【作者】 张石群；

【导师】 姜远英； 曹永兵；

【作者基本信息】 第二军医大学 ， 药理学， 2012， 硕士

【摘要】 近年来，随着器官移植后免疫抑制剂的使用，广谱抗生素的大量使用，系统性真菌感染的发生率急剧升高，尤其以白念珠菌感染最为普遍、严重。开展以深部真菌感染主要病原菌白念珠菌为代表的真菌耐药机制研究，寻找新的抗真菌药物作用靶点，对于创建抗真菌药物筛选新模型，设计开发新型抗真菌药物，从而克服真菌感染及其耐药性，防治日益严重的深部真菌感染具有重要意义。本实验室前期研究发现了新的耐药基因RTA2，并已证明钙离子通过钙调神经磷酸酶通路上调RTA2基因的表达，其编码蛋白Rta2p是白念珠菌鞘脂成分的异位酶，定位于细胞膜脂筏，能将细胞膜内侧鞘脂骨架成分--长链碱转运至细胞膜外侧，从而调节细胞膜内外两侧的鞘脂成分分布，增强脂筏的稳定性。因此，本课题通过进一步研究Rta2p蛋白的结构与功能关系，发现一些与钙调神经磷酸酶通路、与长链碱结合转运相关的生物学活性位点，将会对白念珠菌耐药性形成的分子机制，发现新的抗真菌药物靶标，设计新型抗真菌抑制剂奠定理论基础。通过白念珠菌基因组数据库和酿酒酵母基因组数据库比对分析发现4个同源蛋白(Rsb1p,Yer185w,Rta1p,Ylr046c)；其中Rta2p和Rsb1p具有长链碱二氢鞘氨醇转运活性，其他同源蛋白为七次跨膜的膜蛋白，并且均为脂质转运蛋白家族的成员。通过Blast比对，找出了跨膜区中的保守序列，设计将需要考察的位点突变为结构差异较大，性质相反的氨基酸，本课题共设计16个突变点。首先，据此设计错配引物，用PCR方法扩增突变质粒，并转化至超感受态细胞，抽提质粒得到大量突变质粒，通过基因测序和Blast比对目的突变点，得到14个目的突变质粒。然后，根据同源重组原理，利用LiAC转染法将线性突变质粒转染至RTA2缺失菌JXM101中，并利用RTA2特异性引物和套氏PCR方法鉴定阳性转化菌。将所有Rta2p突变菌通过敏感性相关实验进行筛选，发现突变菌M10-1(G234S)与RTA2回复菌JXM201差异较明显，与空载体回复菌P-B类似，在高钙条件下MIC80值无明显变化，不能诱导真菌耐药性的形成，而在低钙条件下和缺失菌一样，对氟康唑表现得更为敏感；而突变菌M3-1(G158E)与RTA2回复菌JXM201差异并不明显，在钙离子存在时并未完全耐药，不能完全诱导真菌耐药性的形成。其他突变菌和RTA2回复菌JXM201无明显差异，在加入Ca2+后均对氟康唑的敏感性降低。另外，通过各突变菌对氟康唑的生长曲线影响和时间杀菌曲线等实验也可证明突变菌M10-1(G234S)对氟康唑的敏感性有所改变。通过体内动物实验考察突变RTA2对菌株毒力的影响以及FLC治疗效果，发现经氟康唑治疗后，突变菌M10-1感染动物的生存率较JXM201感染动物有所提高。最后，通过考察各突变菌对荧光底物二氢鞘氨醇（NBD-DHS）的外排、摄入情况，以及对鞘胺醇代谢通路的上下游抑制剂敏感性等实验发现M10-1(G234S)对底物的转运能力受到影响，即对NBD-DHS定量摄入后外排释放量减少，并且对鞘脂代谢通路下游抑制剂的敏感性较RTA2回复菌JXM201有明显差异。因此，通过对白念珠菌耐药相关蛋白Rta2p跨膜区保守序列突变后的菌株耐药性和转运功能研究，我们发现跨膜区中高保守区域为RTA2重要功能域，通过考察Rta2p跨膜区不同位点突变前后对钙调神经磷酸酶通路调控的耐药性形成的影响以及对底物长链碱二氢鞘氨醇（NBD-DHS）转运能力的改变，发现突变菌M10-1(G234S)与RTA2回复菌JXM201差异较明显，而与空载体回复株P-B类似，表现为在高钙条件下，不能诱导真菌耐药性的形成；而在低钙条件下，也和缺失菌一样，对氟康唑表现得更为敏感，同时突变菌M10-1(G234S)胞内的NBD-DHS积累情况显著高于JXM201，而且其外排能力也明显下降，证明了G234是影响长链碱转运的重要活性位点。更多还原

【Abstract】 In recent years, with the use of immunosuppressants after organ transplantation, and broad-spectrum antibiotic, the incidence of systemic fungal infections increased rapidly, especially the infections of Candida albicans are most serious. Carrying out research to find new antifungal drug targets of the new model for deep fungal infection resistance and mechanisms of the major pathogen Candida albicans is important for the designment of creation antifungal drugs and development of new antifungal agents, in order to overcome fungal infection and the growing problem of prevention and treatment of deep fungal infection.A new resistance gene RTA2was found in our preliminary studies, and we have proved that Ca2+increased the expression of RTA2by the calcineurin phosphatase pathway. The encoded protein Rta2p, located in cell membrane lipid rafts, was the ectopic enzyme of sphingolipid composition of Candida albicans, and could transport membrane sphingolipid skeleton composition-long chain base from inside to outside, which could regulate cell membrane on both sides of the sphingolipid composition distribution and enhance the stability of lipid rafts. Therefore, if we can study Rta2p protein structure-function relationship furthermore to find the biological active sites related with the calcineurin pathway and the long-chain base binding cassette transporter, we will lay the theoretical foundation of molecular mechanism in Candida albicans resistance, finding new antifungal drug targets, designing new anti-fungal inhibitors.We found four homologous proteins (Rsblp, Yer185w, Rtalp, Ylr046c) through analysis of Candida albicans genome database and the Saccharomyces cerevisiae genome database. By Blast comparison to find conserved sequences in transmembrane region, we designed pointed mutations in the structural differences and contrary nature of amino acids, a total of16mutations. First, based on the mismatch primers, we amplified the mutant plasmids by PCR, transformed into ultra-competent cells, extracted plasmids sequenced and got14pointed mutant plasmids. Then, according to the principle of homologous recombination, we used LiAC transfected method to transfect linear mutant plasmids to the RTA2absence strain JXM101, by using RTA2specific primers and nested PCR to identify positive transformed clone. All of the Rta2p mutant strains were proved by sensitivity experiments. We found that compared with JXM201, the difference of mutant strain M10-1(G234S) was obvious, and was similar with the empty vector strain P-B. The MICso showed no significant changes in high calcium conditions, and M10-1couldn’t induce the formation of fungal resistance in the low calcium conditions and showed more sensitive to fluconazole. While the mutant strain M3-1(G158E) didn’t showed obvious differences in the calcium compared with JXM201, and could not fully induce the formation of fungal resistance. Other mutant strains showed no significant difference with JXM201. In addition, the growth curves of each mutant to fluconazole and time of sterilization curve experiments also proved that the mutant strain M10-1(G234S) to fluconazole was changed. Besides, we found that the mutant strain M10-1(G234S) affected the experiments by the outake and intake of fluorescent substrate (NBD-DHS), and metabolism pathways downstream inhibitor sensitivity in the substrate transport and sensitivity to inhibitors of the sphingolipid metabolic pathway downstream with the difference of JXM201.So, through the study of resistant strains and the transport function on the transmembrane domain of resistance-associated protein Rta2p, we found a highly conserved region of the transmembrane region is an important function domain of RTA2. By studying different sites mutations of Rta2p transmembrane region we found that the difference of mutant strain M10-1(G234S) and wild strains JXM201was more obvious, and it was similar with the empty vector strain P-B in the high Ca2-conditions, which couldn’t induce the formation of fungal resistance. While in the low Ca2+conditions it showed more sensitive to fluconazole. Besides we indicated that the G234is an important active site of transporting long-chain base.更多还原