钙调神经磷酸酶通路及其靶基因RTA2在白念珠菌耐药性形成中的作用

发布时间：2018-09-05 20:17

【摘要】：白念珠菌依然是造成免疫功能低下病人感染的最主要的致病性真菌,临床上由于唑类药物尤其是氟康唑的长期应用,引起白念珠菌耐药日趋严重,使得临床白念珠菌感染的治疗更加困难和复杂。白念珠菌耐药性的形成是由多因素造成,已有研究发现钙调神经磷酸酶通路参与了白念珠菌耐药性的形成,是白念珠菌的致病因子之一。本实验室前期利用基因修饰技术在体外证明RTA2基因是钙调神经磷酸酶通路调节白念珠菌对唑类药物的耐药性产生的必需基因,但并未从体内证实RTA2基因是否与白念珠菌耐药性形成有关。因此本课题在实验室构建菌株及临床菌株中,对于钙调神经磷酸酶通路及RTA2基因与白念珠菌耐药性形成的关系及作用在体内外进行深入研究。为进一步研究与证实钙调神经磷酸酶通路在白念珠菌耐药性中形成的作用,本课题选取来自不同地域、不同病患的45株临床白念珠菌,利用微量液基稀释法的体外药敏实验,外源性给予人血清内类似浓度的离子钙激活钙信号通路,考察菌株对氟康唑敏感性的变化,发现45株临床菌中37.8%的菌株由敏感变为剂量依赖性耐药,13.3%的菌株由敏感变为耐药,从体外间接证实钙调神经磷酸酶通路的激活,降低了菌株对药物的敏感性,参与了菌株耐药性的形成。另一方面,在血清内类似浓度的离子钙环境下,临床白念珠菌对药物的敏感性的降低有可能意味着临床治疗过程中,菌株对氟康唑的实际敏感性与体外测定的敏感性不一致的情况发生。而采用同浓度钙离子激活该通路后,利用real time RT-PCR法测定RTA2基因表达水平的变化,66.7%的菌株RTA2表达上调,证明大部分菌株中钙调神经磷酸酶通路的激活促使了RTA2基因的表达上调。为分析同浓度离子钙条件下,体内外白念珠菌对药物的敏感性是否一致,钙调通路及RTA2基因对菌株毒力的影响及耐药性形成中的作用,我们将体外MIC值及RTA2表达水平两指标进行分类,共选取8株临床白念珠菌及前期构建的RTA2基因缺失菌、转录因子编码基因CRZ1基因缺失菌、回复菌及野生菌进行小鼠体内毒力及药物治疗效果评价,考察小鼠生存率,肾菌落数并进行病理切片分析,发现体外加钙后测定MIC值变化小、未耐药菌株感染小鼠后,经氟康唑治疗效果优于MIC值变化大、产生耐药及RTA2表达上调的菌株;而RTA2基因缺失菌及CRZ1基因缺失菌系统性真菌感染造模后,经氟康唑治疗效果也优于基因回复菌及野生菌。但各组菌株感染小鼠后未经治疗,生存时间及肾菌落数没有差异。以上结果表明RTA2基因与菌株的毒力无关,但是与菌株体内对氟康唑药物敏感性相关,并且以依赖钙调神经磷酸酶通路的方式参与了白念珠菌耐药性的形成。为探究钙调神经磷酸酶通路激活后,小部分临床菌株RTA2基因表达水平没有变化的原因,本课题首先对部分菌株的RTA2基因上游启动区1500bp序列进行测序,发现六个位点的碱基突变,继而采用报告基因法,将含有不同突变位点不同长度片段与报告基因质粒pBES116-rluc-act1连接,转化入RTA2缺失菌JXM101中,菌株提蛋白后加入荧光素酶底物,通过测定发光值确定荧光素酶基因的表达,来验证启动区不同突变位点是否有意义。我们发现,含-531bp、-652bp碱基突变的两菌株未测出荧光素酶活性,推测两位点的碱基突变导致钙调神经磷酸酶通路的转录因子Crz1p不能正常识别启动区,影响了RTA2基因的表达。综上所述,本课题在体内外共同验证了钙调神经磷酸酶通路及其靶基因RTA2在白念珠菌耐药性的形成中发挥重要作用。
[Abstract]:Candida albicans is still the most important pathogenic fungi causing infection in immunocompromised patients. The long-term use of azoles, especially fluconazole, has led to increasingly serious drug resistance of Candida albicans, making the treatment of clinical Candida albicans infection more difficult and complex. Previous studies have shown that the calcineurin pathway is involved in the formation of drug resistance in Candida albicans and is one of the pathogenic factors of Candida albicans.In our laboratory, gene modification technique was used to prove that RTA2 is an essential gene for calcineurin pathway to regulate the resistance of Candida albicans to azole drugs in vitro. Therefore, the relationship between calcineurin pathway, RTA2 gene and the formation of drug resistance of Candida albicans and its role in vitro and in vivo were studied in this study.
In order to further study and confirm the role of calcineurin pathway in the formation of drug resistance in Candida albicans, 45 clinical Candida albicans strains from different regions and different patients were selected in this study. Microdilution in vitro drug sensitivity test was used to investigate the calcium-activated signaling pathway of exogenous calcium ions with similar concentration in human serum. The changes of susceptibility to fluconazole showed that 37.8% of the 45 clinical strains changed from susceptibility to dose-dependent drug resistance, 13.3% from susceptibility to drug resistance, indirectly confirmed the activation of calcineurin pathway in vitro, reduced the sensitivity of the strains to drugs, and participated in the formation of drug resistance. The decrease of susceptibility of clinical Candida albicans to fluconazole in the presence of intracellular calcium may mean that the actual susceptibility to fluconazole is inconsistent with the in vitro susceptibility during clinical treatment. The expression level of RTA2 was up-regulated in 66.7% of the strains, indicating that the activation of calcineurin pathway in most strains promoted the up-regulation of RTA2 gene expression.
In order to analyze the susceptibility of Candida albicans in vitro and in vivo to the same concentration of ionic calcium, the effects of calcium regulation pathway and RTA2 gene on the virulence of Candida albicans and the formation of drug resistance, we classified the MIC value and RTA2 expression level in vitro and selected 8 clinical Candida albicans and the deletion of RTA2 gene constructed earlier. The toxicity of CRZ1 gene deleted bacteria, recombinant bacteria and wild bacteria in vivo and the effect of drug treatment in mice were evaluated. The survival rate of mice, the number of colonies in kidney and the pathological section were observed. It was found that the MIC value changed little after calcium was added in vitro. After infection with non-resistant bacteria, the therapeutic effect of fluconazole was better than that of MIC value. The results showed that there was no difference in the survival time and the number of colonies in the kidney between RTA2 gene deleted strains and CRZ1 gene deleted strains. The virulence of the strain was not related to the susceptibility of the strain to fluconazole, but was related to the susceptibility of the strain to fluconazole.
To explore the reason why the expression level of RTA2 gene in a small number of clinical strains did not change after activation of calcineurin pathway, we first sequenced the 1500bp sequence of the upstream promoter region of RTA2 gene in some strains, and found six mutation sites. Then we used the reporter gene method to find the different length slices containing different mutation sites. Segment was linked to reporter gene plasmid pBES116-rluc-act1 and transformed into RTA2-deficient strain JXM101. The luciferase substrate was added after protein extraction. Luciferase gene expression was determined by luminescence value to verify the significance of different mutation sites in the promoter region. It is speculated that the two-point mutation of the base leads to the inability of the transcription factor Crz1p in the calcineurin pathway to recognize the promoter region and affects the expression of RTA2 gene.
In conclusion, this study demonstrated that calcineurin pathway and its target gene RTA2 play an important role in the formation of drug resistance in Candida albicans in vitro and in vivo.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R379

【相似文献】