Abstract
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.
Highlights
In nature, the vast majority of microorganisms exist in association with surfaces in structured communities known as biofilms
We have optimized a functional genomics platform for massively parallel analysis of fitness using generation sequencing to quantify the relative proportion of each barcoded strain present in the gene replacement and conditional expression (GRACE) collection
A pooled collection of 1,481 GRACE strains covering ~25% of the genome was grown in YPD in the presence or absence of 0.5 μg/ml doxycycline for 6 hours in order to repress the expression of the target genes
Summary
The vast majority of microorganisms exist in association with surfaces in structured communities known as biofilms. Fungal biofilms are a major cause of human mortality and are highly recalcitrant to most treatments due to intrinsic drug resistance, necessitating costly surgical procedures to remove contaminated implanted devices [1,2]. Biofilms of C. albicans contaminate and grow on medically implanted devices such as catheters, pacemakers and prostheses and are the third leading cause of intravascular catheter-related infections [2,5]. They can colonize mucosal surfaces from which they can seed systemic infections. Biofilm formation is a key virulence trait for this opportunistic pathogen and poses a significant threat to human health
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