Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
Core Unit Systems Medicine, University of Würzburg, Würzburg, Germany.
Cell Rep. 2021 Feb 2;34(5):108722. doi: 10.1016/j.celrep.2021.108722.
A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.
充分了解小 RNA（sRNA）对细菌毒力的贡献需要了解其在感染相关条件下的靶标。在这里，我们采用一种综合的方法来捕获 Hfq 相关 sRNA PinT 的靶标，PinT 是沙门氏菌两种主要毒力程序的已知转录后定时器。使用 MS2 亲和纯化和 RNA 测序（MAPS），我们在模拟感染周期特定阶段的体外条件下和在巨噬细胞内生长的细菌中鉴定出 PinT 配体。这揭示了 PinT 介导的分泌效应激酶 SteC 的翻译抑制，这在以前的靶标搜索中未被注意到。使用遗传、生化和显微镜检测，我们提供了 PinT 介导的 steC mRNA 抑制的证据，最终延迟了感染宿主细胞中的肌动蛋白重排。我们的研究结果支持 PinT 作为沙门氏菌毒力的中央转录后调节剂的作用，并说明了需要互补的方法来揭示 sRNA 的全部靶标。
