探討剔除白色念珠菌ENG1之影響

Effect of ENG1 null mutations in Candida albicans

蔡馨儀(Hsin-Yi Tsai)

指導教授 ： 楊昀良

國立交通大學/生物科技學院/分子醫學與生物工程研究所/碩士(2009年)

https://doi.org/10.6842/NCTU.2009.00869

摘要

白色念珠菌為人類常見之共生菌，也是一種伺機性病原菌。白色念珠菌之致病機轉與其型態變化的能力是相關的；當失去菌絲型和酵母菌型之型態互換的能力時，此白色念珠菌也失去造成毒性的能力。β-glucan是真菌細胞壁中最主要的成分，研究指出白色念珠菌細胞壁的β-glucan與調節宿主免疫機轉相關。白色念珠菌之CaENG1可轉譯出β-1,3-glucan內切，此基因與啤酒酵母菌之ENG1有很高的同源性。因此本文為探討CaENG1對於白色念珠菌型態轉變扮演之角色。實驗策略以抗藥性篩選標記SAT1 flipper剔除白色念珠菌雙套CaENG1，再以南方點墨法和RNA表現量確認建構之單套CaENG1突變株、雙套Caeng1突變株以及補償單套CaENG1之補償株；檢驗符合預期之突變株進行下一步性狀分析。然而至目前所進行的實驗，在生長曲線測定、芽管試驗、誘發菌絲生長觀察型態變化和侵犯力試驗中，Caeng1雙套突變株與野生株相比，性狀上沒有明顯的差異，指出以目前的結果分析，CaENG1並不會影響白色念珠菌型態轉變之過程；在化學藥品感受性實驗中也發現Caeng1雙套突變株生長表現也與野生株無明顯差異；在細胞分裂時，觀察到Caeng1雙套突變株有少數細胞分裂不完全，形成四、五顆細胞聚集的現象。然而，將Caeng1雙套突變株從a/α基因型誘發為a/a之a-type型態之菌株，可發現在添加血清誘發菌絲生長時，a-type型態的Caeng1雙套突變株有3/5機率，其菌絲生長能力下降。而當中一a-type型態的Caeng1雙套突變株對於0.1％ SDS有高度敏感性，推測此突變株之細胞壁組成有缺陷，進而提高對SDS的感受性。

關鍵字

白色念珠菌 ； 細胞壁 ； ENG1 ； 1,3-beta-glucan ； 化學藥品感受性 ； 染色體缺失

並列摘要

Candida albicans is a commensal fungus and a major opportunistic pathogen. The pathogenicity of C. albicans is related to its ability to switch between yeast and filamentous forms and cells defective in the switching is avirulent. β-glucan is a major structural component of fungal cell wall and several studies have suggested that the β-glucan of C. albicans has immunomodulatory effects on the host defense system. CaENG1 shares homology with Saccharomyces cerevisiae ENG1, which encodes an endo-1,3-β-glucanase. Hence, it is interested to know the role of CaENG1 in the morphogenesis and virulence. I used the SAT1 flipper, a drug resistance marker to disrupt CaENG1 in C. albicans. The genomic constructions of the heterozygous, homozygous knock-out mutants and a rescued strain have been confirmed by Southern blot and RNA expression analysis. However, there is no significant difference between the wild-type strain and the Caeng1 null mutant strains in all the morphology analyses conducted, including growth rate, germ tube assay, colony morphology observation and invasion assay. These results indicate that CaENG1 is not involved directly in hyphal morphogenesis in C. albicans under experimental conditions. In various chemical susceptibility tests, the null mutants displayed the same phenotypes as the wild type. I also analyzed the cell morphology of the null mutant cells, and few could form small clusters of cells that were defective in completing cellular separation. However, after the Caeng1 null mutants were induced to form a-type cells from initial heterozygous a/α type, in 3/5 chance the a-type Caeng1 null mutants have reduced hyphal formation in the presence of serum. An a-type Caeng1 null mutant, EKP2-3a, defective in hyphal growth was also hypersensitive to 0.1％ SDS. It is suggested that this mutant strain was defective in cell wall composition or organization to become more sensitive to SDS.

並列關鍵字

Candida albicans ； cell wall ； ENG1 ； 1,3-beta-glucan ； chemical susceptibility ； chromosome loss

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