Functional Genomic Analysis of <i>Candida albicans</i> Adherence Reveals a Key Role for the Arp2/3 Complex in Cell Wall Remodelling and Biofilm Formation

Fungal biofilms are complex, structured communities that can form on surfaces such as catheters and other indwelling medical devices. Biofilms are of particular concern with Candida albicans, one of the leading opportunistic fungal pathogens of humans. C. albicans biofilms include yeast and filamentous cells that are surrounded by an extracellular matrix, and they are intrinsically resistant to antifungal drugs such that resolving biofilm infections often requires surgery to remove the contaminated device. C. albicans biofilms form through a regulated process of adhesion to surfaces, filamentation, maturation, and ultimately dispersion. To uncover new strategies to block the initial stages of biofilm formation, we utilized a functional genomic approach to identify genes that modulate C. albicans adherence. We screened a library of 1,481 double barcoded doxycycline-repressible conditional gene expression strains covering ~25% of the C. albicans genome. We identified five genes for which transcriptional repression impaired adherence, including: ARC18, PMT1, MNN9, SPT7, and orf19.831. The most severe adherence defect was observed upon transcriptional repression of ARC18, which encodes a member of the Arp2/3 complex that is involved in regulation of the actin cytoskeleton and endocytosis. Depletion of components of the Arp2/3 complex not only impaired adherence, but also caused reduced biofilm formation, increased cell surface hydrophobicity, and increased exposure of cell wall chitin and β-glucans. Reduced function of the Arp2/3 complex led to impaired cell wall integrity and activation of Rho1-mediated cell wall stress responses, thereby causing cell wall remodelling and reduced adherence. Thus, we identify important functional relationships between cell wall stress responses and a novel mechanism that controls adherence and biofilm formation, thereby illuminating novel strategies to cripple a leading fungal pathogen of humans.真菌生物膜（fungal biofilms）是一类结构复杂的群落，可在导管及其他留置医疗器械表面形成。白色念珠菌（Candida albicans）作为人类主要的机会致病性真菌病原体之一，其相关生物膜尤其受到关注。白色念珠菌生物膜包含被细胞外基质包裹的酵母态与丝状态细胞，且天然固有耐药于抗真菌药物，因此治愈此类生物膜感染通常需要通过手术移除受污染的装置。白色念珠菌生物膜通过受严格调控的过程形成：依次经历表面黏附、丝状化、成熟，最终发生分散。为了探寻阻断生物膜形成初始阶段的新策略，我们采用功能基因组学方法，筛选能够调控白色念珠菌黏附过程的基因。我们对覆盖约25%白色念珠菌基因组的、由1481株双条形码、多西环素（doxycycline）可阻遏的条件性基因表达菌株组成的文库进行了筛选。最终鉴定出5个转录阻遏后会显著削弱黏附能力的基因，分别为ARC18、PMT1、MNN9、SPT7以及orf19.831。其中转录阻遏ARC18时观察到最严重的黏附缺陷，该基因编码肌动蛋白相关蛋白2/3（Arp2/3）复合物的一个亚基，参与调控肌动蛋白细胞骨架与胞吞过程。敲低肌动蛋白相关蛋白2/3复合物的组分不仅会削弱黏附能力，还会导致生物膜形成减少、细胞表面疏水性升高，同时使细胞壁几丁质与β-葡聚糖的暴露量增加。肌动蛋白相关蛋白2/3复合物功能受损会破坏细胞壁完整性，并激活Rho1介导的细胞壁应激反应，进而引发细胞壁重塑与黏附能力下降。综上，我们揭示了细胞壁应激反应与调控黏附及生物膜形成的新机制之间的重要功能关联，从而为攻克人类主要致病性真菌病原体提供了全新的干预策略。