EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity.
EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity.
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DOI：
10.1371/journal.ppat.1000957
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发表时间：
2010-06-24
期刊：
影响因子：
6.7
通讯作者：
Fortune SM
中科院分区：
文献类型：
--
作者：
Garces A;Atmakuri K;Chase MR;Woodworth JS;Krastins B;Rothchild AC;Ramsdell TL;Lopez MF;Behar SM;Sarracino DA;Fortune SM
结核分枝杆菌（Mtb）需要ESX1专门的蛋白分泌系统的毒力，触发胞质免疫监视途径，并引发最佳的CD8+ T细胞反应。这表明ESX1可能主要通过破坏细菌周围的吞噬体膜的稳定来发挥作用。然而，鉴定ESX1系统的主要功能一直很困难，因为任何底物的缺失都会抑制所有已知底物的分泌，从而消除所有ESX1活性。在这里，我们证明了ESX1底物EspA在分泌后形成二硫键连接的同源二聚体。通过破坏EspA二硫键的形成，我们已经将毒力与其他已知的ESX1介导的活性分离开来。EspA二硫键形成的抑制不抑制ESX1分泌、感染的巨噬细胞中胞质模式受体的ESX1依赖性刺激或Mtb引发对ESX1底物的适应性免疫应答的能力。然而，阻断EspA二硫键形成严重削弱了Mtb在小鼠中存活和致病的能力。引人注目的是，我们发现，EspA二硫键形成的抑制也显着损害分枝杆菌细胞壁的稳定性，因为ESX1基因座或ESX1系统的单个组件的删除。因此，我们证明EspA是ESX1介导的毒力的主要决定因素，独立于其在ESX1分泌中的功能。我们认为ESX1和EspA在体内Mtb的毒力中起着核心作用，因为它们改变了分枝杆菌细胞壁的完整性。从结核病疫苗BCG的研究中，我们知道结核分枝杆菌需要一个专门的蛋白质分泌系统ESX1才能在人体内引起疾病。ESX1是Mtb所需的，以吸收细菌所在的宿主细胞，这被认为解释了它在毒力中的核心作用。然而，其他数据表明ESX1在细菌本身中起着重要作用，改变了生物体的细胞壁。然而，很难确定这些ESX1相关功能的相对重要性，因为删除该装置的任何部分都会完全消除所有ESX1活性。在这里，我们使用一种简单的方法来确定ESX1分泌的蛋白质之一EspA的功能重要目标。我们突变EspA，使ESX1系统仍然分泌其底物，但细菌不再引起疾病。减毒EspA突变体在其细胞壁中具有缺陷，但在体外与宿主细胞的相互作用中没有缺陷。我们认为ESX1系统及其分泌的蛋白质对于结核分枝杆菌的生存和在人体中引起疾病非常重要，因为它们可以确保细菌细胞壁的完整性。
Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall. From studies of BCG, the tuberculosis vaccine, we know that Mycobacterium tuberculosis requires a specialized protein secretion system, ESX1, to cause disease in people. ESX1 is required for Mtb to co-opt the host cells in which the bacterium resides and it is thought that this explains its central role in virulence. However, other data suggests that ESX1 serves an important role in the bacterium itself, altering the organism's cell wall. It has been difficult to determine the relative significance of these ESX1-associated functions, however, because deletion of any piece of the apparatus completely abolishes all ESX1 activities. Here we use a simple approach to pinpoint the functionally significant target of one of the proteins secreted by ESX1, EspA. We mutate EspA such that the ESX1 system still secretes its substrates but the bacterium no longer causes disease. The attenuated EspA mutant has defects in its cell wall but not in its interactions with host cells in vitro. We propose that the ESX1 system and the proteins it secretes are important for Mtb to survive and cause disease in people because they act to ensure the integrity of the bacterial cell wall.
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