干酪乳杆菌对斑马鱼抵御气单胞菌感染的分子机制研究

发布时间：2018-11-16 08:02

【摘要】：疾病暴发,如气单胞菌引起的鱼类出血病,已经成为目前限制鱼类养殖业健康发展的主要问题。益生菌在促进动物生长、维持机体肠道健康、增强免疫防御功能及保护宿主免受病原菌感染等方面起重要作用。本文通过转座子基因突变文库的构建、目的基因筛选、基因敲除、转录组、无菌斑马鱼模型以及菌群转接模型等方法分别对气单胞菌的感染机制、益生菌促生长及其抗气单胞菌感染机制进行了研究。主要研究内容和结果如下:一、气单胞菌感染机制的研究2009-2014期间从中国华南地区出血病爆发塘口的病鱼中分离得到的病原菌一共有五种:维氏气单胞菌(A.veronii)、嗜水气单胞菌(A.hydrophila)、简氏气单胞菌(A.jandei)、舒氏气单胞菌(A.schubertii)及水生气单胞菌(A.aquariorum),其中维氏气单胞菌和嗜水气单胞菌是二种最重要的病原菌。另外,维氏气单胞菌和嗜水气单胞菌的混合感染是引起华南地区鱼类败血症爆发的主要原因,维氏气单胞菌是造成感染的前提条件。采用浸浴模型和腹腔注射模型对气单胞菌毒力进行评价,结果发现,在浸浴模型中维氏气单胞菌表现出最强的毒力,而嗜水气单胞菌的毒力相对较弱;而在腹腔注射模型中,嗜水气单胞菌的毒力远远高于维氏气单胞菌,注射嗜水气单胞菌后10-18h内斑马鱼死亡率为100%,而注射维氏气单胞菌96h后死亡率仅为10-40%;在混合感染过程中,由维氏气单胞菌II型分泌系统分泌的气溶素(aerolysin)可以对斑马鱼头部口腔周围以及肠道部位造成严重的损伤,使嗜水气单胞菌等其他细菌通过损伤部位进入到斑马鱼体内,产生败血症。用气溶素基因敲除的维氏气单胞菌斑马鱼感染,不会产生肠道损伤及死亡;RtxA基因的ACD结构域敲除后,嗜水气单胞菌毒力明显降低(P0.01),证明了 Aer和RtxA分别在维氏气单胞菌和嗜水气单胞菌的致病性上起着关键作用。二、益生菌促进斑马鱼生长作用的机制研究益生菌的促生长效果:评价了五种乳杆菌,包括鼠李糖乳杆菌LGG(L.rhamnosus LGG)、鼠李糖乳杆菌 20300(L.rhamnosus 20300)、干酪乳杆菌 BL23(L.casei BL23)、植物乳杆菌1149(1.plantarum JCM 1149)及嗜酸乳杆菌 1132(L.acidophilusJCM1132)对斑马鱼的促生长效果,结果发现干酪乳杆菌BL23和鼠李糖乳杆菌20300以106CFU/mL连续浸浴21d或35d后可以显著提高斑马鱼的体重(P0.05);进一步研究发现,BL23活菌(106CFU/mL)连续浸浴处理斑马鱼幼鱼14d或28d后,可显著提高其体重(P0.05),而热灭活BL23不能提高斑马鱼的体重;数字基因表达谱分析(DGE)发现BL23活菌可以参与调节斑马鱼PPAR信号通路、氨基酸代谢、脂肪酸代谢以及固醇类激素的合成等,从而促进斑马鱼生长发育。三、益生菌促进斑马鱼抵御气单胞菌感染的机制研究干酪乳杆菌BL23浸浴处理刚孵化的斑马鱼3d后,再用维氏气单胞菌攻毒,结果发现BL23可以显著提高斑马鱼的存活率(P0.01),结合无菌斑马鱼模型和菌群转接实验表明,BL23抗气单胞菌感染的机制与改变肠道菌群无关,而与其菌体本身相关。进一步研究发现,BL23细胞提取物胞外多糖-蛋白复合物(EPSP)由40-45KD大小蛋白和α-由鼠李糖(α-Rha),α-葡萄糖(α-Glc),β-N乙酰葡萄糖(β-GlcNAc)和β-N乙酰半乳糖(β-GalNAc)组成的胞外多糖所组成。用10-20 μg/mlEPSP处理无菌斑马鱼,3d后用维氏气单胞菌攻毒,结果发现EPSP处理组无菌斑马鱼的死亡率显著低于对照组(P0.001);另外EPSP可以上调斑马鱼肝脏细胞系tlr1和tlr2的表达,并调节促炎因子和抑炎因子基因的表达(P0.05)。以上结果表明,乳酸菌BL23 EPSP的抗感染作用与其通过TLR1/TLR2信号通路增强斑马鱼免疫应答密切相关。
[Abstract]:The outbreak of the disease, such as the disease of the fish caused by Aeromonas, has become the main problem to restrict the healthy development of the fish industry. The probiotics play an important role in promoting animal growth, maintaining the intestinal health of the body, enhancing the immune defense function and protecting the host from the infection of pathogenic bacteria, and the like. The infection mechanism of Aeromonas, the growth of probiotics and the infection mechanism of Aeromonas were studied by the construction of the transposon gene mutation library, the target gene selection, the gene knock-out, the transcriptome, the sterile zebrafish model and the colony transfer model. The main contents and results are as follows: 1. The infection mechanism of Aeromonas hydrophila is divided into five types: A. veronii, A. hydrophila and A. jandei. Aeromonas sp. (A. schuertii) and Aeromonas hydrophila (A. aquariorum), which are two of the most important pathogens of Aeromonas sp. and Aeromonas hydrophila. In addition, the mixed infection of D. aeromonas and Aeromonas hydrophila is the main cause of the outbreak of the fish septicemia in South China. The virulence of Aeromonas was evaluated by the immersion bath model and the intra-abdominal injection model. The results showed that the virulence of Aeromonas hydrophila was the strongest in the bath model, and the virulence of Aeromonas hydrophila was relatively weak. In the model of intraperitoneal injection, The virulence of Aeromonas hydrophila is much higher than that of the D. aeromonas, and the mortality of the zebrafish is 100% in 10-18h after injection of Aeromonas hydrophila, and the mortality of the injection of the Aeromonas hydrophila 96h is only 10-40%; during the mixed infection process, Agersolin, which is secreted by the type II secretion system of Aeromonas, can cause serious damage to the oral cavity of the head of the zebrafish and the part of the intestinal tract, and the other bacteria such as Aeromonas hydrophila can enter into the zebra fish body through the damaged part, and the septicemia is generated. after the ACD domain of the RtxA gene is knocked out, the virulence of the Aeromonas hydrophila is obviously reduced (P0.01), It is proved that Aer and RtxA play a key role in the pathogenicity of Aeromonas and Aeromonas hydrophila, respectively. 2. The mechanism of probiotics to promote the growth of zebrafish study the growth-promoting effect of probiotics: five kinds of lactobacillus were evaluated, including Lactobacillus rhamnosus LGG, Lactobacillus rhamnosus 20300 (L. rhamnosus 20300), and Lactobacillus casei BL23 (L. casei BL23). The effects of Lactobacillus plantarum 1149 (1. planaricum JCM 1149) and Lactobacillus acidophilus 1132 (L. acidopilusJCM1132) on the growth-promoting effect of zebrafish were found. The results showed that Lactobacillus casei BL23 and Lactobacillus rhamnosus 20300 could significantly improve the body weight of the zebrafish after 21d or 35d (P0.05). BL23 live bacteria (106CFU/ mL) were treated with a continuous dip bath for 14d or 28d of the zebrafish, and the body weight was significantly increased (P0.05), while the heat-inactivated BL23 could not increase the body weight of the zebrafish. The digital gene expression profiling (DGE) found that the BL23 living bacteria could participate in the regulation of the PPAR signal pathway and amino acid metabolism of the zebrafish. the fatty acid metabolism and the synthesis of sterol hormones, and the like, so as to promote the growth and development of the zebrafish. The results showed that BL23 could significantly improve the survival rate of zebrafish (P0.01). The results showed that the mechanism of the infection of the BL23 against Aeromonas was not related to the change of the intestinal flora, but it was related to the cell itself. Further study found that the extracellular polysaccharide-protein complex (EPSP) of the BL23 cell extract was composed of 40-45KD protein and HCO3-derived from rhamnose (L-Rha), L-glucose (P-Glc), The extracellular polysaccharide consists of 1-N-glyoxyglucose (HCO3-GlcNAc) and 1-N ethylidene-galactose (HCO3-GalNAc). Aseptic zebrafish were treated with 10-20 & mu; g/ ml EPSP, and after 3days, the toxicity of the sterile Zebrafish was detected by D. aeromonas, and the results showed that the death rate of the sterile zebrafish in the EPSP treatment group was significantly lower than that of the control group (P 0.001), and the EPSP can increase the expression of the zebrafish liver cell lines tLr1 and tLr2. and the expression of the pro-inflammatory factor and the anti-inflammatory factor gene was adjusted (P0.05). The results showed that the anti-infection of BL23 EPSP was closely related to the immune response of zebrafish through TLR1/ TLR2 signal pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S941.4

【相似文献】