沙门氏菌生物菌膜中特征性非编码小RNA的鉴别及调控机制

Biofilm is the main contamination source of Salmonella spread in food-processing industry, Salmonella biofilm has been directly associated with many food-borne outbreaks and food recalls, it was very essential to reveal the mechanism of Salmonella biofilm formation under food processing environments. Non-coding small RNA (sRNAs) was found as a new regulatory element in bacteria. Previous studies have found that several sRNAs have involved in regulation of maturation of Salmonella biofilm, However, which characteristic sRNAs played key roles in biofilm formation, and the concrete regulation pathways were still unknown. In the present study, Salmonella Enteritidis was tested as the target bacteria, and the food-grade stainless steel surface was used for biofilm formation. As compared with a standard growth medium (TSB), a meat thawing-loss broth (MTLB) was used for Salmonella growth. The novel sRNAs (3-6) regulating biofilm formation of Salmonella in MTLB were genome-wide identified with RNA-seq and bioinformatics technologies. On the basis of confirming the sequences sites and conservation, the molecular regulation mechanisms of target sRNAs in biofilm formation were clarified by constructing the deleted mutant strains of novel sRNAs and analyzing the differences of adhesion phenotypes between mutants and wild-type Salmonella. Our study will perfect the scientific theory of biofilm formation, and also establish the theoretical foundation for developing effective prevention and control measures of Salmonella biofilm.

生物菌膜是食品加工中沙门氏菌污染的重要传播根源，其与食物中毒和食品召回有密切关联，探明食品加工环境中生物菌膜的形成机制非常必要；非编码小RNA（Non-coding small RNA，sRNAs）是一类新型的细菌调控因子，前期研究发现sRNAs参与在沙门氏菌生物菌膜的成熟调控中，但何种标志性sRNAs发挥着关键调控作用，以及具体的调控途径均不清楚；本研究拟以肠炎沙门氏菌为对象，以食品级不锈钢为接触面，以肉汁液（meat thawing-loss broth，MTLB）和标准培养液（TSB）为生长基质对比研究，利用RNA-seq技术结合生物信息学挖掘生物菌膜中的特征性sRNAs（3-6个），在探明其序列位点和保守性的基础上，通过构建特征性sRNAs的缺失突变株，结合粘附表型差异，揭示sRNAs对生物菌膜的调控机制；该成果可完善生物菌膜的科学理论，并为研发生物菌膜的防控技术奠定理论基础。

沙门氏菌已成为全球范围内危害公众健康的首要食源性致病菌，生物菌膜是食品加工中沙门氏菌的重要污染根源，可引发大规模食物中毒和食品召回。该项目以肠炎沙门氏菌为研究对象，以食品级不锈钢为接触面，以肉汁液（MTLB）和标准培养液（TSB）为生长基质，研究了非编码小RNA对沙门氏菌生物菌膜形成的调控作用。研究发现肠炎沙门氏菌在不锈钢表面可形成多于7 Log cfu/cm2的生物菌膜，MTLB诱导19个差异表达的sRNA，部分sRNA序列在沙门氏菌属内高度保守，部分sRNA序列依赖血清型；依据潜在功能，选择了6个sRNA进行深入研究。利用RT-qPCR验证出各sRNA真实存在于基因组中，并通过序列比对确定了各自的具体起始位点。利用自杀质粒法和Red同源重组法构建出6株肠炎沙门氏菌sRNA突变株；发现sRNA突变菌株在不同pH、渗透压、氧化、温度条件下的生长特性与野生菌株相比无显著差异，但营养条件可对突变株和野生株的生长产生显著影响。同时，突变株的聚集、泳动性能、表面疏水性和生物菌膜形成能力与野生株存在显著差异，且野生株的粘附能力显著依赖于培养时间和粘附表面类型，其中突变株Δs282在微孔表面展现出极强的生物菌膜形成能力；此外，发现，sRNA缺失对菌体的毒力和粘附产生了显著影响。综上，该研究发现部分非编码小RNA在沙门氏菌粘附过程中发挥负调控作用。该成果可完善生物菌膜的科学理论，并为研发生物菌膜的防控技术奠定理论基础。