IscR Is Essential for <i>Yersinia pseudotuberculosis</i> Type III Secretion and Virulence

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coliiron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.III型分泌系统（Type III secretion systems, T3SS）对数十种病原菌的致病力至关重要，但在宿主外生长时并非必需。因此，多数细菌的III型分泌系统受到严格的调控。为加深对III型分泌系统调控分子机制的理解，我们针对食源性病原菌假结核耶尔森菌（Yersinia pseudotuberculosis）开展了转座子筛选实验，以鉴定对其III型分泌系统功能发挥关键作用的基因。我们在YPTB2860基因中发现了两处独特的转座子插入位点，该基因与大肠杆菌（E. coli）来源的铁硫簇调控蛋白IscR（iron-sulfur cluster regulator）的同源性达79%。我们构建了假结核耶尔森菌iscR基因的框内缺失突变株（ΔiscR），该突变株无法正常分泌Ysc型III型分泌系统效应蛋白，且无法通过III型分泌系统靶向巨噬细胞。为解析IscR调控Ysc型III型分泌系统的分子机制，我们通过转录组学与生物信息学分析，鉴定了假结核耶尔森菌中受IscR调控的基因。我们在假结核耶尔森菌yscW-lcrF操纵子的上游区域发现了一个推定的IscR结合基序。鉴于LcrF可调控耶尔森菌中多个关键III型分泌系统基因的转录，我们推测耶尔森菌的IscR可能通过LcrF实现对Ysc型III型分泌系统的调控。实验结果证实，纯化的IscR可与我们鉴定出的yscW-lcrF启动子基序特异性结合；且在缺失IscR的假结核耶尔森菌中，lcrF以及另外24个III型分泌系统基因的mRNA水平均显著下调。值得注意的是，经口感染ΔiscR突变株的小鼠，其派尔集合淋巴结（Peyer's patches）、肠系膜淋巴结（mesenteric lymph nodes）、脾脏及肝脏中的细菌载量均显著降低，这表明IscR在假结核耶尔森菌的致病过程中发挥不可或缺的关键作用。本研究首次完成了耶尔森菌IscR的功能表征，并证实IscR通过直接调控III型分泌系统的主调控因子LcrF，对病原菌的致病力与III型分泌系统功能至关重要。