基于Tn-Seq技术的阪崎克罗诺杆菌耐胆盐分子机制研究

Cronobacter sakazakii is an important opportunistic foodborne pathogen. It could cause life-threatening disease in neonates or infants such as necrotic enterocolitis, meningitis after being ingested through contaminated powdered infant formula. To successfully establish the infection, the pathogen need to cope with the antibacterial action of bile salt, although currently genetic determinants involved in C. sakazakii bile salt tolerance remain largely unknown. In this proposal, C. sakazakii strain highly resistant to bile salt will be selected from strains previously isolated from food, and a saturated transposon insertion mutant library will be constructed by using a mariner transposon. The library will be grown in a normal growth environment or a bile salt-containing environment, then transposon insertion frequency in each gene in two libraries grown in two different environments will be compared and analyzed by using transposon insertion sequencing (Tn-Seq) method, through which potential bile resistance genes will be determined. Genes that have novel bile resistant mechanism will be selected and the genes’ contribution to bile resistance will be confirmed by gene deletion and gene complementation experiments. Meanwhile, the effect of these bile salt resistance genes on tolerance to other stresses will be investigated. In addition, their influences on the ability of the pathogen to adhere and invade intestinal epithelial cells and on in vivo pathogenicity will also be tested. The findings from this project will decipher the molecular mechanism of bile salt resistance in C. sakazakii, broaden current understanding of its stress tolerance, and provide theoretic basis for future development of more effective control strategy to prevent or reduce C. sakazakii associated diseases.

阪崎克罗诺杆菌是一种重要的食源性条件致病菌，婴幼儿食用污染该菌的奶粉后可出现坏死性肠炎、脑膜炎等严重疾病。该菌致病的重要前提之一是其必须能抵抗肠道中胆盐的杀菌作用，然而目前对该菌耐胆盐的分子机制尚未明确。本项目拟从食源性菌株中筛选高胆盐耐受的菌株，再利用Mariner型转座子在筛出的菌株基础上建立突变株文库。将文库在有无胆盐的环境下分别培养，通过转座子插入突变结合高通量测序（Tn-Seq）技术，对各个基因中转座子插入频率在两种环境下培养的文库中的差异进行分析，从而确定与耐胆盐相关的基因。选取具有新型耐胆盐机制的基因通过基因敲除和基因互补实验验证其功能；同时分析耐胆盐基因对该菌耐受其他环境压力的影响；最后分析耐胆盐基因对该菌粘附和侵入肠道上皮细胞能力以及对小鼠致病能力的影响。本研究将阐明阪崎克罗诺杆菌的胆盐耐受的分子机制，从而为深入了解该菌的逆境存活机制以及制定更有效的控制措施奠定理论基础。

阪崎克罗诺杆菌是一种重要的食源性条件致病菌，该菌致病的重要前提之一是其必须能抵抗肠道中胆盐的杀菌作用。本项目旨在确定与耐胆盐相关的基因并确定其在细菌环境耐受中的功能。项目通过Tn-Seq技术确定与阪崎克罗诺杆菌耐胆盐相关的基因，并通过基因敲除和基因互补实验验证其功能；同时分析耐胆盐基因对该菌耐受其他环境压力的影响；最后分析耐胆盐基因对该菌粘附和侵入肠道上皮细胞能力以及对小鼠致病能力的影响。主要结果如下：（1）phoPQ基因与胆盐耐受能力成正相关，hfq基因与胆盐耐受能力成负相关；（2）phoPQ基因缺失使阪崎克罗诺杆菌对多粘菌素B、热、氧化和渗透压的耐受能力降低；（3）PhoPQ系统可能通过调控细菌生物膜及毒力相关基因的表达来调控其生物膜形成以及对肠上皮细胞粘附和侵入能力；（4）phoPQ基因缺失使阪崎克罗诺杆菌对新生小鼠的肠道损伤作用及其在回肠中的定殖能力减弱；（5）phoPQ基因缺失使阪崎克罗诺杆菌在细菌分泌系统、鞭毛组装、细菌趋化性及脂多糖生物合成方面的基因转录水平发生显著影响。本项目阐明了阪崎克罗诺杆菌的胆盐耐受的部分分子机制，为深入了解该菌的逆境存活机制以及制定更有效的控制措施奠定理论基础。