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

分枝杆菌利用III型(ESX)分泌系统将底物输出到其复杂的细胞膜上。 分枝杆菌编码多个平行的ESX系统(结核分枝杆菌编码五个ESX系统)，其中 已经进化到执行非冗余功能。尽管进行了近20年的研究，但只有ESX-1、-3和 已对毒力、铁摄取和细胞壁完整性进行了表征和功能分配， 分别进行了分析。对ESX系统的作用、它们的底物和 细胞外靶点代表了我们对分枝杆菌生物学的理解的一个重大差距。 在这项提案中，我们将重点关注ESX-4，它被认为是ESX的前体分泌系统，因为它 在所有的分枝杆菌中都存在。Esx4基因座基因表达的缺失使以前的esx-4基因混乱 分析并防止其功能表征。然而，我们演示了ESX-4的表达是 由细胞间的直接接触触发，激活胞浆外Sigm因子，启动 保守的核心esx4基因转录。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保守，也 由Sigm控制。通过SIGM直接控制ESX-4表达的能力提供了一个独特的机会 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对于耻垢分枝杆菌的配偶DNA转移是必不可少的，并且ESX-4诱导 毛菌落形态和细胞壁组成的变化。在脓肿分枝杆菌中，ESX-4在 发病机制：巨噬细胞和阿米巴细胞内生存所需，阻断吞噬小体 酸化并促进受感染巨噬细胞的吞噬小体破裂。通过扩展，我们假设ESX-4 在其他致病分枝杆菌中发挥重要作用，由ESX-4分泌效应分子介导 以及细胞壁的重塑。在这个提案中，我们将比较和对比M。 结核、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。 使用生化、化学生物学、遗传学和分子技术的组合，我们将定义 SIGM调节子和ESX-4分泌体。我们预计能识别出效应分子 促进分枝杆菌细胞壁的重塑，以及那些针对其他细菌或宿主的重塑 巨噬细胞。总而言之，这项工作将提供对ESX-4的首次全面分析，重要的是， 对这一祖先分泌器官的保守和物种特有功能的重大新见解。

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.