Department of Microbiology, Immunology, and Cancer Biology, University of Virginiagrid.27755.32 School of Medicine, Charlottesville, Virginia, USA.
mSphere. 2022 Dec 21;7(6):e0048522. doi: 10.1128/msphere.00485-22. Epub 2022 Nov 8.
Shigella flexneri uses a type 3 secretion system (T3SS) apparatus to inject virulence effector proteins into the host cell cytosol. Upon host cell contact, MxiE, an S. flexneri AraC-like transcriptional regulator, is required for the expression of a subset of T3SS effector genes encoded on the large virulence plasmid. Here, we defined the MxiE regulon using RNA-seq. We identified virulence plasmid- and chromosome-encoded genes that are activated in response to type 3 secretion in a MxiE-dependent manner. Bioinformatic analysis revealed that similar to previously known MxiE-dependent genes, chromosome-encoded genes and contain a regulatory element known as the MxiE box, which is required for their MxiE-dependent expression. The significant AT enrichment of MxiE-dependent genes suggested the involvement of H-NS. Using a dominant negative H-NS system, we demonstrate that H-NS silences the expression of MxiE-dependent genes located on the virulence plasmid (.8 and ) and the chromosome ( and ). Furthermore, we show that MxiE is no longer required for the expression of , , and when H-NS silencing is relieved. Finally, we show that the H-NS anti-silencer VirB is not required for and expression upon MxiE/IpgC overexpression. Based on these genetic studies, we propose a model of MxiE-dependent gene regulation in which MxiE counteracts H-NS-mediated silencing. The expression of horizontally acquired genes, including virulence genes, is subject to complex regulation involving xenogeneic silencing proteins, and counter-silencing mechanisms. The pathogenic properties of Shigella flexneri mainly rely on the acquisition of the type 3 secretion system (T3SS) and cognate effector proteins, whose expression is repressed by the xenogeneic silencing protein H-NS. Based on previous studies, releasing H-NS-mediated silencing mainly relies on two mechanisms involving (i) a temperature shift leading to the release of H-NS at the promoter, and (ii) the virulence factor VirB, which dislodges H-NS upon binding to specific motifs upstream of virulence genes, including those encoding the T3SS. In this study, we provide genetic evidence supporting the notion that, in addition to VirB, the AraC family member MxiE also contributes to releasing H-NS-mediated silencing in S. flexneri.
福氏志贺菌利用 III 型分泌系统（T3SS）装置将毒力效应蛋白注入宿主细胞质中。在与宿主细胞接触后，福氏志贺菌 AraC 样转录调节因子 MxiE 需要表达大型毒力质粒上编码的一组 T3SS 效应基因。在这里，我们使用 RNA-seq 定义了 MxiE 调控组。我们鉴定了 T3 分泌依赖的 MxiE 依赖性方式激活的毒力质粒和染色体编码基因。生物信息学分析表明，与先前已知的 MxiE 依赖性基因类似，染色体编码基因和包含一个称为 MxiE 盒的调节元件，该元件是其 MxiE 依赖性表达所必需的。MxiE 依赖性基因的显著 AT 富集表明 H-NS 的参与。使用显性负 H-NS 系统，我们证明 H-NS 沉默位于毒力质粒（和）和染色体（和）上的 MxiE 依赖性基因的表达。此外，我们表明，当 H-NS 沉默被解除时，MxiE 不再需要表达、和。最后，我们表明，当 MxiE/IpgC 过表达时，VirB 作为 H-NS 反沉默因子不是和表达所必需的。基于这些遗传研究，我们提出了 MxiE 依赖性基因调控的模型，其中 MxiE 对抗 H-NS 介导的沉默。水平获得基因的表达，包括毒力基因，受到涉及异种沉默蛋白和反沉默机制的复杂调控。福氏志贺菌的致病特性主要依赖于 III 型分泌系统（T3SS）和同源效应蛋白的获得，其表达受到异种沉默蛋白 H-NS 的抑制。基于先前的研究，释放 H-NS 介导的沉默主要依赖于两种机制，包括（i）温度变化导致在启动子处释放 H-NS，以及（ii）毒力因子 VirB，其在结合到毒力基因上游的特定基序时会使 H-NS 移位，包括那些编码 T3SS 的基因。在这项研究中，我们提供了遗传证据支持这样一种观点，即除了 VirB 之外，AraC 家族成员 MxiE 也有助于释放福氏志贺菌中的 H-NS 介导的沉默。
