Department of Medical Microbiology and Infection Control, VU University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Dis Model Mech. 2011 Jul;4(4):526-36. doi: 10.1242/dmm.006676. Epub 2011 Mar 3.
The hallmark of tuberculosis (TB) is the formation of granulomas, which are clusters of infected macrophages surrounded by additional macrophages, neutrophils and lymphocytes. Although it has long been thought that granulomas are beneficial for the host, there is evidence that mycobacteria also promote the formation of these structures. In this study, we aimed to identify new mycobacterial factors involved in the initial stages of granuloma formation. We exploited the zebrafish embryo Mycobacterium marinum infection model to study initiation of granuloma formation and developed an in vivo screen to select for random M. marinum mutants that were unable to induce granuloma formation efficiently. Upon screening 200 mutants, three mutants repeatedly initiated reduced granuloma formation. One of the mutants was found to be defective in the espL gene, which is located in the ESX-1 cluster. The ESX-1 cluster is disrupted in the Mycobacterium bovis BCG vaccine strain and encodes a specialized secretion system known to be important for granuloma formation and virulence. Although espL has not been implicated in protein secretion before, we observed a strong effect on the secretion of the ESX-1 substrates ESAT-6 and EspE. We conclude that our zebrafish embryo M. marinum screen is a useful tool to identify mycobacterial genes involved in the initial stages of granuloma formation and that we have identified a new component of the ESX-1 secretion system. We are confident that our approach will contribute to the knowledge of mycobacterial virulence and could be helpful for the development of new TB vaccines.
结核分枝杆菌（TB）的标志是肉芽肿的形成，即受感染的巨噬细胞簇被额外的巨噬细胞、中性粒细胞和淋巴细胞包围。尽管长期以来人们一直认为肉芽肿对宿主有益，但有证据表明分枝杆菌也促进了这些结构的形成。在这项研究中，我们旨在确定参与肉芽肿形成初始阶段的新分枝杆菌因子。我们利用斑马鱼胚胎分枝杆菌感染模型来研究肉芽肿形成的起始，并开发了一种体内筛选方法，以选择不能有效诱导肉芽肿形成的随机分枝杆菌突变体。在筛选了 200 个突变体后，有三个突变体反复引发肉芽肿形成减少。其中一个突变体被发现 espL 基因缺陷，该基因位于 ESX-1 簇中。ESX-1 簇在牛分枝杆菌卡介苗疫苗株中缺失，并编码一种专门的分泌系统，已知该系统对肉芽肿形成和毒力很重要。尽管 espL 以前没有被牵连到蛋白质分泌中，但我们观察到它对 ESX-1 底物 ESAT-6 和 EspE 的分泌有强烈的影响。我们得出结论，我们的斑马鱼胚胎分枝杆菌筛选是一种有用的工具，可以识别参与肉芽肿形成初始阶段的分枝杆菌基因，并且我们已经确定了 ESX-1 分泌系统的一个新组件。我们相信我们的方法将有助于了解分枝杆菌的毒力，并可能有助于新的结核病疫苗的开发。
