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

分枝杆菌利用VII型（ESX）分泌系统将底物输出穿过其复杂的细胞包膜。 分枝杆菌编码多种旁系同源ESX系统（M.结核病编码五个ESX系统）， 已经进化到执行非冗余功能。尽管近二十年的研究，只有ESX-1，-3和 -5已被表征并在毒力、铁吸收和细胞壁完整性方面被赋予功能， 分别缺乏对ESX系统、其基底和 细胞外靶点代表了我们对分枝杆菌生物学的理解中的一个重大空白。 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。esx 4基因座基因表达的缺失混淆了之前的ESX-4 分析并阻止其功能表征。然而，我们证明ESX-4表达是 通过直接细胞-细胞接触触发，其激活细胞质外σ因子SigM， 保守的核心esx 4基因的转录。我们的数据还表明，SigM致力于监管 一个扩展的ESX-4基因组，其中许多在其他分枝杆菌物种中是保守的， 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在M.而ESX-4诱导 总菌落形态和细胞壁组成的变化。In M.因此，ESX-4在以下方面发挥着直接作用： 发病机制：它是巨噬细胞和阿米巴细胞内生存所必需的，阻断吞噬体 酸化并促进感染的巨噬细胞中的吞噬体破裂。通过扩展，我们假设ESX-4 在其他致病性分枝杆菌中起重要作用，由ESX-4介导分泌效应分子 和细胞壁的重塑。在本提案中，我们将比较和对比M. tuberculosis，M. marinum，M. P. smegalloy，利用SigM激活ESX-4的能力。 使用生物化学、化学生物学、遗传学和分子技术的组合，我们将定义 每个物种的SigM调节子和ESX-4分泌体。我们期望识别出效应分子， 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重塑 巨噬细胞总之，这项工作将提供ESX-4的第一个全面分析，重要的是， 重要的新的见解保守和物种特异性功能的这一祖先分泌装置。

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.