Illingworth Melissa, Hooppaw Anna J, Ruan Lu, Fisher Derek J, Chen Lingling
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
Department of Microbiology, Southern Illinois University, Carbondale, Illinois, USA.
J Bacteriol. 2017 May 25;199(12). doi: 10.1128/JB.00844-16. Print 2017 Jun 15.
Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigm chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of and homologs, paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three genes, (), , and We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel -specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the essentiality of the three genes through targeted mutagenesis in ChGroEL hydrolyzed ATP at a rate 25% of that of EcGroEL and bound with high affinity to ChGroES, and the ChGroEL-ChGroES complex could refold malate dehydrogenase (MDH). The chlamydial ChGroEL was selective for its cognate cochaperonin, ChGroES, while EcGroEL could function with both EcGroES and ChGroES. A P35T ChGroES mutant (ChGroESP35T) reduced ChGroEL-ChGroES interactions and MDH folding activities but was tolerated by EcGroEL. Both ChGroEL-ChGroES and EcGroEL-ChGroESP35T could complement an EcGroEL-EcGroES mutant. Finally, we successfully inactivated both paralogs but not , leading to minor growth defects in cell culture that were not exacerbated by heat stress. Collectively, our results support novel functions for the paralogs and solidify ChGroEL as a bona fide chaperonin that is biochemically distinct from EcGroEL. is an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL and chaperonin paralog ChGroEL2 have been associated with survival under stress conditions, and ChGroEL is linked with immunopathology elicited by chlamydial infections. However, their exact roles in bacterial survival and disease remain unclear. Our results further substantiate the hypotheses that ChGroEL is the primary chlamydial chaperonin and that the paralogs play specialized roles during infection. Furthermore, ChGroEL and the mitochondrial GroEL only functioned with their cochaperonin, in contrast to the promiscuous nature of GroEL from and , which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle.
伴侣蛋白在正常和应激条件下对细胞生长至关重要，因此是最保守且最古老的蛋白质类别之一。典型的伴侣蛋白EcGroEL及其共伴侣蛋白EcGroES通过依赖ATP的机制协助蛋白质折叠。除了存在同源物外，在许多细菌（包括病原体）中还发现了旁系同源物，它们进化出了尚不清楚的物种特异性功能。沙眼衣原体是专性胞内细菌，其基因组虽已缩减，但仍包含三个groEL基因，即cgtA（ChGroEL）、cgtB和cgtC。我们假设ChGroEL是真正的伴侣蛋白，而旁系同源物具有新的衣原体特异性功能。为了验证我们的假设，我们研究了ChGroEL及其共伴侣蛋白ChGroES的生化特性，并通过对沙眼衣原体进行靶向诱变来探究这三个groEL基因的必要性。ChGroEL水解ATP的速率是EcGroEL的25%，并与ChGroES具有高亲和力结合，且ChGroEL-ChGroES复合物可以使苹果酸脱氢酶（MDH）重新折叠。衣原体的ChGroEL对其同源共伴侣蛋白ChGroES具有选择性，而EcGroEL可以与EcGroES和ChGroES都发挥作用。P35T ChGroES突变体（ChGroESP35T）减少了ChGroEL-ChGroES相互作用和MDH折叠活性，但EcGroEL可以耐受。ChGroEL-ChGroES和EcGroEL-ChGroESP35T都可以互补EcGroEL-EcGroES突变体。最后，我们成功使两个旁系同源物失活，但未使cgtA失活，导致细胞培养中出现轻微的生长缺陷，热应激并未加剧这些缺陷。总体而言，我们的结果支持旁系同源物具有新功能，并巩固了ChGroEL作为一种在生化上与EcGroEL不同的真正伴侣蛋白的地位。沙眼衣原体是包括肺炎、性传播感染和沙眼在内的人类疾病的重要病因。衣原体伴侣蛋白ChGroEL和伴侣蛋白旁系同源物ChGroEL2与应激条件下的存活有关，且ChGroEL与衣原体感染引发的免疫病理学有关。然而，它们在细菌存活和疾病中的具体作用仍不清楚。我们的结果进一步证实了以下假设：ChGroEL是主要的衣原体伴侣蛋白，旁系同源物在感染过程中发挥特殊作用。此外，ChGroEL和线粒体GroEL仅与其共伴侣蛋白发挥作用，这与大肠杆菌和嗜热栖热菌的GroEL的混杂性质形成对比，这可能表明在从自由生活生物向专性胞内生活方式转变过程中GroEL的进化分歧。
