Integration Host Factor Modulates the Expression and Function of T6SS2 in Vibrio fluvialis.
Integration Host Factor Modulates the Expression and Function of T6SS2 in Vibrio fluvialis.
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整合宿主因子调节T6SS2在河弧菌中的表达和功能
DOI：
10.3389/fmicb.2018.00962
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发表时间：
2018
影响因子：
5.2
通讯作者：
Liang W
中科院分区：
文献类型：
--
作者：
Pan J;Zhao M;Huang Y;Li J;Liu X;Ren Z;Kan B;Liang W
河流弧菌是一种新出现的引起公众健康关注的食源性致病菌，它含有编码VI型分泌系统(T6SS)的两个不同的基因簇，T6SS是革兰氏阴性菌中最新发现的分泌途径。以前我们已经证明了两个T6SS簇中的一个，即VflT6SS2，是活性的，并与抗菌活性有关。然而，它的活动是如何受到监管的还没有完全弄清楚。在这里，我们报道了全球调节因子整合宿主因子(IHF)通过共同调节VflT6SS2的主簇和tssD2-tssI2(也称为hcp-vgrG)孤儿簇，正向调控VflT6SS2的表达，从而调节VflT6SS2的功能。具体来说，报告基因活性分析表明，IHF反式激活了VflT6SS2的主簇和孤儿簇，而编码IHF亚基的基因ihfA或ihfB的缺失降低了它们的启动子活性和所选主簇基因的tssB2、Vash和tssM2以及所选孤儿簇基因的tssD2和tssI2的mRNA水平。随后，通过凝胶迁移率改变分析(EMSA)证实了IHF与主要和孤立簇的启动子区域的直接结合。定点突变结合报告基因活性分析或EMSA定位了IHF在主要和孤立簇启动子中的准确结合部位，在主簇启动子上有两个位点，与EMSA中观察到的两个移位条带一致。功能研究表明，在ΔihfA和ΔihfB缺失突变体中，溶血素协同调节蛋白(Hcp)的表达和分泌以及VflT6SS2介导的抗菌毒力被严重抑制，但在它们的反式互补质粒引入后又恢复了表达和分泌，这表明ihf主要通过直接结合和调节VflT6SS2的反式活性和功能来促进河流弧菌的环境生存。
Vibrio fluvialis, an emerging foodborne pathogen of increasing public health concern, contains two distinct gene clusters encoding type VI secretion system (T6SS), the most newly discovered secretion pathway in Gram-negative bacteria. Previously we have shown that one of the two T6SS clusters, namely VflT6SS2, is active and associates with anti-bacterial activity. However, how its activity is regulated is not completely understood. Here, we report that the global regulator integration host factor (IHF) positively modulates the expression and thus the function of VflT6SS2 through co-regulating its major cluster and tssD2-tssI2 (also known as hcp-vgrG) orphan clusters. Specifically, reporter gene activity assay showed that IHF transactivates the major and orphan clusters of VflT6SS2, while deletion of either ihfA or ihfB, the genes encoding the IHF subunits, decreased their promoter activities and mRNA levels of tssB2, vasH, and tssM2 for the selected major cluster genes and tssD2 and tssI2 for the selected orphan cluster genes. Subsequently, the direct bindings of IHF to the promoter regions of the major and orphan clusters were confirmed by electrophoretic mobility shift assay (EMSA). Site-directed mutagenesis combined with reporter gene activity assay or EMSA pinpointed the exact binding sites of IHF in the major and orphan cluster promoters, with two sites in the major cluster promoter, consisting with its two observed shifted bands in EMSA. Functional studies showed that the expression and secretion of hemolysin-coregulated protein (Hcp) and the VflT6SS2-mediated antibacterial virulence were severely abrogated in the deletion mutants of ΔihfA and ΔihfB, but restored when their trans-complemented plasmids were introduced, suggesting that IHF mostly contributes to environmental survival of V. fluvialis by directly binding and modulating the transactivity and function of VflT6SS2.
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