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

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.

The Role of Candida albicans Secreted Polysaccharides in Augmenting Streptococcus mutans Adherence and Mixed Biofilm Formation: In vitro and in vivo Studies

“…However, as described by others and confirmed by us, the key available mutants are aberrant in adhesion, hyphal and biofilm formation and virulence due to pronounced structural changes in their cell walls (Southard et al, 1999;Munro et al, 2005;Mitchell et al, 2015;Lee et al, 2016). Therefore, given these limitations, it was not possible to use these mutants to confidently determine the impact of the absence of the relevant genes on S. mutans adherence and retention in a mixed biofilm.…”

Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

“…Cultures were then moved to 37°C for 1 hr to induce hyphae in the presence of caspofungin. To measure the effect of caspofungin on β(1,3)-glucan levels, C. albicans hyphae-infected wells were stained with 0.05% aniline blue ( EVANS et al, 1984 ; Lee et al, 2016 ) overnight.…”

The Role of Secretory Pathways in Candida albicans Pathogenesis

“…There is now indirect evidence for a Rho1p-mediated endocytic pathway in C. albicans, which is not dependent on Arp2/3, revealing unexpected differences from clathrin-independent endocytosis in S. cerevisiae [296,297]. Based upon a forward genetic screen for filamentation-defective mutants, a Ca arp2∆/∆ mutant was found to be viable but unable to form hyphae; Ca arp2∆/∆ and arp2∆/∆ arp3∆/∆ mutants were endocytosis-competent for Lucifer Yellow, and had only a modest delay of FM4-64 endocytosis to the vacuole [298].…”