Gao Xiating, Liu Yang, Liu Huan, Yang Zhen, Liu Qin, Zhang Yuanxing, Wang Qiyao
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
School of Food and Biological Engineering, Shanxi University of Science and Technology, Xi'an, China.
J Bacteriol. 2017 Sep 19;199(20). doi: 10.1128/JB.00252-17. Print 2017 Oct 15.
In species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in , a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of and the master quorum-sensing (QS) regulator DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the promoter region overlaps that of LuxR-binding site I, which mediates the positive control of promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in In this work, AphB is determined to play essential roles in the expression of genes associated with QS, physiology, and virulence in , a pathogen for marine animals and humans. AphB was found to bind directly to 20 genes and control their expression by a 17-bp consensus binding sequence. Among the 20 genes, the gene itself was identified to be positively autoregulated, and AphB also positively controlled and expression. Taken together, these findings improve our understanding of the roles of AphB in controlling physiological adaptation and QS-controlled virulence gene expression.
在某些物种中，AphB对于通过感知低pH值和厌氧信号来激活毒力级联反应至关重要；然而，其调控子在很大程度上仍不为人所知。在此，发现AphB是一种对海洋动物和人类致病的病原体中的关键毒力调节因子。染色质免疫沉淀结合高通量DNA测序（ChIP-seq）能够检测到该病原体基因组中20个含有AphB结合峰的位点。通过电泳迁移率变动分析（EMSA）证实了AphB特异性结合共有序列，并使用定量实时PCR分析证明了此类结合位点侧翼基因的调控情况。AphB直接与其自身启动子结合，并在生长后期阶段正向调控其自身表达。AphB还通过直接结合和的启动子区域来激活外毒素Asp的表达。DNA酶I足迹分析在这些启动子中发现了不同的AphB结合位点（BBS）。此外，启动子区域中的一个BBS与LuxR结合位点I重叠，该位点介导AphB对启动子活性的正向调控。这项研究为AphB调控子提供了新的见解，并揭示了AphB在该病原体中调节生理适应和群体感应（QS）控制的毒力的机制。在这项工作中，确定AphB在与QS、生理和毒力相关的基因表达中发挥着重要作用，该病原体可感染海洋动物和人类。发现AphB直接与20个基因结合，并通过一个17bp的共有结合序列控制它们的表达。在这20个基因中，基因自身被鉴定为正向自我调控，并且AphB也正向调控和的表达。综上所述，这些发现增进了我们对AphB在控制生理适应和QS控制的毒力基因表达中作用的理解。
