环境胁迫调控副溶血弧菌整合结合元件自主传播的分子机理

Vibrio parahaemolyticus contamination in aquatic products was the major contributors to food safety issues caused by pathogenic bacteria in China. However, the mechanism underlying outbreak and prevalence of foodborne disease caused by V. parahaemolyticus has been largely unknown. In this project, the interdisciplinary methodology will be employed to investigate the impact of environmental stresses on the regulation of an integrative and conjugative element ICEVpCn1 with multiple drug resistance gene cassettes in V. parahaemolyticus MnSCn1, which was isolated from aquatic products in China. The draft genome map of V. parahaemolyticus MnSCn1 has been obtained. The genes involved in integration, conjugation and regulation of ICEVpCn1 will be investigated at a global level by transcriptome analyses under stress conditions with or without conjugation receptors. The differential expression regulation genes of ICEVpCn1 will be knocked out, and proteome of wild and mutant V. parahaemolyticus grown under stress conditions will be analyzed to uncover signal transmission pathways of environmental stress. Based on the results of this project, a three dimensional (host-ICEVpCn1-receptor) gene regulation web mediated by environmental stresses will be constructed, which will lead to new insights into molecular mechanisms underlying reemerging infectious diseases and suggest the future research directions. The outputs of this project will also provide technical support to early warning systems of foodborne infectious diseases in China.

副溶血弧菌(Vibrio parahaemolyticus)污染引发的水产品安全事件在我国微生物性食品安全事件中位居首位。然而,副溶血弧菌介导食源性疾病爆发流行的规律至今尚未阐明。本课题研究探索环境胁迫调控副溶血弧菌整合结合元件自主传播的分子机理,以副溶血弧菌MnSCn1及其携带的具有多药抗性的整合接合元件ICEVpCn1为研究对象,运用转录组学技术,分析环境胁迫在全局性水平对ICEVpCn1整合、切离、结合等基因转录的影响;研究不同环境条件下结合受体对ICEVpCn1自主转移活性的影响;利用基因敲除技术与蛋白质组学方法,获取环境因子调控ICEVpCn1的信号传导通路信息;初步构建环境胁迫调控ICEVpCn1自主传播的多维(宿主-整合结合元件-受体)基因调控网络。本课题的开展将为副溶血弧菌污染造成的食源性疾病的爆发流行及其预警的研究提供理论基础和技术支持,进而提升我国在该领域的水平。

副溶血弧菌(Vibrio parahaemolyticus)污染引发的水产品安全事件在我国微生物性食品安全事件中位居首位。该菌隶属于γ-变形菌纲、弧菌科、弧菌属，广泛分布于近岸海水、河口、水产养殖水体等环境中,并常见于鱼、虾、贝类等水产品中,是引发我国沿海地区食物中毒和腹泻疾病的首要致病菌。本项目以水产动物源副溶血弧菌CHN25及其携带的整合接合元件ICEVpaChn1为研究对象, 运用食品微生物学、生物信息学、转录组学和蛋白组学等研究方法与技术,在前期研究工作基础上，已完成副溶血弧菌CHN25全基因组序列的缺口封闭和注释，获得了该菌株全长基因组序列5,443,401 bp，GC含量45.2%，预测蛋白质编码基因4,724个；获得了ICEVpaChn1元件的全长DNA序列88,453 bp，预测蛋白质编码基因77个。序列分析发现，ICEVpaChn1元件位于副溶血弧菌CHN25基因组的1号环状染色体上，含有SXT/R391家族整合接合元件保守的维持、散播和调控模块的核心基因，其可变区基因序列具有杂合的进化起源。基于上述研究结果，定制了基因组DNA微列阵芯片，从全局调控视角分析了环境胁迫条件，包括酸性pH、温度变化、抗生素(链霉素、氨苄西林、磺胺甲恶唑)暴露，以及重金属(氯化镍、氯化镉、氯化镉和氯化铜)暴露处理，介导副溶血弧菌CHN25及其ICEVpaChn1元件基因差异表达的转录谱。比较转录组分析结果显示，环境胁迫条件分别诱导了副溶血弧菌CHN25对数生长期细胞中多条代谢途径发生了显著变化。以此为基础，首次构建获得了差异表达的调节子VpaCspA、VpaCspD编码基因的缺失突变株ΔVpacspA、ΔVpacspD、ΔVpacspAD，并获得了野生型和三株突变型菌株响应低温胁迫条件的转录组数据；证明了VpaCspA是副溶血弧菌主要的冷激蛋白，并且负调控低温生长的抑制子VpaCspD。此外，首次构建获得了副溶血弧菌CHN25整合接合元件缺失突变株△ICEVpaChn1，并获得了野生型和△ICEVpaChn1突变型菌株的蛋白质组数据。综上所述，本项目研究结果揭示了副溶血弧菌CHN25及其ICEVpaChn1元件响应环境胁迫的复杂多维的分子调控网络，发现了大量不依赖于细胞生长期或环境胁迫条件的调节子，为副溶血弧菌污染造成的食源性疾病的研究提供了必要的理论基础和技术支撑。