研究白色念珠菌REP3突變株的功能性補救

Abstract

伺機性病原菌—白色念珠菌 (Candida albicans) 是造成人體黴菌感染最常見的病原。Azole類藥物常用來治療白色念珠菌的感染。然而，廣泛使用fluconazole使得抗藥性白色念珠菌菌株之案例大增，進而導致臨床上治療失敗的機會增高。因此，瞭解白色念珠菌抗藥機轉對於克服黴菌感染是很重要的。排藥幫浦CDR1和CDR2的過量表現常見於抗藥性臨床菌株，然而其表現被如何調控卻所知甚少。實驗室先前經由在Saccharomyces cerevisiae 所進行之library screening 發現在白色念珠菌中，REP3 (Regulator of Efflux Pump) 能增加CDR1 Ym990348啟動子-lacZ 的 □-galactosidase 活性約四倍。再者根據藥物敏感性測試的結果顯示REP3突變株對azole類藥物的感受性有提高的現象，但REP3 突變補救株對藥物感受性卻沒有恢復。本研究重新建構突變株，但結果仍同，經Real-time PCR檢測顯示REP3 mRNA 確實存在rep3/rep3::REP3 突變補救株中且表現量與野生株SC5314相近。意外的是，在rep3/rep3 突變株、rep3/rep3::REP3突變補救株及野生株SC5314中CDR1 的mRNA量相近。再者，在藥物誘導下，另一個距REP3基因下游處266 個核酸的orf19.3926表現量在rep3/rep3::REP3 突變補救株中比rep3/rep3 突變株及野生株提高了3.5倍。即使利用SAT1的策略再次重新建構rep3/rep3::REP3突變補救株，以減少額外載體DNA序列介入rep3/rep3 突變株中可能造成的干擾，仍然無法補救rep3/rep3 突變株對藥物敏感的表現型。由於在rep3/rep3 突變株中，CDR1 之mRNA並未下降，因此，對於REP3 是否透過其他非調控CDR1的路徑而影響白色念珠菌的藥物抗藥性，以及rep3突變株的功能難以補救回復的原因，將來需做更進一步的研究。

Candida albicans is an opportunistic fungal pathogen and is the most common cause of deep mycoses in humans. Azole therapy is commonly used to treat C. albicans infections. However the widespread use of azoles led to an increased frequency of treatment failure due to azole-resistant C. albicans in clinical setting. Therefore, understanding the molecular mechanisms of drug resistance in C. albicans is important to render the fungal infection. Over expression of drug efflux pumps— CDR1 and CDR2 (Candida Drug Resistance 1 and 2) in azole resistant C. albicans is commonly observed but the regulatory mechanism is poorly understood. Previously in the laboratory, REP3 (Regulator of Efflux Pump) was isolated from Candida genomic library due to its ability to increase the □-galactosidase activity of CDR1YM990348 promoter-lacZ about four folds in Saccharomyces cerevisiae in the presence of miconazole. According to the results of the drug susceptibility tests, rep3/rep3 homozygous mutant seems to be more susceptible to miconazole, itraconazole, ketoconazole, fiuconazole and voriconazole than the SC5314 wild-type strain in spite of rep3/rep3::REP3 rescued strains not showing a restoration of drug susceptibility phenotype. In this study, REP3 rescued strains were re-constructed and the outcome remained the same. No REP3 mRNA could be detected in rep3/rep3 mutant and the real-time PCR results showed that the REP3 mRNA could be detected in the rep3/rep3::REP3 rescued strains in similar quantity as that of the SC5314 wild-type strain. Surprisingly, the expressions of CDR1 were similar in the rep3/rep3 mutants, rep3/rep3::REP3 rescued strains, and SC5314 wild type strain. Furthermore, the expression level of the orf19.3926, which locates at the down stream 266 bps from REP3, increased about 3.5 folds in the REP3 rescued strain than that of the wild-type strains and rep3/rep3 mutants in the presence of miconazole. Even though the REP3 rescued strains were reconstructed through SAT1 flipping method to eliminate the potential interference from the integration of vector DNA fragment in C. albicans, the regenerated rep3/rep3::REP3 rescued strains still cannot restore completely the drug susceptibility phenotype of rep3/rep3 mutant. Since the mRNA level of CDR1 in rep3/rep3 mutant was the same as that in the wild type. REP3 may be involved in drug resistance through pathways other than CDR1 in C. albicans. There is no clear reason as the why the rep3/rep3::REP3 rescued strain could not restore the REP3 phenotype. Both will require further studies.