剖析MAP kinase Cek1調控網絡對於白色念珠球菌型態變化、費洛蒙反應以及致病力的作用

Dissecting the roles of MAP kinase Cek1 regulatory network in phenotypic transitions, pheromone responses and pathogenicity in Candida albicans

鄧福勝(Fu-Sheng Deng)

指導教授 ： 林晉玄

國立臺灣大學/生命科學院/生化科技學系/博士(2020年)

https://doi.org/10.6342/NTU202001903

摘要

MAPK路徑為真核細胞中廣泛存在且具重要性的訊息傳遞路徑，透過磷酸化方式導入外部訊息，並藉以活化特定反應或基因。在本篇研究中，我們證實白色念珠菌磷酸水解酶Cpp1可作為MAP激酶Hog1與Cek1之間連接橋樑，以調控菌體在不同環境刺激下的型態變化以及細胞反應。根據qPCR結果，CPP1基因在hog1∆突變株中的表現量相較於野生株有顯著下降的情形；Western blotting結果亦顯示Cek1激酶蛋白的磷酸化在hog1∆以及cpp1∆突變株中皆有明顯地提升，說明Cpp1作為Cek1磷酸化的抑制子可受Hog1調控基因表現並藉以控制Cek1蛋白磷酸活性。透過進一步的型態測試發現，CPP1基因的剔除可顯著提升菌體white-to-opaque轉換比率、誘導菌絲生成、以及異常地改變費洛蒙誘導的反應型態，而這些的現象亦會隨著CEK1基因的剔除而消失。目前已知Cek1激酶活化為白色念珠菌活化費洛蒙吸附反應的必要因子，本篇研究亦有興趣尋找Cek1下游受轉錄因子Cph1所調節費洛蒙吸附反應的關鍵基因。透過基因轉錄的比較分析，我們系統性地篩選出至少四個受費洛蒙誘導表現的未知基因。利用對突變株的型態測試，我們發現ORF19.1539、ORF19.1725、ORF19.2430以及ORF19.5557，其基因缺失大幅降低白色念珠菌費洛蒙誘導的吸附力，但對於交配反應與交配效率並無造成明顯影響。說明white型態的白色念珠菌在費洛蒙誘導下，可利用Cek1活化下游特定的white-specific基因以啟動菌體吸附反應。此外，qPCR分析亦發現，ORF19.1725基因表現同時受轉錄因子Cph1與Tec1所調控，且其基因缺失對於菌體的生物膜、菌絲生成以及致病力皆有嚴重的影響。這些結果顯示此基因對於白色念珠菌的致病相關機制十分重要。綜合本篇研究的結果，我們對於白色念珠菌MAPK間訊息調控以及下游牽涉的重要基因功能皆有突破性的了解，期許這些成果未來可進一步發展作為臨床上抗真菌藥物開發的重要契機。

關鍵字

白色念珠菌 ； MAPK路徑 ； Cek1激酶 ； Hog1激酶 ； Cpp1磷酸水解酶 ； 費洛蒙反應

並列摘要

MAPK (Mitogen-activated protein kinase) cascades are prevalent in nature and are important signaling transduction pathways in Candida albicans. They can be stimulated by external signals through phosphorylation and subsequently activate many response genes. In this study, we demonstrated that a dual-specificity phosphatase Cpp1 serves as a bridge between MAP kinases Cek1 and Hog1. Quantitative PCR revealed that expression of the CPP1 gene was significantly reduced around 30% in hog1∆. In addition, western blotting showed an obvious elevation of Cek1p phosphorylation levels in cpp1∆ and hog1∆ mutants. Further investigation demonstrated that cpp1∆ and hog1∆ showed similar morphology in Lee’s GlcNAc medium on plates, in which both mutant strains exhibited 100% white-to-opaque switching and hyphae formation. These phenotypes disappeared after deletion of the CEK1 gene. The results indicate that Cpp1 is an inhibitor of Cek1p and its gene expression is regulated by Hog1p. We further found that deletion of the CPP1 gene in C. albicans also resulted in abnormal phenotypes during pheromone treatment. Given that Cek1 activity is essential for C. albicans pheromone responses, we found that cpp1∆, compared to the wild-type, exhibited an abnormal elevation in the length of mating projections and the numbers of adherent cells. Through comparative transcriptomic analysis, we identified four target genes that were significantly upregulated in C. albicans white cells during pheromone treatment. Subsequently, we validated Orf19.1539, Orf19.1725, Orf19.2430, and Orf19.5557 as novel factors affecting pheromone-stimulated adhesion in white cells but not mating efficiency in opaque cells, indicating that these genes contribute to the phase- and pheromone-specific responses in C. albicans white cells. Furthermore, ORF19.1725, which encodes an adhesin-like protein, is co-regulated by both transcription factors Cph1 and Tec1. Similar to tec1∆, ORF19.1725 gene deletion significantly inhibited hyphae development, biofilm formation, cell adhesion and pathogenicity in C. albicans. Taken together, we established a model to explain how C. albicans accurately mediates Cek1 activation to regulate its morphogenesis and pheromone responses. Furthermore, our research provides a potential target for new anti-Candida drug development in the future, given the important role of ORF19.1725 in virulence-associated function.

並列關鍵字

C. albicans ； MAP kinase ； Cek1 ； Hog1 ； Cpp1 ； pheromone responses

參考文獻

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