【作者】 贾鑫明；

【导师】 姜远英；

【作者基本信息】 第二军医大学 ， 药理学， 2008， 博士

【摘要】 由于临床上唑类抗真菌药物（尤其是氟康唑）的广泛和长期使用,导致了白念珠菌耐药性产生,这是临床治疗白念珠菌感染失败的首选原因。白念珠菌耐药性的获得是多基因在多层级水平变异积累的复杂过程;目前已明确药物外转运蛋白的过度表达是白念珠菌耐药性产生的重要原因,这类蛋白包括能量依赖性ABC转运蛋白（Cdrlp和Cdr2p）和易化载体超家族蛋白（Mdrlp和Flulp）。本研究利用氟康唑和氟奋乃静联合长期诱导已知耐药蛋白编码基因（CDR1,CDR2,CaMDR1和FLU1）缺失的白念珠菌DSY1024,得到对唑类药物（包括氟康唑、酮康唑、伊曲康唑、益康唑和活力康唑）高度耐药的菌株DSF28;然后利用基因表达谱芯片在DSF28中筛选得到13条上调基因和22条下调基因,其中多数为未知功能基因（49%）;其余基因的功能涉及RNA的转录和合成（14%）、钙调神经磷酸酶通路（9%）、氨基酸和碳水化合物代谢（9%）、小分子转运蛋白（9%）、能量生成（5%）和未分类功能基因（5%）。利用实时定量RT-PCR考察了DSF28耐药性的产生过程中,13条上调基因的动态表达水平;利用非监督聚类分析对这13条基因的动态表达样式进行聚类,发现这些基因具有3类表达样式,其中RTA2,MXR1,CPA2,ARO3和BAT22的表达样式与已知耐药基因ERG11,CDR1和CaMDR1在耐药产生过程中的表达样式类似,而且在耐药性产生过程中均保持较高的表达水平。本研究又利用基因敲除和异位高表达技术成功构建了RTA2基因在白念珠野生菌RM1000和已知耐药基因缺失菌DSY1024中的缺失菌和高表达菌。利用微量液基稀释法和Spot assay方法发现RTA2基因在白念珠菌RM1000和DSY1024中缺失均导致对唑类药物包括氟康唑、益康唑、伊曲康唑、酮康唑和活力康唑敏感性不同程度增加;而RTA2基因在白念珠菌RM1000和DSY1024中高表达均导致对上述唑类药物敏感性下降,证明RTA2基因参与白念珠菌对唑类药物耐药性的产生。利用实时定量RT-PCR我们发现26株临床分离的白念珠耐药菌中有18株菌中RTA2基因不同程度过度表达,但在18株临床分离的敏感菌中RTA2基因高表达水平变化很小;说明RTA2基因参与白念珠菌耐药性形成具有临床意义。另外,有研究报道高浓度的氯化钙能够诱导RTA2基因的表达上调,而且这种上调作用依赖于钙调神经磷酸酶（calcinetJrin,CaN）及其转录因子Crzlp;而CaN通路能够被氯化钙激活,并参与白念珠菌耐药性的形成,但其参与耐药形成的靶基因目前尚不明确。本研究通过加入CaCl₂、钙离子螯合剂EGTA以及CaN抑制剂（FK506和CsA）考察了唑类药物（氟康唑和酮康唑）对白念珠菌RM1000和RTA2基因缺失菌JXM101的抗真菌活的变化,发现CaN通路通过其靶基因RTA2调节对唑类药物的耐药性,我们进一步考察了CNA（编码CaN催化亚单位）缺失菌、CNA回复菌、CRZ1（编码CaN通路转录因子Crzlp）缺失菌和CRZ1回复菌对唑类药物（氟康唑和酮康唑）的抗真菌活性变化,得到了与RTA2基因单独缺失一致的结果。我们又利用实时定量RT-PCR在RTA2基因转录水平证明CaCl₂能够以CaN和Crzlp依赖的方式上调RTA2基因的表达。证明CaN通路调节白念珠菌对唑类药物的耐药性产生依赖于RTA基因,而RTA2基因的表达水平的上调依赖于CaN通路。利用透射电镜考察白念珠菌超微结构发现:RTA2基因缺失使得白念珠菌的细胞膜容易受到氟康唑的损伤;而氯化钙诱导的RTA2基因高表达能够减弱氟康唑对细胞膜的损伤作用。生物信息学分析发现RTA2基因编码产物是脂质转运蛋白。利用激光共聚焦技术发现RTA2基因敲除后导致细胞内鞘脂成分一长链碱二氢鞘氨醇向细胞外转运减少;利用鞘脂合成通路特异性抑制剂烟曲霉毒素B1和多球壳菌素,进一步确证RTA2基因编码蛋白参与转运细胞内外的长链碱。综上所述,RTA2基因是CaN通路调节白念珠菌对唑类药物的耐药性产生的必需基因。更多还原

【Abstract】 Widespread and repeated use of azoles,particularly fluconazole,has led to the rapid development of azole resistance in Candida albicans.Overexpression of CDR1,CDR2, CaMDR1 and FLU1 has been reported contributing to azole resistance in C.albicans.In this study,hyper-resistant C.albicans mutant,with the above 4 genes deleted,was obtained by exposure to fluconazole and fluphenezine for 28 passages.Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; Among the 35 genes,most were of unknown function（49%）,followed by those involved in transcription and RNA processing（14%）,calcineurin stress-response pathway（9%）, amino acid and carbohydrate metabolism（9%）,small molecule transport（9%）,energy generation（5%）,and those not classified（5%）.Moreover,expression pattern of RTA2 was similar to that of other known resistance-related genes（CDR1,CaMDR1,and ERG11）.The deletion of RTA2 in C.albicans strains with and without deletions of CDR1,CDR2, CaMDR1 and FLU1,was found to increase their susceptibility to azoles;meanwhile,we found that ectopic overexpression of RTA2 in the rta2 mutants conferred resistance to azoles.RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans.The calcineurin pathway has also been reported to participate in azole resistance in C.albicans.In the present study,CaCl₂-induced-activation of the calcineurin pathway in wild-type C.albicans promoted resistance to azoles;it was also found that the Ca²⁺ chelator（EGTA）,calcineurin inhibitors（FK506 and cyclosporin A） and the deletion of RTA2 blocked the resistance-promoting effects of CaCl₂.Furthermore,we found that the addition of CaCl₂ up-regulated RTA2 in a calcineurin-dependent manner.Ultra-structure analysis by transmission electronic microscopy showed the depletion of RTA2 make the cell membrane of C.albicans liable to be destroyed by fluconazole and Ca²⁺-induced-upregulation of RTA2 attenuated the destroying effects.Bioinformatics assay revealed that Rta2p is a lipid transporter.Our study found that the depletion of RTA2 decreased the translocation of sphingolipid dihydrosphingosine to outside of the cell membrane in C. albicans.In conclusion,our findings suggest that RTA2 is responsible for the development of calcineurin-mediated azole resistance in C.albicans.更多还原