【作者】 李俊；

【导师】 王豪举； 方维焕；

【作者基本信息】 西南大学 ， 微生物学， 2011， 硕士

【摘要】 副溶血弧菌是一种弧菌科弧菌属革兰氏阴性嗜盐短杆菌,菌体呈弧状、杆状或丝状。能在较宽的盐浓度与pH值范围内生长,最早于1950年在日本发生的一次暴发性食物中毒中发现并分离成功。此菌广泛分布于海湾、海岸线区域、盐湖及海产品中,能够感染鱼、虾和贝类等,引起虾红体病、鱼类皮肤溃疡等。在沿海地区,经常由于食用污染副溶血弧菌而又未良好加工处理的海产品引起突发性食物中毒事件,患者出现腹泻、恶心、呕吐、发热,甚至脱水、昏迷等症状。耐热直接溶血素(TDH)与耐热直接溶血素相关溶血素(TRH)被认为是副溶血弧菌的主要毒力因子,它们与副溶血弧菌的致病能力密切相关。随着水产养殖业的发展,弧菌病的暴发越来越频繁,传播速度越来越快,给全世界水产养殖业带来了巨大的经济损失。副溶血弧菌还是污染海产品的主要细菌之一,我国出口的海产品曾多次因副溶血弧菌超标而被退货、销毁。欧盟368决议(97/368EC)和587号决议(97/587/EC)更是要求对原产于中国的水产品逐批检验副溶血弧菌。我国食源性疾病监测网1998年以来的数据显示,副溶血性弧菌引起中毒的发生规模及人群暴露规模呈明显上升趋势,均超过沙门氏菌食物中毒而跃居首位。1、丝氨酸/苏氨酸激酶和酪氨酸激酶是两个主要的蛋白激酶超家族成员。丝氨酸／苏氨酸蛋白激酶(serine/threonine protein kinase, STPK)是一类能使特定底物蛋白中丝氨酸或苏氨酸羟基磷酸化的酶。通过催化多种功能蛋白(如酶、受体、运输蛋白、调节蛋白、核内蛋白等)的磷酸化,STPK可以改变功能蛋白的构象,导致功能蛋白活性、性质的变化,从而调节细胞多种生命活动过程。丝氨酸/苏氨酸特异性蛋白激酶和蛋白磷酸酶的可逆性磷酸化在信号传递过程中起切换开关的作用。蛋白激酶催化蛋白质上磷酯键的形成,细胞蛋白质活性发生极大变化,形成的磷酯键性质非常稳定,去除时需要蛋白磷酸酶水解。细胞通过调节蛋白激酶与蛋白磷酸酶的动态平衡,产生一个限定时空的信号,并以一种高度特异性方式来影响蛋白质的活性状态,调节细胞功能。本实验室通过生物信息学分析发现,在副溶血弧菌中存在两个丝氨酸／苏氨酸蛋白激酶,分别位于染色体Ⅰ和Ⅱ上,命名为Vpk1和Vpk2,一个蛋白磷酸酶位于染色体Ⅱ上,命名为Vpp2。2、Ⅵ型分泌系统(TypeⅥsecretion system, T6SS)是最近发现的一种新的分泌系统,广泛存在于革兰氏阴性菌变形菌门细菌中,主要由构成分泌系统的结构蛋白、形成跨膜管道结构的转位蛋白、分泌蛋白以及一些对分泌系统起辅助功能的蛋白组成。T6SS能够增强细菌对外界环境的适应性,介导细菌对宿主细胞的致病力等。因此,T6SS的结构、功能和调控等都是亟待深入研究的课题。在含有T6SS基因簇的细菌上清液中,能检测到两类蛋白质——溶血素共调节蛋白(Hcp)和缬氨酸-甘氨酸重复蛋白G (VgrG),他们共同构成了分泌蛋白的管道结构,辅助分泌蛋白跨膜转运并最终到达宿主细胞(即转位蛋白)。本实验通过生物信息学分析发现,在副溶血弧菌中存在两套T6SS,分别位于染色体Ⅰ和染色体Ⅱ上,其中转位蛋白Hcp1和Hcp2分别位于染色体Ⅰ和染色体Ⅱ上。3、本试验针对vpk1、vpk2、vpp2、hcpl和hcp2等5个目的基因及其外围序列分别设计A、B、C、D、E和F等6条引物,首先利用普通PCR扩增同源臂AB、CD或EB、CF,然后通过融合PCR得到同源臂融合片段vpk1-AD (1041bp)、vpk2-AD (1261bp)、vpp2-AD (896bp)、hcp1-AD (767bp)和hcp2-AD(746bp)。为了构建基因缺失载体,我们酶切此融合片段后连接至pYAK1质粒,再利用大肠杆菌性菌毛的接合作用获得同源重组基因缺失株HZ-△vpk、HZ-△vpk2、HZ-△vpp2、HZ-△hcp1和HZ-△hcp2。并利用引物E、F对构建的基因缺失株进行了鉴定,成功获得同源重组基因缺失株。4、基于同源重组技术的发展,本文采用的融合PCR技术是直接在X-B、X-C的5’端添加黏性末端,让它们在第二轮中以X-A、X-D为引物直接融合。同时以pYAK1质粒为供体,亲本株HZ为受体,pRK2013-HB101为辅助菌进行大肠杆菌性菌毛的接合转移试验,即将供体菌、受体菌和辅助菌混匀后点样在0.22μM的滤膜上,为有效接合的发生提供了良好平台。由于一次重组效率比较低,所以我们采用10%蔗糖培养基进行二次重组筛选阳性重组子,同时在接合转移过程中加入辅助质粒pRK2013或加入10mM的Mg2+以增加细胞膜的通透性来提高接合转移效率。结果成功地将质粒pYAK1接合转移到HZ株中,得到相应的基因缺失株,为深入研究丝氨酸／苏氨酸蛋白激酶和磷酸酶对V1分泌系统转位蛋白Hcp表达量的影响奠定基础。5、为了研究丝氨酸／苏氨酸蛋白激酶和磷酸酶对T6SS的影响,本试验分别利用实验室制备的鼠源单抗和兔源多抗,通过免疫印记的方法分析了不同缺失株对转位蛋白Hcpl和Hcp2表达量的影响。本实验采用高镁低钙的BHI培养基(BHI’),以减少细菌生长过程中的差异,同时它也可以增加细菌细胞的通透性,便于分泌蛋白的分泌与转位。对培养时间和温度进行了优化,细菌在28℃条件下培养6h和14h适于分析Hcp2转位蛋白的变化。原核表达蛋白Hcp2和RpoB时,以低温长时诱导方法即15-20℃诱导12h,可以得到可溶性较好的蛋白。免疫印记结果分析,得出以下结论：1)亲本株HZ及Avpk1、Avpk2、Avpp2的上清和沉淀中都没有检测到Hcp1,而在互补hcpl后的菌体沉淀中能检测到Hcp1,表明T6SS1的转位蛋白在正常情况下低表达或者不表达。2)在△vpk1、△vpk2、△vpp2的培养上清中检测不到Hcp2,而在△vpk2、△vpp2菌体沉淀中能检测到,说明丝氨酸/苏氨酸激酶系统对T6SS转位蛋白Hcp2有调控作用。3)△vpp2缺失株中Hcp2的量明显高于亲本株HZ,说明该丝氨酸/苏氨酸磷酸酶Vpp2下调Hcp2的表达。4)Vpk1激酶位于染色体Ⅰ,却对位于染色体Ⅱ的转位蛋白Hcp2的转位和表达都有调节作用,表明副溶血弧菌丝氨酸/苏氨酸激酶系统对两套T6SS可能因为空间结构的复杂性存在交叉调节,也说明丝氨酸/苏氨酸激酶系统对T6SS的调控存在多样性。更多还原

【Abstract】 Vibrio parahaemolyticus (VP) is a gram-negative halophilic bacterium, which can grow in a wide range of pH and salt concentration. It was first discovered and successfully isolated from a food-borne illness in Japan in 1950. It is widely distributed in seafood products and can infect fish, shrimp and shellfish resulting in shrimp red body disease and fishes’skin ulcers, etc. The outbreaks of food poisoning were almost exclusive with consumption of contaminated or inadequately cooked seafood. The common symptoms are usually presented as gastroenteritis characterized mainly by watery diarrhea and abdominal cramps, sometimes a dysentery-like illness with bloody or mucoid stoos. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) were known as the major virulent factors, and they were correlated firmly with the pathogenecity of vibrio parahaemolyticus.With the development of the aquaculture industry, vibriosis outbreaks are more and more frequently, and spreads faster and faster, which bring a huge economic losses to the world aquaculture. It is the main bacterial which can contaminate seafood, and several times returned and destroyed were happened because of Vibrio parahaemolyticus excessive during China’s exports. The EU resolution 368 (97/368/EC) and 587 (97/587/EC) required to test batches of Vibrio parahaemolyticus on the origin of aquatic products in China. Data of food-borne disease showed, since 1998 the outbreaks of Vibrio parahaemolyticus have increased significantly and been to the first rank instead of Salmonella.1. Serine/threonine kinase (STPK) and tyrosine kinase are numbers the two main protein kinase superfamily. STPK makes serine or threonine hydroxy-phosphorylation of special substrate proteins. Through catalyzing a variety of functional proteins (such as enzymes, receptors, transport proteins, regulatory proteins, nuclear proteins, etc.) phosphorylation, STPK can change conformation of functional proteins, leading to functional proteins activity and properties changes to regulate various cell life activities. Reversible phosphorylation of serine/threonine protein kinases and protein phosphatases plays a switch role on signaling processes. Protein kinases catalytic the formation of phosphate ester bonds, cell protein activity occurred significantly change, and the properties of phosphate ester bonds are very stable. The bioinformatics analysis found that Vibrio parahaemolyticus processes two serine/threonine protein kinase, which are located on chromosomesⅠandⅡ, named Vpk1 and Vpk2, respectively, and a protein phosphatase, which is located on chromosomeⅡ, named Vpp2.2. TypeⅥsecretion system (T6SS) is a new secretion system discovered recently. It is widely found in gram-negative bacteria, and consists of structural proteins, translocon proteins, secreted proteins and some auxiliary functions proteins. T6SS can enhance the adaptability of bacteria to the external environment, and mediate bacterial virulence on host cells, etc. T6SS plays an important role for gram-negative bacteria, but plays different roles in different bacteria. Therefore, its structure, function and regulation are all urgent further study. The laboratory analyze by bioinformatics to find that Vibrio parahaemolyticus processes two sets of T6SS, which locates on chromosomeⅠand chromosomeⅡ, respectively. Hemolysin co-regulated protein (Hcp) and valine-glycine repeat protein G (VgrG) can be detected in the supernatant of T6SS gene cluster. They together constitute the pipeline structure of secreted proteins, assisting secreted protein transmembrane transport and eventually reach the host cell. Hcp1 and Hcp2 locates on chromosomeⅠand chromosomeⅡ, respectively.3. To reveal the regulation effect serine/threonine kinase system on T6SS, we designed 6 primers (A, B, C, D, E and F) according to vpk1, vpk2, vpp2, hcp1 and hcp2 genes. First, a common PCR was carried to amplify AB, CD or EB, CF, and then we obtained vpk1-AD, vpk2-AD, vpp2-AD, hcpl-AD, hcp2-AD through the fusion PCR. Next, these fusion fragments were digested and connected pYAK1 vector to construct gene deletion vectors. We successfully obtained mutants HZ-△vpk1, HZ-△vpk2, HZ-△vpp2, HZ-△hcp1,HZ-△hcp2 through conjugation. Primers E and F were used to identify these mutants.4. In present study, we linked the fusion fragment into pYAK1 plasmid by homologous recombination, and transformed into host bacteria CC118λpir. The fusion PCR technique we used overcomes the disadvantage of traditional homologous recombination, and eliminates the digestion process of connecting to compare with the digested ligation. In this study, we utilized plasmid pYAK1 as the donor, the parent strain HZ as a receptor, and pRK2013-HB101 as an auxiliary bacteria in conjugation. Because the recombination rate is relatively low, we used 10%sucrose medium for the second re-screened positive recombinants. To optimize conjugation by Escherichia coli fimbriae, we mixed the donor strain, recipient strain and auxiliary bacteria before dropping in the filter membrane of 0.22μM, and the helper plasmid transfer pRK2013 or 10mM Mg2+was added to increase bacterial cell membrane permeability to increase with the transfer efficiency. The results showed that we successfully transferred the plasmid pYAKl into Vibrio parahaemolyticus strain HZ and obtained mutants HZ-△vpk1, HZ-△vpk2, HZ-△vpp2, HZ-△hcp1, HZ-△hcp2. This lays the foundation for further research the effect of serine/threonine protein kinase and phosphatase on expression of Hcp protein.5. We analyzed different gene deletion mutant effect on Hcp1 and Hcp2 expression by western blotting. In order to decrease the differences on bacterial growth, we used the BHI medium containing high magnesium and low calcium (named BHI’). Meanwhile, BHI’medium can also increase the permeability of bacterial cells to facilitate secretion of protein secretion and translocation. Besides, through a series experiments of temperature and incubation time, the translocation of Hcp2 was optimized at 28℃for 6h and 14h. A relatively high soluble Hcp2 and RpoB were obtained in 15-20℃for 12h in prokaryotic expression E.coli. Western blotting results showed that 1) we failed to detect Hcp1 in the parent HZ strain and△vpk1,△vpk2,△vpp2 deletion mutant in precipitation and supernatant protein, however, Hcp1 protein was detected after complementing hcpl in precipitation, showed that the T6SS1 translocation protein was low expressed under normal conditions.2) Hcp2 was not detected in the supernatant of bacteria with absence of vpkl, vpk1, vpp2, showed that the serine/threonine kinase Vpk1, Vpk2 and phosphatase Vpp2 played important roles on the translocation of Hcp2 protein. But we detected Hcp2 protein in cell precipitation with absence vpk2, vpp2.3) Hcp2 expression was significantly higher than the parent HZ strain in△vpp2, showing that the serine/threonine phosphatase downregulate Hcp2 expression.4) In△vpkl bacteria precipitation, we did still not detect Hcp2 protein, indicating that the serine/threonine kinase Vpkl not only regulates the translocation of Hcp2, but also increases Hcp2 protein. Taken together, two T6SS in Vibrio parahaemolyticus may be cross-regulated by STPKs, and it also showed that serine/threonine kinase syetem regulate T6SS in the diversity ways.更多还原