探討基因FLO11在台灣之Saccharomyces cerevisiae臨床分離菌株中對Mat形成的影響

Abstract

生物膜的形成機制中第一步就是菌體黏附在一表面上，而生物膜的形成常伴隨著抗藥性的產生，因此於臨床上，研究如何阻擋微生物的黏附性以及抗藥性成為臨床上重要的議題。Saccharomyces cerevisiae能形成生物膜的初始型態結構mats，其黏附上塑膠表面能力的重要因子是FLO11基因，過去針對實驗室菌種∑1278b中FLO11基因的研究，可分為二個方向： FLO11的調控機制及FLO11的中央串聯重覆序列如何影響其表現型。因此於本論文中，我們的研究目標是1. 藉由比較實驗室菌種∑1278b與研究臨床菌種的mats表現型及其FLO11的表現，找出FLO11的可能調控機制，2. 針對FLO11基因型與mats表現型兩者之間的關係作分析，3. Mats表現型與抗黴菌藥物抗藥性的關係。 我們先從三株台大臨床分離株S. cerevisiae中找到能產生mats典型表現型的菌株，並分析其15組四分孢子的mats表現型，指出mats表現型為數量性狀 (quantitative trait)，可能受多基因調控，並且找到一種被我們稱做“hyper mats”的特殊表現型。在黏附能力試驗中，結果顯示細胞的黏附能力與mats的形成能力有正相關，然而不同表現型之間的黏附能力的差異在此試驗中則不明顯。我們利用北方點墨法分析各菌株間的FLO11表現，發現FLO11於對數生長期的表現對於mats的形成是必須的，然而其量的變化與mats的表現型並沒有太大關係。為了分析∑1278b與YYC1間FLO11表現量的差異，我們對FLO11的基因的3.1 kb 啟動子作定序，但並沒有找到有意義的突變點。另一方面，我們針對FLO11的中央串聯重覆序列研究，證實此段序列於YYC1中比∑1278b縮短了約1 kb 大小，指出YYC1與∑1278b的mats表現型差異源由可能在此。此外，我們對此三株台大臨床分離株作抗藥性篩檢，然而就目前篩檢的菌種中，我們並未能找出mats的形成與抗藥性有直接關係。 總括來說，在本研究中，我們的研究結果指出mats的形成依靠FLO11的表現，然而其表現量與mats的表現型沒有太大的關係，真正影響mats表現型的因子，可能是FLO11的中央串連序列的結構所導致的Flo11p多樣性。而抗黴菌藥物的測試，我們發現S. cerevisiae臨床分離株中，生物膜的形成與抗藥性之間並無直接的關聯性，未來可朝著mats的生成對整體菌落而言是否能對抗黴菌藥物產生抗藥性去研究。

The first step of biofilm formation is surface attachment for microbes, and the consequence of biofilm formation is highly correlated to drug resistance. That is why many studies focus on how to prevent microbes’ adhesion and drug resistance. Saccharomyces cerevisiae cells form the initial structure of biofilm called mats which is dependent on FLO11gene. The previous studies on FLO11 have been focused on lab strain ∑1278b in two directions: the regulation of FLO11 and how the FLO11 phenotype is affected by its central tandem repeat. In this study, in order to find out the regulation of FLO11, first, mats phenotypes and the expression levels of FLO11 were compared in clinical isolates of S. cerevisiae with lab strain ∑1278b. Second, we wanted to find out the relationship between FLO11 genotypes and mats phenotypes. Third, we want to study about the relationship between mats formation and antifungal drug resistance. We found that one of our National Taiwan Hospital clinical isolates, named YYC1, could form typical mats phenotype. After further screening the mats phenotypes in YYC1 segregants from 15 tetrads, the result indicated that mats phenotype might be a quantitative trait. We also found a special phenotype called “hyper mats” in our study. In the 96-well adherence assay, we found that the adhesion ability of strains was positively related to mat formation. Nevertheless, this assay might not be sensitive enough to distinguish the differences between the typical mats phenotype and “hyper mats.” The results of Northern blot analysis showed that the expression of FLO11 in log phase cells is important for mat formation. However, the change of FLO11 expression levels does not seem to relate to different mats phenotypes. In order to analyze the different expression levels of FLO11 between YYC1 and ∑1278b, we sequenced 3.1 kb of the FLO11 promoter regions in both strains. However, we could not find out any significant change in FLO11 promoter of YYC1 compared to ∑1278b. On the other hand, we surprisingly found that the length of the tandem repeats in FLO11 coding regions in YYC1 was about 1 kb shorter than that in ∑1278b. These results indicated that the different mats phenotypes between YYC1 and ∑1278b might be due to the length of this tandem repeat sequences. In addition, we have screened the antifungal drug resistance on our S. cerevisiae clinical isolates. However, we could not find out the direct relationship between mat formation and antifungal drug resistance in S. cerevisiae. In conclusion, in our study, we found that mat formation is dependent on FLO11 expression, although higher expression levels of FLO11 did not seem to relate to “hyper mats” phenotype. Our data also inferred that central tandem repeat sequences of FLO11 perhaps affect mats phenotypes in strains we analyzed. We found that the antifungal drug resistance might not directly correlate with mats formation in S. cerevisiae.