I型群体感应系统调控大肠杆菌耐酸性的重要信号通路分子挖掘研究

Pathogenic Escherichia coli (E. coli) can survive under extreme acidic conditions through its complex acid-resistant system with capability of overcoming the acidic stomach environment and play their pathogenic roles. In quorum sensing 1 system（QS-I）, E. coli encode a single LuxR homolog named SdiA, but does not have the LuxI homolog expressing the acyl-homoserine lactone (AHL) synthase, which produces AI-1. In previous study, we applied the improved screen method to isolate AHLs positive bacteria such as Pseudomonas aeruginosa strain in cattle rumen and Aeromonas hydrophilia strain in pig intestine, which would be helpful in further study of interaction between E. coli QS system and exogenous AHLs. Transformation of the target E. coli O157:H7 with the P. aeruginosa genes lasI and RhlI encoding proteins that produce short- and long-side chain acyl-homoserine lactones respectively, conferred upon the E. coli O157:H7 the ability to synthesize acyl-homoserine lactones and affected E. coli gadA gene expression of 2 folds up-regulation consistent with the phenotype of acid tolerance increased. And likewise transformation of E. coli F18ab(107/86 )with the target A. hydrophilia genes AhyI, encoding proteins that produce short- side chain AHLs conferred upon the E. coli the ability to synthesize AHLs and did affect E. coli gadA gene expression of 4 folds up-regulation consistent with the phenotype of acid resistance increased. But unlike both E. coli O157:H7 and E. coli F18ab, extra-intestinal avian pathogenic E. coli) (APEC) strains did not work..The applicant and his research team members had tested QS-1 affect AR2 acid resistance system, influence BarA/UvrY regulator in AR1 acid resistance system, and have screened E. coli strains with different acid tolerance phenotypes under QS regulation. Based on the previous experimental data involved in QS-I regulation function on acid tolerance of E.coli, will further focus on screening AHL molecular species that directly regulate the acid resistance of E. coli, establishing AR1/AR2 mutants platform to explore regulatory mechanism, analyzing the molecular genetic mechanisms of phenotypic differences in acid resistance both intra-intestinal and extra-intestinal E. coli strains under QS-1 signals through omics techniques. This would help well understand the regulatory networks and mechanisms of QS--acid resistance--bacterial virulence and survivability.

病原大肠杆菌E.coli通过复杂的耐酸系统在极端酸性条件下仍可存活并能克服胃部酸性环境，发挥其病原致病作用，而E.coli缺乏合成I型群体感应系统QS-1自体诱导物分子AI-1的luxI同源基因，本身无法产生AI-1。鉴于已从不同动物源胃肠道成功分离出能合成AI-1（高丝氨酸内酯，AHL）的阳性细菌，且该不同类阳性菌中表达AHL分子的luxI同源基因能赋予产肠毒素大肠杆菌F18ab和出血性大肠杆菌O157：H7耐酸性能力，并发现耐酸性表型不受QS-1影响的肠外禽致病性E.coli APEC菌株。申请人拟在确定QS-1影响AR2耐酸系统、影响AR1耐酸系统BarA/UvrY调控子基础上，筛选直接调控大肠杆菌耐酸性的AHL分子种类，建立AR1/AR2缺失株平台探讨调控机制，利用组学技术分析针对QS不同耐酸性应答的表型差异E.coli菌株，探析群体感应—耐酸性—细菌毒力及存活能力调控分子与机制。

病原大肠杆菌E.coli适应复杂的耐酸（AR）系统在极端酸性条件下仍可存活并能克服胃部酸性环境，发挥其病原致病作用。虽然E.coli缺乏合成I型群体感应系统QS-1自体诱导物分子AI-1的luxI同源基因，本身无法产生AI-1（高丝氨酸内酯，AHL），鉴于我们已从不同动物源胃肠道成功分离出能合成AI-1的阳性细菌，且该不同类阳性菌中表达AHL分子的luxI同源基因能赋予肠道病原如产肠毒素大肠杆菌F18ab和出血性大肠杆菌O157：H7耐酸性能力，并发现肠外禽致病性E.coli（APEC）菌株没有耐酸性表型。因此，筛选直接调控大肠杆菌耐酸性的AHL分子种类，建立AR1/AR2缺失株平台探讨调控机制，探析群体感应—耐酸性—细菌毒力及存活能力调控分子与机制至关重要。通过外源添加或内源合成AHL 分子的方式，本项目成功筛选到与耐酸性相关的关键基因rpoS和gadA作为AHL信号通路的下游信号分子，在酸性条件下外源添加AHL分别上调rpoS和gadA转录量3.83倍和3.37倍，且O157：H7 rpoS缺失株的存活率显著下降至42%，提示rpoS对O157：H7在极端酸环境下的存活有重要意义。转录组技术验证了上述结果，首次发现核糖体调控因子rmf密切参与到大肠杆菌耐酸性调控。深入解析了 rmf、rpoS、sdiA之间具有相互调控作用，通过构建O157：H7 rmf和rpoS基因缺失株，发现在酸性条件下，O157：H7 rmf和rpoS基因缺失株中sdiA基因转录分别降至24%和60%，这表明rmf和rpoS在耐酸性方面对sdiA基因的转录有调控作用。综上所述，本项目探索和筛选到酸性条件下I型群体感应分子AHL调控病原大肠杆菌耐酸通路的关键信号分子，揭示了这些关键分子相互之间的调控关系，为进一步解析细菌耐酸机制提供了理论基础和研究模式。