AI-2/LuxS介导的QS系统调控禽肠炎沙门氏菌耐药性与致病力的研究

Quorum sensing (QS) is involve in regulate bacterial pathogenesis, which is expected to being the novel drug targets. The luxS gene of QS systems was detected in a number of multi-resistant S. enterica serovar Enteritidis isolates in our previous works. The multidrug resistance efflux pump AcrAB has been shown to involve in multidrug resistance of various bacterial, but it is unknown whether multidrug resistance mediated by AcrAB pump is regulated by QS systems. Therefore, based on AI-2/LuxS QS of S. Enteritidis, the study was to clarify the association between AI-2/LuxS and AcrAB pump through assessing the difference of antibiotic susceptibility and expression of acrA and acrB among S. Enteritidis WT, luxS deletion mutants, and complementary strains. Then, the difference of antibiotic susceptibility after inactivation of the acrA and acrB, and luxS overexpression was analyze to identify resistance mediated by AcrAB pump result from AI-2/LuxS system regulation. The transcription and protein levels of acrA and acrB after inactivation of the luxS and luxS overexpression in the WT strains was analyze to identify the regulation levels of AcrAB pump by AI-2/LuxS system. The difference of growth, LD50, motility, histopathology and cytokines was determined to explore the effect of AI-2/LuxS on pathogenesis of S. Enteritidis strains. The above studies would provide new idea for further study on antibiotic resistance regulated by QS, as well as the choice of novel drugs targets against resistant pathogens.

群体感应(QS)因调控细菌群体的致病能力有望成为新型药物靶标。我们在已分离的多重耐药禽肠炎沙门氏菌中发现QS系统基因luxS。外排泵AcrAB与许多细菌多重耐药有关，其介导的多重耐药是否受AI-2/LuxS系统调控，目前尚不清楚。为此，本项目通过比较luxS基因缺失前后药物敏感性及外排泵基因acrA/acrB的表达水平差异，分析luxS与外排泵的关系；通过比较acrA/acrB缺失及过量表达luxS时的药物敏感性差异，明确luxS通过外排泵调控耐药；通过比较luxS缺失前后及野生株过量表达luxS时acrA/acrB的转录和蛋白表达水平，阐明luxS对AcrAB泵的调控。通过比较luxS缺失前后菌株生长速率、LD50、运动性、组织病理学及细胞因子差异，浅析luxS对禽肠炎沙门氏菌致病性的影响。对AI-2/LuxS系统调控细菌耐药和致病力的研究为新型药物靶标选择和耐药病原菌防控提供新思路。

肠炎沙门氏菌是食品源沙门氏菌病最常见的病原菌。抗生素大量应用导致沙门氏菌的耐药性逐年增强。因此，如何有效预防肠炎沙门菌感染已经成为食品公共安全关注的焦点。LuxS/AI-2型群体感应系统因调控细菌群体的致病力有望为肠炎沙门菌的防控提供新思路。本项目对禽沙门氏菌的LuxS/AI-2开展了研究，完成了申请书的主要研究内容，获得如下重要结果：1）LuxS/AI-2系统影响肠炎沙门氏菌的生物学特性：构建了禽肠炎沙门氏菌luxS缺失株，对缺失株与野生株的生物学特性研究显示：luxS缺失株能够稳定遗传缺失的△luxS基因；体外生长速率略快；生物被膜形成能力明显增强；对DF-1细胞和Caco-2细胞粘附侵袭能力均显著增加，这些结果表明luxS对肠炎沙门氏菌的生物学特性具有重要作用。2）LuxS/AI-2系统提高肠炎沙门氏菌的药物敏感性：与野生株相比，除氨基糖苷类无变化外，luxS缺失株对喹诺酮类的敏感性提高128-256倍，对−内酰胺类提高8倍，四环素类提高 2-4倍，氯霉素类提高4-8倍，表明luxS基因缺失能提高肠炎沙门氏菌对部分药物的药物敏感性，可显著提高其对喹诺酮类药物的敏感性。3）LuxS/AI-2系统通过AcrAB外排泵调控肠炎沙门氏菌对喹诺酮类药物敏感性：AcrAB-TolC外排泵过度表达是介导细菌对喹诺酮类耐药的主要机制，与野生株相比，luxS突变后AcrAB外排泵基因acrA和acrB基因的转录水平显著降低。GST-Pull-down结果显示LuxS与AcrA蛋白存在特异的相互作用。表面等离子共振进一步证实了LuxS与AcrA蛋白存在强烈的相互作用。本研究明确了LuxS/AI-2系统对肠炎沙门氏菌生物学特性及药物敏感性的影响，发现LuxS蛋白与AcrAB外排泵的AcrA蛋白存在体外的相互作用，暗示LuxS/AI-2系统可能通过AcrAB外排泵调控肠炎沙门氏菌对喹诺酮类药物的敏感性。