阪崎克罗诺杆菌鞭毛介导的运动性及生物膜形成的微环境响应及LuxS调控研究

Abstract：Cronobacter sakazakii is an important foodborne pathogen associated with neonatal meningitis by using the powdered infant formula as the main transmission vehicle, and its biofilm is the key factor causing peristent contimination of powdered milk. Our group revealed that growing environmental factors played important effects on biofilm formation. However,mechanism of regulation and environmental responses to biofilm formation mediated by flagella were largely unknown. Consequently, taking the motilities and biofilm formation mediated by flagella as the starting point, the project will create flagella-associated proteins mutants of C. sakazakii, the mechanism of actions of flagella mediating motilities and biofilm formation were studied. Then, combined with swimming, swarming, and twitching, and detection of biofilm formation by scanning electron microscopy and confocal laser scanning microscopy, the micro-environmental responses on motilities and biofilm formation of C. sakazakii was further explored. Finally, the LuxS mutant was created and regulationary roles of LuxS on motilities and biofim formation was studied. The expected findings will reveal the action mechanism of flagella on motilities of C. sakazakii, and further clarify the micro-environmental responses mechanism and LuxS regulation of motilities and biofilm formation of C. sakazakii. The results will enrich the regulatory theory of motilities and biofilm formation of pathogens, and also provide novel ideas and ways for control and precaution of C. sakazakii contamination in production of powdered milk.

阪崎克罗诺杆菌（Cronobacter sakazakii）是以婴幼儿奶粉为主要传播媒介的重要食源性致病菌，其在设备及环境表面形成生物膜是奶粉被持续污染的关键因素。本课题组前期研究证实该菌生物膜形成受生长的微环境影响，但生物膜形成的微环境响应与调控机制尚不清楚。本项目以鞭毛介导细菌运动与生物膜形成为切入点，拟通过：1）构建鞭毛关键蛋白基因突变株，研究鞭毛蛋白介导细菌运动及生物膜形成的作用机制；2）基于微环境下细菌运动、生物膜形成及鞭毛蛋白基因表达分析，探讨鞭毛介导运动及生物膜形成的微环境响应机制；3）构建LuxS突变株，开展野生株、突变株及回复株运动及生物膜形成分析，探讨LuxS调控细菌运动及生物膜形成的分子机制。预期成果将揭示鞭毛在介导运动及生物膜形成中的作用机制，阐明阪崎克罗诺杆菌运动及生物膜形成的微环境响应及LuxS调控机理，为防控奶粉中阪崎克罗诺杆菌生物污染提供新思路和新途径。

阪崎肠杆菌（Enterobacter sakazakii），又名克罗诺杆菌（Cronobacter），包含有7个种。细菌生物膜是指细菌附着于惰性或者活性实体的表面，繁殖、分化并分泌一些多糖基质，将菌体群落包裹其中而形成的细菌聚集体膜状物，是食品在生产加工与储藏过程中被持续污染及抗逆境胁迫的关键因素。克罗诺杆菌形成生物膜能力较强，本项目在已有分离菌株的基础上，开展生长微环境（二价离子浓度、pH、培养温度、培养时间、NaCl浓度等）对生物膜形成的影响研究；基于克罗诺杆菌浮游态和生物膜态细菌的转录组学分析，发现LuxS等一系列基因在生物膜状态下较浮游态高表达；通过敲除克罗诺杆菌群体感应调控基因，系统开展LuxS等相关基因对克罗诺杆菌生物膜形成的影响，并在此基础上，开展LuxS对氧化应激、渗透压胁迫及酸胁迫下的作用及生物膜形成的影响，为阐明克罗诺杆菌生物膜形成及调控奠定了良好基础，对预防和控制该菌污染导致的微生物安全具有重要的理论意义。探讨了外膜蛋白W在克罗诺杆菌逆境胁迫中的作用及对生物膜形成的影响。同时，克罗诺杆菌形成生物膜能力很强，增加了食品加工过程中被持续污染的风险，本项目进一步开展克罗诺杆菌及其生物膜形成的抑制或消除作用研究，重点开展壳聚糖、过氧化氢等对克罗诺杆菌的作用进行探讨，为该菌污染危害形成的预防与控制提供重要的基础数据。