白色念珠菌CaGPM1醣解酵素基因對細胞型態及抗藥性之影響

The involvement of the glycolytic pathway gene, CaGPM1, in the morphology and drug resistance in Candida albicans

李淑萍(Shu-Ping Li)

指導教授 ： 楊昀良

國立交通大學/生物科技學院/生物科技系所/碩士(2009年)

https://doi.org/10.6842/NCTU.2009.00881

摘要

白色念珠菌為人類重要的伺機性病源菌。先前本實驗室利用抑制刪除雜交法 ( Suppression Subtractive Hybridization , SSH ) 經過定序後的分析比對，得到在野生株 ( SC5314 ) 與失去致病力的efg1/efg1 cph1/cph1 雙基因突變株 ( HLC54 ) 兩者之間表現量有差異的基因。其中有一個是醣解作用中的基因CaGPM1。利用北方墨點法分析，結果顯示CaGPM1基因在efg1/efg1 cph1/cph1雙突變株的表現量大於野生株。此外，在篩選影響抗藥性表現的基因時，發現CaGPM1的表現受CaNDT80之影響，因此本實驗研究目標想要了解醣解酵素基因 CaGPM1與細胞型態及抗藥性是否有關系。
首先利用同源重組置換方式將CaGPM1基因剔除並且將CaGPM1單套基因重新置入Cagpm1/Cagpm1雙套基因破壞株進行基因補救，接著進行這些突變株之特性描述，分析其與型態及抗藥性，結果顯示於Etest試驗Cagpm1/Cagpm1雙套基因破壞株中對於caspofungin、amphotericin B兩種藥物的敏感具有提高現象，Cagpm1/Cagpm1雙套基因破壞株影響初期的芽管形成，長菌絲型態介於野生株SC5314與失去致病力HLC54之間，Cagpm1/Cagpm1雙套基因破壞株在固態YPGE/uridine 培基上，細胞菌落小，細胞生長緩慢，因此證實CaGPM1與藥物敏感性、型態變化有所相關並且會影響細胞生長。

關鍵字

白色念珠菌 ； CaGPM1

並列摘要

Candida albicans is an important pathogen of humans. It has been reported that the cph1/cph1 efg1/efg1 double mutant strain 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. Genes isolated from these experiments may be involved in virulence and / or filamentous growth. Among them, there was a glycolytic pathway gene, CaGPM1. The result of northern analysis showed that CaGPM1 expressed at higher level in cph1/cph1 efg1/efg1 double mutant strain than that in the wide-type strain. In addition, it is found that CaNDT80 influenced the expression of CaGPM1. The purpose of present study was to elucidate the involvement of the CaGPM1 gene on drug resistance and morphogenesis.
I have constructed a Cagpm1/Cagpm1 null strain by gene replacement using ARG4 and URA3 as the selection markers to obtain homozygous knockout strains. The homozygous knockout strain was then rescued with single copy of the wild-type CaGPM1 gene. The phenotypes of mutant strains were characterized and analyzed for the association with morphology and drug resistance. I found that in Etest, Cagpm1/Cagpm1 homozygous mutants seemed to be more susceptible to caspofungin and amphotericin B. Cagpm1/Cagpm1 strains showed significant alteration on germ tube formation and hyphal growth when compared to the wild type and cph1/cph1 efg1/efg1 double mutant strain. Disruption of the CaGPM1 gene has dramatic effects on growth rate of the strain by growth curve analysis. Besides, on YPGE/uridine agar, the colony of Cagpm1/Cagpm1 mutant strain is smaller than the other strains. According to these results, it was suggested that Gpm1p may be involved in the drug resistance, morphogenesis and cell growth.

並列關鍵字

candida albicans ； CaGPM1

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