Nucleoid structure and energy metabolism in chlamydial gene expression

描述（由申请方提供）：衣原体属细菌是专性细胞内寄生虫，包括常见的人类病原体，如沙眼衣原体，这是性传播疾病和致盲性沙眼的主要原因。与大多数细菌病原体不同，C.沙眼衣原体在两种生理上不同的细胞形式之间交替以建立感染：感染性初级体（EB）和复制性网状体（RB）。两种组蛋白相关蛋白HctA和HctB在发育形式之间的过渡期间调节衣原体染色质的压缩和松弛，并控制RNA聚合酶进入DNA。衣原体复制生态位的生物发生依赖于EB萌发成RB细胞类型时蛋白质表达的重新启动。在分化过程中控制衣原体基因表达的因素仍然是一个重大的知识差距。2-C-甲基-D-β-2，4-环二磷酸（MEC）是类异戊二烯合成的甲基β-磷酸（MEP）途径的中间体，其促进衣原体组蛋白从分离的EB染色质结构解离。MEP途径起始于糖酵解中间体甘油醛3-P和丙酮酸的缩合，因此可能受糖酵解活性调节。衣原体磷酸己糖转运蛋白UhpC在形态分化开始时的表达以及最近证实的体外葡萄糖6-P对EB的代谢活化表明，糖酵解和病原体能量代谢的活化与染色质解凝在时间上相关。我们假设组蛋白通过与染色体上的特定位点或结构元件结合，通过影响调控蛋白进入参与分化控制的启动子和基因，从而有序控制早期基因表达。我们进一步预测，代谢的启动是控制组蛋白释放的关键因素。本提案的目的是确定染色质结构在发育调控中的作用，并在感染后立即建立EB能量代谢启动和EB染色质结构调控之间的联系。将使用高通量DNA和RNA-seq技术测定染色质结构和从头基因转录之间的关系，以测量细菌在含有葡萄糖6-P的宿主无细胞培养基中孵育后和早期分化期间DNA可及性和RNA转录的变化。将体内发育早期EB染色质结构的调节与体外活化EB的染色质结构变化进行比较，将为EB萌发机制提供前所未有的见解。此外，由于MEP途径不存在于哺乳动物细胞中，了解代谢在激活衣原体致病循环中的新作用将为预防或治疗衣原体感染提供潜在的途径。

DESCRIPTION (provided by applicant): Bacteria of the genus Chlamydia are obligate intracellular parasites and include common human pathogens such as Chlamydia trachomatis, a leading cause of sexually transmitted diseases and blinding trachoma. Unlike most bacterial pathogens, C. trachomatis alternates between two physiologically distinct cell forms to establish infection: the infectious Elementary Body (EB) and replicative Reticulate Body (RB). Two histone related proteins, HctA and HctB, regulate compaction and relaxation of chlamydial chromatin during transition between developmental forms and control RNA polymerase access to the DNA. Biogenesis of the chlamydial replication niche depends on the re-initiation of protein expression as the EB germinates into the RB cell type. The factors controlling chlamydial gene expression during differentiation remains a significant knowledge gap. 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC) is an intermediate of the methylerythritol phosphate (MEP) pathway for isoprenoid synthesis that promotes dissociation of chlamydial histones from isolated EB chromatin structures. The MEP pathway starts with condensation of the glycolytic intermediates glyceraldehyde 3-P and pyruvate and is therefore likely to be regulated by glycolytic activity. Expression of the chlamydial hexose phosphate transporter UhpC at the onset of morphological differentiation and recently demonstrated metabolic activation of EBs by glucose 6-P in vitro suggests that activation of glycolysis and pathogen energy metabolism is temporally linked to chromatin decondensation. We hypothesize that the histones, by binding to specific sites or structural elements on the chromosome, allow ordered control of early gene expression by affecting the access of regulatory proteins to promoters and genes involved in the control of differentiation. We further predict that initiation of metabolism is a key element in controlling histone release. The aim of this proposal is to determine the role of chromatin structure in developmental regulation and to establish a link between initiation of EB energy metabolism and regulation of EB chromatin structure immediately upon infection. The relationship between chromatin structure and de novo gene transcription will be determined using high throughput DNA- and RNA-seq techniques to measure changes in DNA accessibility and RNA transcription upon incubation of bacteria in a host cell-free medium containing glucose 6-P, and during early differentiation. Comparing the regulation of the EB chromatin structure early in development in vivo to chromatin structure changes of activated EBs in vitro will provide unprecedented insight into the mechanisms of EB germination. Additionally, as the MEP pathway is not present in mammalian cells, understanding the novel role of metabolism in activating the chlamydial pathogenic cycle will provide potential avenues for preventing or treating chlamydial infections.