MET5 同源基因為隱球菌生長、有性生殖及致病性所必須

Abstract

常見的含硫胺基酸包括半胱胺酸（cysteine）與甲硫胺酸（methionine），此 二者對於生物細胞的正常生理活動有重要貢獻。硫酸鹽同化途徑（sulfate assimilation pathway，SAP），包含有一系列的還原步驟，將無機氧化態硫素進行 同化，並合成含硫胺基酸。在真菌界的各物種中，SAP 的主要步驟相當類似，參 與其中基因的蛋白質序列，也有很高的保守性。以啤酒酵母菌（Saccharomyces cerevisiae）為例，環境中的硫酸鹽類被 Sul1p 與 Sul2p（sulfate transporter）運送 至細胞內，經 Met3p（ATP sulfurylase）催化成 5’-adenylylsulfate（APS）。接 著，經 Met14p（APS kinase）作用，加上一個磷酸根，後成為 3’-phospho-5’- adenylylsulfate （ PAPS ） ， 再經 Met16p （ PAPS reductase ） 轉為亞硫酸鹽 （sulfite）。最後，由 Met10p 與 Met5p（sulfite reductase α and β subunit）組成的 酵素複合體，將亞硫酸鹽還原成負二價硫離子（sulfide），硫離子即可繼續作為 合成含硫胺基酸或其他含硫化合物之用。本研究之目的，為探討 MET5 基因於隱 球菌之生理角色，首先利用 in vitro transposition 反應製備得突變載體，再以基因 槍轉殖技術，將其送入隱球菌野生菌株內。經南方雜合分析及細胞內硫離子含量 測定，確認隱球菌 met5 突變株。met5 突變株造成半胱胺酸營養缺陷型、生長速 率緩慢、生殖菌絲減少、無黑色素形成，以及於替代性昆蟲宿主體內之致病力大 幅下降等情形。上述 met5 突變株的缺陷，在重新轉殖 MET5 基因至突變株後， 回復至野生株之性狀。本研究之結果與前人研究一致，證實硫酸鹽同化途徑，對 於隱球菌的生理作用有極大重要性，後續之深入研究，有潛力發展出抗真菌藥劑 之可能標的。

Sulfur-containing amino acids such as cysteine and methionine are important for cellular physiology. Sulfate assimilation pathway (SAP) is a reduction sequence involved in the biosynthesis of these amino acids from the inorganic oxidized sulfur source. In Fungi, sequential steps of SAP are similar, and enzymes involved in this pathway are also highly conserved. In Saccharomyces cerevisiae, exogenous sulfate is transported from the environment into yeast cells by sulfate transporter, Sul1p and Sul2p, and catalyzed by ATP sulfurylase, Met3p, to form 5’-adenylylsulfate (APS). APS is subsequently phosphorylated by Met14p, APS kinase, to produce 3’-phospho- 5’-adenylylsulfate (PAPS), and then reduced by Met16p (PAPS reductase) to generate sulfite. Finally, the sulfite reductase enzyme complex consisted of α subunit Met10p and β subunit Met5p further reduces sulfite to sulfide. The reduced sulfur ion can then be incorporated into sulfur-containing amino acids or compounds. In this report, we study the roles of the MET5 homologue in the human fungal pathogen Cryptococcus neoformans. The gene disruption construct was created by in vitro transposition and delivered into the wild-type strain by biolistic transformation. Gene disrupted mutants were verified and characterized. C. neoformans met5 mutants were auxotrophic for cysteine, reduced the growth rate, severely attenuated for mating differentiation, failed to produce melanin in vitro, and lost virulence in the alternative insect host model. All the defects were reverted to the wild-type by reintroduction of the intact copy MET5 gene. Consistent with previous reports, our results showed that the components of SAP play important roles in the physiological processes of C. neoformans and maybe serve as potential targets for antifungal therapy.