白色念珠菌組蛋白 H3 第四離胺酸去甲基酶 Jhd2 的功能探討

白色念珠菌 (Candida albicans) 為多型態的伺機性真菌，當個體免疫力低落或生理狀況劇烈改變時能伺機性感染宿主，一些嚴重的院內感染也使得病人康復的時間延後，影響醫療品質及投入更多代價。目前抗真菌藥物對人體的副作用與抗藥性的問題，使新藥物的持續開發仍有需求，近年來發展出小分子的抗真菌的標靶藥物顯示全面而深入了解白色念珠菌致病分子機制可供持續開發更多具專一性的標靶藥物。白色念珠菌的多型態生長特性以及形成生物膜的能力影響其致病力及抗藥能力，而生物膜相關的特性能幫助牢固群體，白色念珠菌藉此附著於各介質以及散布，其細胞表面特性 (adhesion) 具有廣大適應性能適應各種介質，白色念珠菌受環境刺激後透過何種途徑能影響到細胞表面特性目前仍不清楚。目前已知CaCDC4 為菌絲型態的負調控者，由於其泛素化蛋白質的角色，實驗室已利用蛋白質親和力純化法找出可能與CaCdc4有交互作用的相關性蛋白質，CaORF19.5651(CaJHD2) 為其中之一，Jhd2 (JmjC domain-containg demetylase 2)於啤酒酵母菌(Saccharomyces ceravisiae)及其他物種中為組蛋白 H3K4 demethylase，CaJhd2 蛋白質序列具高度保留性，功能上也可能與其他物種相同，而已知白色念珠菌 H3K4 的甲基修飾可以影響其在特殊環境下的菌絲生長能力以及 adhesion能力，透過本研究探討 CaJhd2 的蛋白質功能以及觀察 CaJHD2 的缺失對白色念珠菌的影響。目前白色念珠菌組蛋白H3 K4 修飾的研究不多，也未曾有白色念珠菌 H3K4 去甲基酶(demethylase) 被報導過，藉本研究了解 CaJhd2 為白色念珠菌 H3K4 mono-demethylase，而是否扮演 H3K4 di-,tri-demethylase 的角色仍未知，白色念珠菌 CaJHD2 的剔除株 jhd2Δ/Δ 在含mannitol 及phosphate 的Spider 環境的誘導中觀察到細胞聚集現象的增加，可能對細胞與細胞或其它非生物介質的adhesion能力有關，雖然其對生物膜形成能力的影響仍無法下定論。

關鍵字

白色念珠菌；組蛋白修飾；組蛋白去甲基酶

並列摘要

Candida albicans is an opportunistic human fungal pathogen. Invading and damaging host tissues when the equilibrium of individual environment is disturbed. Nosocomial infection prolong the discharge time of patients, which increase personal and economic cost. The harmful side effect to human cell and resistance to antifungal drugs have made continuous development of new antifungal drugs necessary. Recent progress in developing small molecules to fungal cellular targets suggests that comprehensive understanding the molecular mechanism underlying Candida albicans virulence is essential to continunous exploration of drugs specific to cellular targets of Candida albicans. The transition of C. albicans among diverse morphological states and ability to form biofilm are the key determinants of its ability to cause systemic candidiasis and resistance to antifungal drugs. The biofilm structure prevents cells from being washed away and support the spread of cells. C. albicans exploits their capacity to adhere to many surfaces. However, it is remain incompletely understood as to how the environment cues are intertwined with genetic factors determines the alteration of adhesion properties. Our previous study reveals that CaCdc4, functioning through the E3 ubiquitin ligase, participates in the repression in filamentous growth of C. albicans. We have identified CaORF19.5651(CaJHD2) as one of the CaCdc4 associated proteins by affinity purification. Jhd2 is histone H3 lysine 4 (H3K4) demethylase in Saccharomyces cerevisiae and other species. CaJhd2 possesses a highly conserved JmjC domain-containg demetylase 2 that is known to require for demethylation of H3K4. It is known that the H3K4 methylation contribute to the capacity of adhesion and morphogenesis in C. albicans. As a result, I aim to investigate the H3K4 demethylase activity of CaJhd2 and the functional consequence of CaJHD2 deficiency. The demethylase of H3K4 of C. albicans has not been verified to date, and the H3K4 methylated modification remains poorly known. To investigate whether the CaJhd2 is a demethylase specifically for H3K4, I constructed a homozygous jhd2 null mutant jhd2Δ/Δ using wild-type strain SC5314 and have examined the mono-, di-, and trimethylation levels of H3K4 of the mutant in comparison with those of the wild-type strain. I confirmed that the CaJhd2 is a H3K4 mono-demethylase of C. albicans but the di-, tri- demethylase activity specifically for H3K4 remains undetermined. In addition, I observed increase of cellular flocculation of jhd2Δ/Δ in relation in spider medium that contains mannitol and phosphate. This suggests that the deficiency of CaJHD2 may be involved in adhesion of cell-to-cell and cell to nonbiotic materials, although its effect to the ability of biofilm formation in C. albicans remains inconclusive.

並列關鍵字

Candida albicans ； H3K4 demethylase ； histone modification

參考文獻

Aoki, W., Kitahara, N., Miura, N., Morisaka, H., Kuroda, K., and Ueda, M. (2012). Profiling of adhesive properties of the agglutinin-like sequence (ALS) protein family, a virulent attribute of Candida albicans. FEMS Immunol Med Microbiol 65, 121-124.

Arnaud, M.B., Costanzo, M.C., Skrzypek, M.S., Binkley, G., Lane, C., Miyasato, S.R., and Sherlock, G. (2005). The Candida Genome Database (CGD), a community resource for Candida albicans gene and protein information. Nucleic Acids Res 33, D358-363.

Barchiesi, F., Schimizzi, A.M., Caselli, F., Novelli, A., Fallani, S., Giannini, D., Arzeni, D., Di Cesare, S., Di Francesco, L.F., Fortuna, M., et al. (2000). Interactions between triazoles and amphotericin B against Cryptococcus neoformans. Antimicrob Agents Chemother 44, 2435-2441.

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白色念珠菌組蛋白 H3 第四離胺酸去甲基酶 Jhd2 的功能探討

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