Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA,VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.     

硫糖铝对抗幽门螺杆菌重组VacA卵黄抗体的保护作用

目的观察硫糖铝对抗幽门螺杆菌重组细胞空泡毒素抗原(VacA)卵黄抗体(IgY)的保护作用,为制备能耐受胃酸和蛋白酶的IgY制剂奠定基础。方法诱导重组菌DH5α-vacA-pQE30,大量表达并纯化VacA重组蛋白,免疫洛曼母鸡,制备VacAIgY,并进行纯化。在不同pH值的IgY液中加入不同浓度的硫糖铝和不同浓度的胃蛋白酶,将含不同浓度硫糖铝的IgY反复冻融7次,将含30%硫糖铝的IgY室温放置1d～4周,ELISA法测定各实验组IgY的相对抗体活性(AT/AC)。结果在pH1.5、2.0和3.0的条件下,含30%～60%硫糖铝的IgY的相对抗体活性高于不加硫糖铝的IgY;在pH1.5条件下,60%硫糖铝可使IgY的相对抗体活性保持68.7%;在pH2.0和3.0条件下,50%和40%以上的硫糖铝几乎可完全保持IgY的相对抗体活性。在pH值为2.0和3.0时,正常胃蛋白酶浓度(0.02mg/ml)下,30%以上的硫糖铝均可有效保护IgY的相对抗体活性。30%以上的硫糖铝可增强IgY的抗冻融能力。室温放置4周后,含30%硫糖铝的IgY仍能保持80%以上的相对抗体活性。结论30%以上的硫糖铝可增强VacAIgY对低pH和胃蛋白酶的耐受能力及抗冻融能力,是较理想的IgY保护剂。     

幽门螺杆菌VacA-HpaA融合蛋白的表达及其免疫学特性

目的构建H.pylori细胞空泡毒素VacA与黏附素HpaA融合基因的原核表达载体,诱导其表达融合蛋白,并检测表达产物的抗原性与免疫原性。方法用PCR从pQE30-VacA质粒扩增出VacA基因,克隆至pTrc99A-HpaA载体中,与HpaA基因融合后,插入原核表达载体pQE30中,再将pQE30-VacA-HpaA转化入大肠杆菌DH5α,诱导表达并提纯融合蛋白,SDS-PAGE检测融合蛋白的表达,Bradford法检测融合蛋白含量,Western blot鉴定特异性。将融合蛋白免疫家兔,得到多克隆抗血清,用双向免疫扩散和ELISA检测免疫原性。结果SDS-PAGE显示融合蛋白相对分子质量约为65000,表达量在35%以上,主要以包涵体形式表达,蛋白含量为0·72mg/ml,具有良好的VacA和HpaA抗原性与免疫原性。结论VacA-HpaA融合蛋白已成功表达,且具有良好的免疫原性,为进一步研究制备H.pylori疫苗创造了条件。     

幽门螺杆菌VacA毒素诱导凋亡

研究了天然VacA细胞空泡毒素蛋白诱导细胞空泡和凋亡的生物学活性.培养空泡毒素阳性的Hp菌株,超声碎菌后提取上清,用SDS—PAGE分析上清中目的条带,测定其蛋白含量,用电镜观察VacA致Hela细胞空泡样变,然后用MTT法检测Hela细胞的生长曲线后,分别用MTT法分析VacA对Hela细胞的生长抑制作用,以及用TUNEL法和流式细胞技术检测VacA蛋白致凋亡的细胞毒活性.实验结果表明,在PAGE上可见相应VacA蛋白条带,电镜下天然VacA毒素致Hela细胞空泡化明显,因此VacA毒素可以抑制Hela细胞的生长,并且诱导其凋亡;在倒置显微镜下观察可见细胞空泡、脱落,胞浆浓缩,胞核皱缩、碎裂,呈现圆形固缩状态.MTT法数据示H.pylori Va-cA蛋白具有浓度依赖性地致细胞毒活性,光镜下可见TUNEL法中可显示棕黄色凋亡细胞,因此证明幽门螺杆菌VacA蛋白有明显的诱导Hela细胞凋亡的作用.