Dissecting and connecting the SigM stimulus and ESX-4 secretory response in mycobacteria

剖析并连接分枝杆菌中的 SigM 刺激和 ESX-4 分泌反应

• 批准号: 10706956
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 40.53万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10706956
• 关键词: Amoeba genus; Bacteria; Binding; Biochemical; Biology; Cell Wall; Cells; Chemicals; Complex; Cytoplasm; DNA; Data; Dedications; Disease; ELF3 gene; Gene Expression; Genes; Genetic; Genus Mycobacterium; Goals; Iron; Learning; Macrophage; Mediating; Molecular; Morphology; Mutagenesis; Mycobacterium abscessus; Mycobacterium marinum; Mycobacterium smegmatis; Mycobacterium tuberculosis; Pathogenesis; Pathogenicity; Phagosomes; Phenotype; Physiology; Play; Protein Secretion; Public Health; Publishing; Regulation; Regulon; Research; Role; Rupture; Sigma Factor; Stimulus; Structure; System; Techniques; Testing; Transcription Initiation; Virulence; Work; cell envelope; cellular targeting; comparative; extracellular; genomic locus; insight; intercellular communication; mutant; mycobacterial; prevent; progenitor; promoter; resilience; response; transcriptome sequencing; uptake

Mycobacteria utilize Type VII (ESX) secretion systems to export substrates across their complex cell envelope. Mycobacteria encode multiple paralogous ESX systems (M. tuberculosis encodes five ESX systems), which have evolved to perform non-redundant functions. Despite almost two decades of research, only ESX-1, -3 and -5 have been characterized and assigned functions in virulence, iron-uptake and cell wall integrity, respectively. The lack of a comprehensive understanding of the roles of ESX systems, their substrates and extracellular targets represents a significant gap in our understanding of mycobacterial biology. In this proposal, we will focus on ESX-4, which is considered the progenitor ESX secretion system because it is found in all mycobacteria. The absence of esx4 locus gene expression had confounded previous ESX-4 analyses and prevented its functional characterization. However, we demonstrated ESX-4 expression is triggered by direct cell-cell contact, which activates the extra-cytoplasmic sigma factor, SigM, to initiate transcription of the conserved core esx4 genes. Our data also show that SigM is dedicated to the regulation of an extended ESX-4-gene set, many of which are conserved in other mycobacterial species and are also controlled by SigM. The ability to directly control ESX-4 expression via SigM provides a unique opportunity to study the role of ESX-4 in both fast- and slow-growing mycobacteria. We have demonstrated ESX-4 is essential for conjugal DNA transfer in M. smegmatis and that ESX-4 induces changes in gross colony morphology and cell wall composition. In M. abscessus, ESX-4 plays a direct role in pathogenesis: it is required for intracellular survival in macrophages and amoebae, blocks phagosome acidification and promotes phagosomal rupture in infected macrophages. By extension, we hypothesize ESX-4 plays an important role in other pathogenic mycobacteria, mediated by ESX-4 secretion of effector molecules and remodeling of the cell wall. In this proposal, we will compare and contrast the ESX-4 systems of M. tuberculosis, M. marinum, M. abscessus and M. smegmatis, utilizing the ability to activate ESX-4 by SigM. Using a combination of biochemical, chemical biology, genetic, and molecular techniques we will define the SigM regulon and ESX-4 secretomes of each species. We anticipate identifying effector molecules that facilitate remodeling of the mycobacterial cell wall, as well as those that target other bacteria or the host macrophage. Together, this work will provide the first comprehensive analysis of ESX-4 and, importantly, significant new insights on the conserved and species-specific functions of this ancestral secretion apparatus.

分枝杆菌利用VII型（ESX）分泌系统在其复杂的细胞包膜上导出底物。 分枝杆菌编码多个寄生虫ESX系统（结核M.编码五个ESX系统），其中 已经演变为执行非冗余功能。尽管研究了将近二十年，但仅ESX -1，-3和 -5在毒力，铁摄取和细胞壁完整性方面的表征和分配功能， 分别。缺乏对ESX系统，其底物和的角色的全面了解 细胞外靶标代表了我们对分枝杆菌生物学的理解的显着差距。 在此提案中，我们将重点关注ESX-4，这被认为是祖细胞ESX分泌系统 在所有分枝杆菌中发现。 ESX4基因座基因表达的缺失使先前的ESX-4混淆了 分析并防止其功能表征。但是，我们证明了ESX-4表达是 由直接细胞 - 细胞接触触发，该接触激活外质Sigma因子Sigm启动 保守核心ESX4基因的转录。我们的数据还表明，SIGM致力于调节 一个扩展的ESX-4基因组，其中许多是在其他分枝杆菌中保守的，也是 由SIGM控制。通过SIGM直接控制ESX-4表达的能力为 研究ESX-4在快速和缓慢增长的分枝杆菌中的作用。 我们已经证明ESX-4对于smegmatis中的链与DNA转移至关重要，ESX-4诱导 总体菌落形态和细胞壁组成的变化。在腹部大分子中，ESX-4在 发病机理：巨噬细胞和变形虫的细胞内存活需要阻塞吞噬体 酸化并促进感染巨噬细胞中的吞噬体破裂。通过扩展，我们假设ESX-4 ESX-4效应分子分泌介导的其他致病性分枝杆菌中起重要作用 并重塑细胞壁。在此提案中，我们将比较和对比M的ESX-4系统。 Tuberculosis，M。Marinum，M。Arbcessus和M. smegmatis，利用SIGM激活ESX-4的能力。 结合生化，化学生物学，遗传和分子技术，我们将定义 每个物种的sigm gemulon和esx-4秘密组。我们预计识别效应子分子 促进分枝杆菌细胞壁的重塑，以及靶向其他细菌或宿主的细胞壁 巨噬细胞。这项工作将共同提供ESX-4的首次综合分析，重要的是 对这种祖先分泌设备的保守和物种特异性功能的重要新见解。