衣原体发育形式之间过渡要求的机械见解。重要性沙眼衣原体是细菌性性传播感染和可预防的感染性失明的主要原因。这种独特的有机体经历了传染性之间的发育过渡,非分裂形式和非传染性,划分形式。因此,衣原体发育周期是衣原体特异性抗生素的一个有吸引力的目标,这将最大限度地减少广谱抗生素对抗生素耐药性在其他生物体中传播的影响。然而,缺乏关于衣原体发育在分子水平上的知识阻碍了特定的鉴定,可下药的目标.这项工作描述了ClpXP的转译和蛋白质组周转的基本过程有助于衣原体分化的机制,衣原体生长和存活的关键方面。鉴于转译和ClpX在真细菌中几乎普遍存在,这种机制可能在其他细菌物种的发育周期中得到保留。此外,这项研究通过强调这些系统在整个细菌进化过程中的功能多样性,扩大了翻译和Clp蛋白酶的领域。
Despite having a highly reduced genome, Chlamydia trachomatis undergoes a complex developmental cycle in which the bacteria differentiate between the following two functionally and morphologically distinct forms: the infectious, nonreplicative elementary body (EB) and the noninfectious, replicative reticulate body (RB). The transitions between EBs and RBs are not mediated by division events that redistribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require bulk protein turnover. One system for targeted protein degradation is the trans-translation system for ribosomal rescue, where polypeptides stalled during translation are marked with an SsrA tag encoded by a hybrid tRNA-mRNA, tmRNA.
ClpX recognizes the SsrA tag, leading to ClpXP-mediated degradation. We hypothesize that
ClpX functions in chlamydial differentiation through targeted protein degradation. We found that mutation of a key residue (R230A) within the specific motif in
ClpX associated with the recognition of SsrA-tagged substrates resulted in abrogated secondary differentiation while not reducing chlamydial replication or developmental cycle progression as measured by transcripts. Furthermore, inhibition of trans-translation through chemical and targeted genetic approaches also impeded chlamydial development. Knockdown of tmRNA and subsequent complementation with an allele mutated in the SsrA tag closely phenocopied the overexpression of ClpXR230A, thus suggesting that
ClpX recognition of SsrA-tagged substrates plays a critical function in secondary differentiation. Taken together, these data provide mechanistic insight into the requirements for transitions between chlamydial developmental forms. IMPORTANCE Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and preventable infectious blindness. This unique organism undergoes developmental transitions between infectious, nondividing forms and noninfectious, dividing forms. Therefore, the chlamydial developmental cycle is an attractive target for Chlamydia-specific antibiotics, which would minimize effects of broad-spectrum antibiotics on the spread of antibiotic resistance in other organisms. However, the lack of knowledge about chlamydial development on a molecular level impedes the identification of specific, druggable targets. This work describes a mechanism through which both the fundamental processes of trans-translation and proteomic turnover by ClpXP contribute to chlamydial differentiation, a critical facet of chlamydial growth and survival. Given the almost universal presence of trans-translation and
ClpX in eubacteria, this mechanism may be conserved in developmental cycles of other bacterial species. Additionally, this study expands the fields of trans-translation and Clp proteases by emphasizing the functional diversity of these systems throughout bacterial evolution.