戊糖乳杆菌HC-2与副溶血弧菌竞争对虾肠道粘附位点的相关基因挖掘及其作用机理研究

Shrimp culture is currently suffering from serious losses due to the infection diseases caused by acute hepatopancreatic necrosis disease (AHPND). It has been confirmed that the bacterium Vibrio parahaemolyticus is the etiological agent of AHPND. The use of antimicrobial drugs, pesticides and disinfectant in aquaculture disease prevention and growth promotion has led to the evolution of resistant strains of bacteria. The use of lactic acid bacteria for aquaculture is increasing with the demand for environment – friendly sustainable aquaculture. The probiotic mechanism is also concerned, but it is mainly focused on the immunity genes of shrimp, less on the competitive exclusion of pathogenic bacteria. Lactobacillus pentosus HC-2 is a probiotic strain with significant competitive exclusion effect on Vibrio in the intestine of Litopenaeus vannamei, and preliminary experiments showed that its surface molecules may play an important role in this effect. In view of this, we will first sequence the whole genome of HC-2, and then explore the genes and molecular markers associated with adhesion by comparative genomics; the candidate genes involved in competitive exclusion will be screened by genomic homologous analysis of HC-2 and Vibrio parahaemolyticus; the mutant strains will be constructed to verify whether the candidate genes are involved in their adherence to the intestine of shrimp; the effects of co-culture of HC-2 and Vibrio parahaemolyticus on the adhesion ability and expression of candidate genes will be discussed. The above results will uncover the competition mechanism between HC-2 and Vibrio parahaemolyticus, and provide the scientific basis for the screening and application of probiotic lactic acid bacteria in shrimp farming.

副溶血弧菌引起的急性肝胰腺坏死疾病是世界范围内严重影响对虾产量的细菌性疾病，乳酸菌被广泛地用于该疾病的防控，对其益生机理研究诸多，但在对病原菌的竞争排斥机理方面研究较少。戊糖乳杆菌HC-2（HC-2）是一株在对虾肠道对弧菌具有明显竞争排斥作用的益生菌，且前期实验结果表明其菌体表面分子或许在该作用中扮演着重要角色。鉴于此，本课题首先借助HC-2全基因组测序及比较基因组学的手段发掘与粘附相关的基因及分子标记；基于HC-2与副溶血弧菌基因组同源性分析，整合筛选参与竞争排斥作用的候选基因；随后构建突变菌株验证候选基因是否参与其在对虾肠道的粘附；最后通过HC-2与副溶血弧菌野生及突变菌株在对虾肠道粘液中交叉共培养探讨微生物互作对彼此候选基因表达和粘附能力的影响，发掘数据内部联系，进一步阐释HC-2与副溶血弧菌竞争对虾肠道粘附位点的机理，为对虾养殖业益生乳酸菌的筛选和疾病防御等方面的应用提供科学依据。

副溶血弧菌引起的急性肝胰腺坏死疾病是世界范围内严重影响对虾产量的细菌性疾病，乳酸菌被广泛地应用于该疾病的控制，对其益生机理研究诸多，但在对病原菌的竞争排斥机理方面研究较少。戊糖乳杆菌HC-2（HC-2）是一株在对虾肠道对弧菌具有明显竞争排斥作用的益生菌，经本项目研究发现该菌的表面蛋白在凡纳滨对虾抵抗病原菌、提高自身免疫力等益生方面发挥着重要的作用。鉴于此，本课题进一步通过高通量测序和比较基因组学手段挖掘到HC-2特有潜在粘附基因21个，与其他戊糖乳杆菌菌株共有潜在粘附基因15个；通过多基因组线性比对筛选得到HC-2与副溶血弧菌具有同源性的粘附相关候选基因11个; 利用经典同源重组方法构建粘附相关候选基因的突变体，发现ftsH基因在HC-2粘附对虾粘液方面发挥着重要作用，该基因的缺失可以增强HC-2对粘液的粘附特性；随后将突变型菌株与野生型菌株分别进行蛋白质组测序，利用比较蛋白质组分析挖掘到HC-2中与粘附功能相关的5个膜蛋白，可作为筛选高粘附性益生乳酸菌的生物标记物；经HC-2与副溶血弧菌VPE1的共培养实验进一步发现共培养时病原菌的存在可以诱导HC-2粘附相关膜蛋白（G0M1M7、I9L3P8、I9AKC3、A0A2S9W1Y1）基因的高表达，表明这些蛋白在HC-2与病原菌VPE1竞争对虾肠道位点方面发挥着重要的作用。此外，挖掘到HC-2基因组中存在多种碳水化合物降解相关的基因及代谢通路，为该菌能够存活并定殖于对虾及其他动物宿主肠道提供了有力的理论依据。总之，以上实验结果为益生乳酸菌的筛选和作为饲料添加剂在对虾养殖业疾病防御等方面的应用提供了重要的科学依据。