Lee Jason A, Robbins Nicole, Xie Jinglin L, Ketela Troy, Cowen Leah E
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
PLoS Genet. 2016 Nov 21;12(11):e1006452. doi: 10.1371/journal.pgen.1006452. eCollection 2016 Nov.
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.
真菌生物膜是复杂的结构化群落，可在诸如导管和其他植入式医疗设备等表面形成。生物膜尤其与白色念珠菌有关，白色念珠菌是人类主要的机会性真菌病原体之一。白色念珠菌生物膜包括被细胞外基质包围的酵母细胞和丝状细胞，并且它们对抗真菌药物具有内在抗性，因此解决生物膜感染通常需要手术移除受污染的设备。白色念珠菌生物膜通过对表面的粘附、丝状化、成熟以及最终分散的调控过程形成。为了发现阻断生物膜形成初始阶段的新策略，我们利用功能基因组学方法来鉴定调节白色念珠菌粘附的基因。我们筛选了一个包含1481个双条形码强力霉素可抑制的条件性基因表达菌株的文库，该文库覆盖了约25%的白色念珠菌基因组。我们鉴定出五个基因，其转录抑制会损害粘附，包括：ARC18、PMT1、MNN9、SPT7和orf19.831。在ARC18转录抑制后观察到最严重的粘附缺陷，ARC18编码Arp2/3复合体的一个成员，该复合体参与肌动蛋白细胞骨架的调节和内吞作用。Arp2/3复合体成分的缺失不仅损害了粘附，还导致生物膜形成减少、细胞表面疏水性增加以及细胞壁几丁质和β-葡聚糖暴露增加。Arp2/3复合体功能的降低导致细胞壁完整性受损和Rho1介导的细胞壁应激反应激活，从而引起细胞壁重塑和粘附减少。因此，我们确定了细胞壁应激反应与控制粘附和生物膜形成的新机制之间的重要功能关系，从而阐明了削弱人类主要真菌病原体的新策略。
