評估白色念珠菌組蛋白H3第四離胺酸去甲基酶JHD2在毒性相關的特徵

白色念珠菌是一種伺機性的致病性真菌，其多型性生長被認為與致病力相關。菌絲型態的生長對於侵入宿主及破壞組織扮演重要角色，含有各種型態菌體的生物膜也會增加致病力及抗藥能力。已知 CaCDC4 是 E3 泛素連接酶，可抑制生長成菌絲。在尋找 CaCDC4的相關蛋白時我們找到了 JHD2 (C.albicans orf19.5651)。雖然已證實 CaJHD2 與 CaCDC4 之間沒有直接交互作用關係。JHD2 在其他物種中為 H3K4 demethylase，而在白色念珠菌中的角色則未知。先前文獻已知在白色念珠菌中至少有兩個基因參與H3K4的調控，進而改變與毒性相關的型態。我們想要探討 JHD2 在白色念珠菌中是否也是扮演 H3K4 demethylase 角色，以及 CaJHD2 的缺失次否會造成外表型的改變。我們比較 SC5314 及 CaJHD2 突變株的生長速率、菌落型態、聚集能力、絮凝能力、生物膜形成能力以及藥物抗性。我們觀察到剔除或 rescue CaJHD2都不會影響生長速率。在菌絲誘導培養條件下，觀察菌落型態並將其定量，發現CaJHD2 剔除株與 Wild type 有明顯差異。利用 Spider medium 液態培養，發現 CaJHD2 剔除株聚集現象較Wild type 明顯，但觀察其 Flocculation 卻沒有差異。以生醫材料 Silicone 進行生物膜形成測試中，觀察到 CaJHD2 剔除株會提高生物膜的形成能力。而 CaJHD2 的缺失並不會影響抗藥性。接著我們以抗體偵測 CaJHD2 是否會影響 H3K4 位點上甲基化的程度，發現缺乏 CaJHD2 的菌株 H3K4 三甲基化程度會累積。由我們結果可得知，CaJHD2 的蛋白質角色可能為 H3K4 tri-demethylase，透過影響一群基因表現不同造成多種但相關的外表型改變。在菌絲誘導培養條件下，缺乏 CaJHD2 會造成各種細胞型態比例的不同而導致菌落型態的差異、增加細胞聚集的能力及增加生物膜形成能力。

關鍵字

白色念珠菌；組蛋白去甲基酶

並列摘要

Candida albicans is an opportunistic human fungal pathogen. It’s morphological plasticity amonst yeast, hyphae and true hyphae is known to associate with virulence. While yeast cells can distribute the whole body of host via blood stream, hyphal cell are important to invade tissues the host cause damage. Biofilm constituted with various cell types could increase virulence and drug resistance. Our previous study have shown that CaCDC4, encoding an E3 ubiquitin ligase, participates in the repression in filamentous growth of C. albicans. During search for interactors of CaCdc4, we found a protein encoding by JHD2 (C. albicans orf19.5651), but C. albicans Jhd2 protein (CaJhd2p) was later excluded to be directly interacted with CaCdc4. C. albicans JHD2 (CaJHD2) encodes a histone 3 lysine 4 (H3K4) demethylase found in many species but is unknown in Candida albicans. Previous reports have shown that at least two genes involved in regulation of H3K4 in C. albicans exhibit morphological alteration that is associated with virulence. Hence, wehave sought to determine if CaJhd2p acts as a H3K4 demethylase for virulence-associated phenotypes in Candida albicans. We made Cajhd2 homozygous null mutant and assessed in comparison with the wild-type strain the consequence of growth, colony morphology, ability in aggregation, flocculation, biofilm formation, and antifungal resistance. We found that cells lacking CaJHD2 were unaffected on growth. However, cells lacking CaJHD2 appeared to be different on colony morphology. In the hypha-inducing Spider medium, cells of the Cajhd2 homozygous null mutant exhibited greater ability to aggregate but did not similar extent in flocculation. In addition, cells lacking CaJHD2 increase their ability to biofilm assayed on the biocompatible silicon. Cells deficient in CaJHD2 appeared to be unaltered on antifungal drug brefeldin A (BFA) resistance. Antibodies specific to mono-, di-, tri-methylation of H3K4 revealed that cells lacking CaJHD2 accumulate tri-methyl on H3K4. We suggest that CaJHD2 may act as a H3K4 tri-demethylase, through which alters the expression of a set of C. albicans genes involved in adhesion of cells, morphological transition, and biofilm formation upon stimulation by filament-inducing agents.

並列關鍵字

Candida albicans ； H3K4 Demethylase ； CaJHD2

參考文獻

Atir-Lande A, Gildor T, Kornitzer D. (2005) Role for the SCFCDC4 ubiquitin ligase in Candida albicans morphogenesis. Mol Biol Cell. 16(6):2772-85.

Bauer J, Wendland J. (2007) Candida albicans Sfl1 suppresses flocculation and filamentation. Eukaryot Cell. 6(10):1736-44.

Bayly JC, Douglas LM, Pretorius IS, Bauer FF, Dranginis AM. (2005) Characteristics of Flo11-dependent flocculation in Saccharomyces cerevisiae. FEMS Yeast Res. 5(12):1151-6.

Beck-Sagué C, Jarvis WR. (1993) Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. J Infect Dis 167(5):1247-51.

Berman J, Sudbery PE. (2002) Candida Albicans: a molecular revolution built on lessons from budding yeast. Nat Rev Genet. 3(12):918-30.

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評估白色念珠菌組蛋白H3第四離胺酸去甲基酶JHD2在毒性相關的特徵

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