Functional Analysis of EspM, an ESX-1-Associated Transcription Factor in Mycobacterium marinum.
Functional Analysis of EspM, an ESX-1-Associated Transcription Factor in Mycobacterium marinum.
复制标题
海分枝杆菌中 ESX-1 相关转录因子 EspM 的功能分析。
DOI：
10.1128/jb.00233-22
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发表时间：
2022
影响因子：
3.2
通讯作者：
Champion,PatriciaA
中科院分区：
文献类型：
--
作者：
Sanchez,KevinG;Prest,RebeccaJ;Nicholson,KathleenR;Korotkov,KonstantinV;Champion,PatriciaA
致病分枝杆菌使用ESX-1分泌系统逃避巨噬细胞吞噬体并在感染中存活。我们证明了ESX-1系统在海洋分枝杆菌（一种非结核性病原体和人类病原体结核分枝杆菌的模型）中受到反馈控制的调节。在存在功能性ESX-1系统的情况下，WhiB 6转录因子上调ESX-1底物基因的表达。在没有组装的ESX-1系统的情况下，保守的转录因子EspM通过特异性结合whiB 6启动子来抑制whiB 6的表达。WhiB 6和EspM共同微调ESX-1底物的水平以响应分泌系统。EspM对ESX-1系统的控制机制尚不清楚。在这里，我们进行了结构和功能分析，以探讨如何EspM的调节。使用生物化学方法，我们测量了体外EspMin的高阶寡聚体的形成。我们证明了体外多聚化可以通过EspM蛋白的多个结构域介导。使用细菌单杂交系统，我们表明，EspM自我协会通过多个域在大肠杆菌。使用这个系统，我们进行了遗传筛选，以识别未能自我关联的EspM变体。筛选产生了四种感兴趣的EspM变体，我们测试了它们在M中的活性。marinum。我们的研究表明，这两个螺旋-转角-螺旋结构域在功能上是不同的。此外，螺旋束结构域是体外野生型多聚化所必需的。我们的数据支持EspM单体或六聚体有助于调节whiB 6表达的模型。重要提示致病性分枝杆菌是对全球人类健康造成巨大负担的细菌。ESX-1分泌系统是致病性分枝杆菌在宿主体内存活并与宿主相互作用所必需的。ESX-1分泌系统的适当功能是通过严格控制分泌的毒力因子的表达来实现的，部分是通过转录调节来实现的。在这里，我们的特点是保守的转录因子EspM，它调节ESX-1毒力因子的表达。我们定义了EspM形成多聚体和结合DNA所需的结构域。这些发现提供了一个初步的表征ESX-1转录因子，并提供其作用机制的见解。
Pathogenic mycobacteria use the ESX-1 secretion system to escape the macrophage phagosome and survive infection. We demonstrated that the ESX-1 system is regulated by feedback control in Mycobacterium marinum, a nontuberculous pathogen and model for the human pathogen Mycobacterium tuberculosis. In the presence of a functional ESX-1 system, the WhiB6 transcription factor upregulates expression of ESX-1 substrate genes. In the absence of an assembled ESX-1 system, the conserved transcription factor, EspM, represseswhiB6expression by specifically binding thewhiB6promoter. Together, WhiB6 and EspM fine-tune the levels of ESX-1 substrates in response to the secretion system. The mechanisms underlying control of the ESX-1 system by EspM are unknown. Here, we conduct a structure and function analysis to investigate how EspM is regulated. Using biochemical approaches, we measured the formation of higher-order oligomers of EspMin vitro. We demonstrate that multimerizationin vitrocan be mediated through multiple domains of the EspM protein. Using a bacterial monohybrid system, we showed that EspM self-associates through multiple domains in Escherichia coli. Using this system, we performed a genetic screen to identify EspM variants that failed to self-associate. The screen yielded four EspM variants of interest, which we tested for activity in M. marinum. Our study revealed that the two helix-turn-helix domains are functionally distinct. Moreover, the helix bundle domain is required for wild-type multimerizationin vitro. Our data support models where EspM monomers or hexamers contribute to the regulation ofwhiB6expression.IMPORTANCEPathogenic mycobacteria are bacteria that pose a large burden to human health globally. The ESX-1 secretion system is required for pathogenic mycobacteria to survive within and interact with the host. Proper function of the ESX-1 secretion system is achieved by tightly controlling the expression of secreted virulence factors, in part through transcriptional regulation. Here, we characterize the conserved transcription factor EspM, which regulates the expression of ESX-1 virulence factors. We define domains required for EspM to form multimers and bind DNA. These findings provide an initial characterization an ESX-1 transcription factor and provide insights into its mechanism of action.