探討台灣臨床分離的菌株Saccharomyces cerevisiae重要的致病因子之一–假菌絲的形成

Abstract

Saccharomyces cerevisiae現在被認為是一個造成伺機性感染的致病原，有研究指出，溫度的抗性及假菌絲的分化是臨床分離菌株的兩個重要致病因子。而S.cerevisiae在缺乏氮源的情況下，會由酵母菌型轉成假菌絲型，至於細胞形態的轉換和菌株致病性之間的關係，目前仍不清楚。我們發現台大附設醫院收集的臨床分離株YYC1, 2及3中，YYC1和3都能在42℃生長，並且在37℃都能形成假菌絲，而YYC2則不具有。我們想利用定序和假菌絲形成相關性的基因如：TEC1、 FLO8、 SSD1，藉以了解基因型和表現型之間的關係。藉由定序的結果和SGD的s288c基因體序列相比，在TEC1中YYC1, 2, 3皆少於2個SNP(single nucleotide polymorphism)；在FLO8最多則有37個SNP；在SSD1中最多也有33個SNP。由定序的結果發現在FLO8其YYC1和3所擁有的SNP異於YYC2，且在此基因上具有異合型，因此我們利用四分孢子分離的技術取得YYC1子代，其假菌絲的生成在15對tetrad中有12對具有2:2 segregation (有兩個spores可形成假菌絲；另外兩個spores沒有) 現象，因此代表有一個扮演關鍵性角色的基因來調控假菌絲的形成，我們分析了6個具有2:2 segregation 現象的tetrad ( 24 segregants )，定序FLO8基因814、2026、2158、2263上的四個位點，發現在此四個位置上分別為A、T、A、T 時對於假菌絲的形成有3/4高比例的正向幫助，反之如為G、C、G、A 時則有負向作用，並且利用基因選殖的方式加以證實。過去針對毒性的測試所做的方法為老鼠活體實驗，但因活體實驗費時，所以我們利用一個簡單又快速就可測知毒性的方法，其做法為利用酵母菌感染小鼠的巨噬細胞，而觀察巨噬細胞釋放的細胞激素，如:TNF-α。藉由我們的結果發現細胞激素的誘導量在已知具有毒性的菌株和不具毒性的菌株相比有兩倍的差異，因此菌株的毒性與細胞激素的表現量上似乎有一個正相關，而YYC1的結果類似於具有毒性的菌株，至於YYC2和3的結果類似於不具有毒性的菌株，因此此方法可做為一個初步篩選毒性的方法。

Saccharomyces cerevisiae is an emerging opportunistic pathogen. Previous work has shown that American clinical isolates exhibit the ability to grow at 42°C and are capable of pseudohyphal formation at 37°C. S. cerevisiae cells grow in low-ammonium solid media mediating a switch from the yeast to the filamentous form. However, morphology switching is not clear about virulence mechanism. We collected three clinical yeast isolates (YYC1, 2, 3) from National Taiwan University Hospital and found that YYC1 and 3 grow at 42°C and are capable of pseudohyphal differentiation at 37°C, whereas YYC2 could not. We analyzed the genes involved in pseudohyphal formation included TEC1, FLO8 and SSD1. Sequence analysis comparing YYC1, 2, 3 to s288c from Saccharomyces Genome Database showed that YYC1, 2 and 3 have 2 single nucleotide polymorphism(SNPs) in TEC1, at most 37 SNPs in FLO8 gene. And at most 33 SNPs in SSD1 gene. FLO8 in YYC2 are different from those in YYC1 and 3. These three clinical isolates all appeared to be heterozygous in FLO8. We used tetrad dissection to separate the segregants of YYC1 and found that 80 percent of the tetrad analyzed showed 2:2 meiotic segregation for the ability to form pseudohyphae at 37°C. We analyzed six tetrad at nucleotide 814, 2026, 2158 and 2263 sites in FLO8 gene, and found that when the haplotype is A, T, A and T on each site respectively, the results of pseudohyphal formation are positive. The FLO8 alleles in YYC1 segregants were cloned and verified in the strain of s288c background. We developed a simple and rapid method for virulence determination because mouse model is relatively complex. The production of cytokine TNF-α was seen when murine macrophage cell lines were stimulated by the yeast cells. Our result suggested that yeast cells with high virulence indeed stimulated higher cytokine levels in macrophages. YYC1 resembled strains with high virulence, while YYC2 and YYC3 resembled avirulent.