探討剔除白色念珠菌 CaORF19.7310 和 CaORF19.173 對白色念珠菌型態變化之影響

Abstract

白色念珠菌是目前人類真菌感染中，最常被分離出來的伺機性致病菌。在免疫健全的個體，能夠造成輕微的感染，但在免疫不全的個體或是體內正常菌叢被破壞時，卻可以造成嚴重的系統性感染甚至死亡。白色念珠菌細胞型態可由酵母菌型轉變為絲狀形式，其中絲狀形式又包含菌絲型與假菌絲型態；這樣的型態轉換，主要是由轉錄因子 Cph1p 和Efg1p 進行調控。在白色念珠菌 cph1/cph1 efg1/efg1 雙剔除株，實驗室培養條件下無法生成菌絲型態並且造成在小鼠中失去致病的能力。顯示白色念珠菌的致病機轉與其型態變化能力之間的重要性。

本文為探討受 Cph1p 或 Efg1p 調控的兩個基因 CaORF19.7310 和 CaORF19.173 對於白色念珠菌型態轉變扮演的角色。實驗策略是利用藥物篩選標記 SAT1 flipper cassette 剔除白色念珠菌目標基因，並以南方點墨法確認單套基因剔除株、雙套基因剔除株和單套基因回復株的構築，再進行性狀分析。在生長曲線、芽管試驗、誘發菌絲生成觀察和侵犯力測試實驗中，Caorf19.7310 和 Caorf19.173 各突變株表現的性狀與野生株 SC5315 皆相同，顯示在上述實驗條件並不會影響菌絲的表現。

Candida albicans is the most frequently isolated opportunistic pathogenic fungus in humans. It causes from minor infections in immunocompetent individuals to life-threatening systemic infections in immunocompromised patients or when the host’s endogenous flora be disturbed. Candida albicans can switch from a yeast form into filamentous forms, including hyphae and pseudohyphae. It is known that the morphological transition is regulated by the transcription factor Cph1p and Efg1p. A cph1/cph1 efg1/efg1 double mutant is defective in filamentous growth in laboratory conditions and is avirluent in a mouse model of infection. Thus, the virulence is related to the morphogenesis in Candida albicans.

In this study, I attempted to characterize the functions of ORF19.7310 and ORF19.173, of which the expressions were affected by transcriptional factors Efg1p and/or Cph1p in Candida albicans. I used the SAT1 flipper, containing a drug resistance marker to disrupt both alleles of these genes to produce knock-out mutants of Candida albicans. The genomic constructions of the heterozygous, homozygous knockout mutants and a rescued strain were confirmed by Southern blotting. However, there is no significant difference among the wild-type and the mutants in various analyses, including growth curve, germ tube assay, colony morphology and invasion assay. The results indicated that both the Caorf19.7310 and Caorf19.173 null mutants did not affect the morphology in Candida albicans in these growth conditions.