RTA2基因降低白念珠菌对氟康唑敏感性的机制研究

发布时间：2018-05-03 23:16

本文选题：白念珠菌 + 钙调神经磷酸酶　；参考：《第二军医大学》2013年博士论文

【摘要】：在白念珠菌（Candida albicans）中，由于越来越多的菌株对药物产生了耐药性，导致临床上用于治疗念珠菌感染的一线药物氟康唑（Fluconazloe,FLC）的治疗产生失败。我们实验室前期的研究发现：在体外，RTA2基因编码的蛋白参与了由白念珠菌钙调神经磷酸酶（calcineurin,CaN）信号通路介导的对氟康唑的耐药。在本研究中，通过药物敏感性实验考察不同基因背景的RTA2基因相关菌株对氟康唑的敏感性差异，发现：在野生型菌株（cnaΔ/Δ::CNA，crz1Δ/Δ::CRZ1，rta2Δ/Δ::RTA2）中加入1mM CaCl2可以激活CaN信号通路，使菌株对唑类药物氟康唑的敏感性显著降低（最低抑菌浓度由0.5μg/ml升高为16μg/ml和64μg/ml）；加入1mM CaCl2激活CaN信号通路，RTA2基因缺失菌（rta2Δ/Δ,cnaΔ/Δ::CNA；rta2Δ/Δ, crz1Δ/Δ::CRZ1）和钙调神经磷酸酶CNA基因缺失菌（cnaΔ/Δ,rta2Δ/Δ::RTA2）以及CaN信号通路转录因子CRZ1基因缺失菌（crz1Δ/Δ, rta2Δ/Δ::RTA2）一样，对氟康唑的敏感性无显著变化。说明在体外钙离子激活的CaN信号通路通过RTA2p降低白念珠菌对氟康唑的敏感性。通过透射电镜实验考察不同浓度的氟康唑对不同基因背景的RTA2基因相关菌株细胞膜超微结构的影响，我们发现：在体外，，钙离子激活钙调神经磷酸酶通路后，可以通过Rta2p来减弱氟康唑对细胞膜的损伤，从而显著的降低了白念珠菌对氟康唑的敏感性。此外，我们构建了小鼠深部真菌感染模型，考察基因RTA2对模型动物生存时间及肾脏荷菌量的影响，发现：RTA2基因本身并不能影响白念珠菌的毒力；但RTA2基因缺失后，感染小鼠接受氟康唑治疗后，生存率显著提高；相反，异位表达RTA2基因显著的降低了氟康唑在宿主中对白念珠菌的疗效。综上所述，我们认为在体内体外钙离子激活的钙调神经磷酸酶通路，通过Rta2p来降低白念珠菌对氟康唑的敏感性。在白念珠菌中，基因RTA2是钙调神经磷酸酶通路调控的一个下游基因，但是其调控机制并不明确。本课题采用海肾荧光素酶报告基因（Renilla Luciferase，RLUC）系统进行研究：将不同长度的RTA2基因启动子片段连接到报告基因载体中，再将构建好的载体分别转入菌株DJY201（Δ/Δcna,Δ/Δrta2）、MJY201（Δ/Δcrz1,Δ/Δrta2）、JXM101（Δ/Δrta2）中，考察加入钙离子前后荧光活性的变化倍数。进一步证明RTA2基因是CaN信号通路的靶基因，其表达水平受到转录因子Crzlp的调控；发现Crzlp的DNA结合序列—钙调神经磷酸酶依赖性应答元件（calcineruin-dependent responseelement,CDRE），可能位于RTA2基因启动子上游-973bp~-920bp的区间内，通过生物信息学分析推测其序列可能为GATGT。我们实验室前期研究发现：在临床白念珠菌中，外源性加入1mMCaCl2激活CaN信号通路，可以不同程度的上调RTA2基因的表达水平。通过小鼠深部真菌感染模型发现，钙离子诱导的RTA2基因高表达的菌株感染的小鼠接受氟康唑治疗后，生存率显著低于钙离子诱导的RTA2基因非高表达的菌株感染的小鼠。本研究通过体外药物敏感性实验和实时定量（Real-Time） PCR实验发现：钙离子诱导的菌株对氟康唑敏感性降低和RTA2基因的表达水平密切相关。在钙离子诱导的RTA2基因高表达的临床菌株0511522中，敲除了RTA2基因。通过体外药物敏感性实验发现， RTA2基因缺失菌RTA2N4（△/△rta2）无法产生钙离子介导的对氟康唑的敏感性降低；通过小鼠深部真菌感染模型发现，敲除RTA2基因并不影响菌株的毒力；但是RTA2基因缺失后，感染动物接受氟康唑治疗以后，生存率显著升高。综上所述，在体内体外钙离子通过激活钙调磷酸酶通路诱导RTA2基因高表达从而降低临床菌株对氟康唑的敏感性。
[Abstract]:In Candida albicans, as more and more strains are resistant to drugs, the treatment of Fluconazloe (FLC), a first-line drug used to treat Candida infection, has failed. Our early laboratory study found that the protein encoded by the RTA2 gene was involved in calcium Candida albicans in vitro. Calcineurin (CaN) signaling pathway mediated resistance to fluconazole. In this study, the sensitivity of RTA2 related strains with different gene backgrounds was investigated by drug sensitivity experiments. It was found that 1mM CaCl2 could be added to the wild type strains (CNA Delta / Delta: CNA, Crz1 Delta / Delta: CRZ1, rta2 Delta / Delta: RTA2). Activation of CaN signaling pathway significantly reduced the sensitivity of the strain to fluconazole (the minimum inhibitory concentration increased from 0.5 mu g/ml to 16 mu g/ml and 64 mu g/ml), and 1mM CaCl2 activated CaN signaling pathway, RTA2 gene deletion bacteria (rta2 Delta / Delta, CNA Delta / Delta: CNA; rta2 Delta / Delta, Delta / delta) and calcineurin gene deletion bacteria Delta / Delta, rta2 Delta / Delta: RTA2), as well as the CaN signaling pathway transcription factor CRZ1 gene deletion bacteria (Crz1 Delta / Delta, rta2 Delta / Delta: RTA2), the sensitivity to fluconazole was not significantly changed. It indicated that the CaN signaling pathway activated by calcium ions in vitro reduced the sensitivity of Candida albicans to fluconazole through RTA2p. The fluorine of different concentrations was investigated by transmission electron microscopy. The effect of conazole on the ultrastructure of the cell membrane of RTA2 gene related strains with different gene background was found. We found that in vitro, after calcium activation of calcineurin pathway, calcium ion can reduce the damage of fluconazole to the cell membrane through Rta2p, thus significantly reducing the sensitivity of Candida albicans to fluconazole. The effect of gene RTA2 on the survival time and kidney load of the model animal was investigated. It was found that the RTA2 gene itself did not affect the virulence of Candida albicans, but after the deletion of the RTA2 gene, the survival rate of the infected mice was significantly increased after the treatment of fluconazole; on the contrary, the ectopic expression of RTA2 gene significantly reduced the fluorine. The effect of conazole on Candida albicans in the host. In summary, we believe that calcium activated calcineurin pathway in vivo and in vivo reduces the sensitivity of Candida albicans to fluconazole by Rta2p.
In Candida albicans, the gene RTA2 is a downstream gene regulated by calcineurin pathway, but its regulatory mechanism is not clear. The subject uses the Renilla Luciferase (RLUC) system of the sea kidney luciferase reporter gene (RLUC) system to connect the RTA2 gene fragment of different lengths to the reporter gene carrier and then construct it. The good carriers were transferred to strain DJY201 (delta / delta cna, Delta / delta rta2), MJY201 (delta / delta Crz1, Delta / delta rta2), JXM101 (delta / delta rta2), and JXM101 (delta / delta rta2). The variation of fluorescence activity before and after the addition of calcium ions was investigated. The RTA2 gene was further proved to be the target gene of the CaN signaling pathway, and its expression was regulated by the Crzlp of the transcription factor. The calcineurin dependent response element (calcineruin-dependent responseelement, CDRE) may be located in the range of -973bp~-920bp in the upstream of the RTA2 gene promoter. It is presumed that the sequence of the RTA2 gene promoter may be GATGT..
Previous studies in our laboratory found that in clinical Candida albicans, exogenous addition of 1mMCaCl2 activates the CaN signaling pathway to increase the expression level of RTA2 genes in varying degrees. The survival rate of mice infected by the calcium induced RTA2 gene highly expressed strain infected mice after the treatment of fluconazole was found. Mice infected with the non high expression of RTA2 gene were significantly lower than calcium ion induced strain. This study found that the decrease of fluconazole induced by calcium ion induced strain was closely related to the expression level of RTA2 gene by drug sensitivity test in vitro and real-time quantitative (Real-Time) PCR test. The high expression of RTA2 gene induced by calcium ion was expressed in this study. In clinical strain 0511522, the RTA2 gene was knocked out. The drug sensitivity test in vitro showed that the RTA2 gene deletion strain RTA2N4 (delta / delta rta2) could not produce calcium ion mediated susceptibility to fluconazole; through the deep fungal infection model in mice, it was found that the knockout of the RTA2 gene did not affect the virulence of the strain; however, the RTA2 gene was missing after the deletion of the RTA2 gene. When the infected animals were treated with fluconazole, the survival rate was significantly increased. In summary, calcium ions in the body and in vivo induced the high expression of RTA2 gene by activating the calcineurin pathway and thus reducing the sensitivity of the clinical strain to fluconazole.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R519.3

【共引文献】