白色念珠菌之致病力/型態變化及抗藥性可經由麥角固醇生合成及醣解酵素基因進行協同調控

Abstract

白色念珠菌是一種伺機性病原菌。在探討致病力的實驗中發現，EFG1及CPH1基因的雙突變株在in vitro實驗中會失去形成菌絲的能力，且對小鼠是不致病的，因而得知白色念珠菌的致病力與其在酵母菌型與菌絲型之間的型態轉變有關。先前本實驗室利用抑制刪除雜交法（SSH）篩選和致病力調控有關的基因時，得到麥角固醇生合成ERG3和ERG11基因。有趣的是，這兩個基因的已知功能是影響念珠菌對於藥物的敏感性。本篇論文之第一個研究目標：探究麥角固醇生合成基因是否和致病力及抗藥性途徑皆有關連。實驗策略以北方墨點法分析ERG3、ERG11基因表現量。結果顯示，ERG3和ERG11在efg1/efg1 cph1/cph1突變株 (HLC54)和efg1/efg1 CPH1/CPH1(HLC52)的mRNA表現量大於野生株(SC5314)。同時也發現白色念珠菌之致病因子Efg1會經由負向調控ERG3基因而改變對真菌藥物的敏感性。再者實驗室曾針對醣解酵素所有生合成途徑進行研究，以北方墨點法檢驗基因之表現，發現其中五個基因ENO1、TPI1、GPM1、PYK1、PGK1其mRNA表現量於酵母菌型菌體(cph1/cph1 efg1/efg1)中及長菌絲型菌體(CPH1/CPH1 EFG1/EFG1)中不同。因此，本篇論文之第二研究目標：經由北方墨點法瞭解此五個醣解酵素基因是否和致病力/型態變化及抗藥性途徑皆有關連。結果顯示，TPI1、GPM1、PYK1、PGK1之表現皆可同時受血清及藥物所影響。為進一步探討其機制，並且選擇TPI1以同源重組的方式將四環黴素調控表現系統置入其啟動子區域作基因功能之研究，目前已知，TPI1與型態變化、細胞生長有關，但和抗藥性途徑的關係仍有待探討。

Candida albicans is an opportunistic fungal pathogen in humans. It has been reported that the cph1/cph1 efg1/efg1 double mutant of C. albicans is defective in filamentous growth and is also avirulent in a mouse model. The ability of C. albicans to switch between a yeast form and a filamentous form has been implicated in its virulence. Previous comparative genomics studies have profiled the expression differences between the wide-type and the cph1/cph1 efg1/efg1 double mutant strains by the method of Suppression Subtractive Hybridization (SSH) and sequence analysis. Two ergosterol genes, ERG3 and ERG11, were isolated. Mutations in ERG3 or ERG11 can alter the susceptibility of antifungal drugs in Candida albicans. Therefore, the first aim of this study was to investigate the coordination of morphogenesis/ virulence and drug resistance via ergosterol biosynthesis genes. The mRNA expression levels of these two genes were assessed by northern blot analysis. The results shown that the mRNA levels of ERG3 and ERG11 were higher in the cph1/cph1 efg1/efg1 double mutant and CPH1/CPH1 efg1/efg1 mutant than that in the wild-type cells, suggesting a negative regulation of ERGs by EFG1. And it has been proven that in addition to virulence, Efg1 was also involved in drug resistance by negatively regulating ERG3 of the ergosterol biosynthesis pathway in C. albicans. Furthermore a previous study has shown that ENO1、TPI1、GPM1、PYK1and PGK1 of glycolytic genes expressed differently in cph1/cph1 efg1/efg1 double mutant and in wide-type strain. Thus, investigating the linkage of morphogenesis/virulence and drug resistance via glycolysis genes was the other purpose of this study. The results indicated that the expressions of TPI1, GPM1, PYK1 and PGK1 could be affected by the presence of goat serum and antifungal drug. Besides, TPI1 gene was subjected to genetic study by introduction of tetracycline-regulated system. These results indicated that TPI1 gene was associated with morphogenesis and also involved in cell growth. Whether TPI1 gene is involved in drug resistance will require further studies.