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) 分泌系统通过其复杂的细胞包膜输出底物。 分枝杆菌编码多个旁系同源 ESX 系统（结核分枝杆菌编码五个 ESX 系统）， 已经发展到执行非冗余功能。尽管进行了近二十年的研究，但只有 ESX-1、-3 和 -5 已被表征并指定了毒力、铁吸收和细胞壁完整性的功能， 分别。对 ESX 系统的作用、其底层和缺乏全面的了解 细胞外靶标代表了我们对分枝杆菌生物学理解的重大差距。 在本提案中，我们将重点关注 ESX-4，它被认为是 ESX 分泌系统的祖先，因为它 存在于所有分枝杆菌中。 esx4 位点基因表达的缺失使之前的 ESX-4 变得混乱 分析并阻止其功能表征。然而，我们证明了 ESX-4 表达是 由细胞与细胞直接接触触发，激活细胞质外 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 致力于监管 扩展的 ESX-4 基因集，其中许多在其他分枝杆菌物种中是保守的，并且也是 由 SigM 控制。通过 SigM 直接控制 ESX-4 表达的能力提供了独特的机会 研究 ESX-4 在快速和缓慢生长的分枝杆菌中的作用。 我们已经证明 ESX-4 对于耻垢分枝杆菌中的配偶 DNA 转移至关重要，并且 ESX-4 诱导 总体菌落形态和细胞壁组成的变化。在脓肿分枝杆菌中，ESX-4 在以下方面发挥直接作用： 发病机制：它是巨噬细胞和阿米巴原虫细胞内生存所必需的，阻断吞噬体 酸化并促进受感染巨噬细胞的吞噬体破裂。推而广之，我们假设 ESX-4 通过效应分子的 ESX-4 分泌介导，在其他致病性分枝杆菌中发挥重要作用 以及细胞壁的重塑。在本提案中，我们将比较和对比 M 的 ESX-4 系统。 结核分枝杆菌、海洋分枝杆菌、脓肿分枝杆菌和耻垢分枝杆菌，利用 SigM 激活 ESX-4 的能力。 结合生物化学、化学生物学、遗传和分子技术，我们将定义 每个物种的 SigM 调节子和 ESX-4 分泌组。我们期望识别出效应分子 促进分枝杆菌细胞壁以及针对其他细菌或宿主的细胞壁的重塑 巨噬细胞。总之，这项工作将提供对 ESX-4 的首次全面分析，而且重要的是， 关于这种祖先分泌装置的保守性和物种特异性功能的重要新见解。