阪崎克罗诺杆菌脂多糖结构对菌膜形成的作用及机制

Foodborne pathogens can aggregate on the surface of food processing equipment to form biofilm, and therefore can strengthen the resistance to sterilization, which extremely threat the control of food safety. Concerning the factors influencing biofilm formation, there are many studies focusing on several kinds of organelles and macromolecules harboring in cell wall, while recently, it was revealed that lipopolysaccharide (LPS) maybe have a more profound effect. As the most abundant macromolecule on cell wall in Gram-negative bacteria, LPS mediated interaction between bacterial cell and many environmental factors, and influenced other components of cell wall and characters of bacterial cell, thus LPS most likely affected biofilm formation in a variety of ways. In order to reveal the role of LPS on biofilm formation and its multiple mechanisms of actions, the foodborne pathogen of Cronobacter sakazakii was employed, firstly, 5 critical genes involving in LPS biosynthesis and modification were identified, and then 8 mutant strain with modified LPS structures, as well as the strain complemented with the gene knocked out, were constructed; secondly, electron microscope was used to monitor biofilm development, in order to compare the differences among the mutant strains, and then the effect of LPS structure on biofilm formation was investigated, finally, the multiple mechanisms of LPS structure affecting biofilm formation were discovered, through investigating influences of change of LPS structure on interaction between LPS and environmental agent, as well as on the components of cell wall and characters of cell that effecting biofilm formation. The work will lay the foundation for improving theory of biofilm development and strategy of biofilm control.

食源性致病菌可在食品加工设备表面聚集形成菌膜，从而增强对杀菌操作的抵抗能力，为食品安全控制带来极大威胁。关于菌膜形成的影响因素,报道较多的为细胞外壁几种细胞器和大分子，而近年发现脂多糖（lipopolysaccharide, LPS）影响可能更为深刻。作为革兰氏阴性菌外壁分布最多的大分子，LPS介导了细菌与多种环境因子的互作，同时影响着细胞外壁中其他组分和细胞性质，因此LPS极可能以多种方式调控菌膜形成。为揭示LPS结构在菌膜形成中的角色及多重机制，拟针对阪崎克罗诺杆菌，首先鉴定LPS合成的5个关键基因，构建8个LPS结构突变株及其基因回补菌株；然后利用电镜等对其菌膜形成进行过程监测和差异比较，揭示LPS结构扮演的调控作用；最后分析LPS结构改变对其与环境因子互作强度、对影响菌膜形成的细胞外壁组分和细胞特性的影响，阐明LPS结构影响菌膜形成的多重机制，为完善菌膜发育理论和控制策略奠定基础。

细菌细胞外壁脂多糖（lipopolysaccharide, LPS）介导了致病菌在宿主细胞表面的黏附，对菌膜形成极可能发挥着关键作用。为揭示结构多变的LPS对菌膜形成的影响及多重机制，针对食源性致病菌阪崎克罗诺杆菌，构建多株LPS突变株并研究显示:在LPS的众多合成基因中，庚糖基转移酶编码基因waaC尤为重要，其缺失将导致LPS核心糖缺失，菌膜形成量增加，归因于细胞特性发生一系列变化：生长速率下降，浮游状态持续的时间变短，胞外多糖分泌量增加等。这些细胞特性变化有助于菌膜形成期的提前，且对细胞粘附有利，从而菌膜形成量增加。而waaC缺失引起的LPS多糖链变短是如何导致这些细胞特性的变化，目前尚不明确。因此我们开展以下工作。.1）.开展差异蛋白组学、代谢轮廓分析、ATP及能荷测定显示：差异蛋白主要涉及糖原合成、ATP及能量代谢、TCA循环、蛋白翻译及折叠。基于这些蛋白功能，推测LPS核心糖缺失引起菌膜形成量增加及细胞特性变化（生长速率下降，浮游状态持续的时间变短，胞外多糖分泌量增加）的可能机制为：LPS核心糖缺失导致大量碳源流向TCA（关键酶Malate dehydrogenase上调），一方面，细菌基于某种调控机制，降低糖原的合成（应对碳源短缺的一种储备机制，Glycogen synthase下调），合成并释放大量的胞外多糖（可促进菌膜的形成），.另外一方面，可产生大量的ATP（ATP synthase epsilon chain上调），以满足胞外多糖的合成，以及相关基因的复制和表达（50S ribosomal protein L4和GTP-binding protein typA/BipA上调），以及多糖合成酶运输和折叠（60 kDa chaperonin上调）。.2）将绿色荧光蛋白编码基因GFP转化C. sakazakii，可利用激光共聚焦显微镜观测菌膜中的微生物细胞分布,支持了waaC缺失株菌膜形成增强的结论。.3）在导致菌膜形成增强的因素中，类胡萝卜素的角色不容忽视，其对细胞对抗菌膜内过氧化胁迫压力有利。C．sakazakii的类胡萝卜素合成基因簇为crtE-idi-XYIBZ，本研究鉴定了C．sakazakii 类胡萝卜素合成途径中关键基因idi，其对于细胞增强氧胁迫响应和菌膜形成能力正在进一步研究。