Virulence of Candida is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitro and in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to interact physically. Surprisingly, a previously identified polysaccharide of functional importance, β-1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Functional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of aggregate matrix with the antifungal fluconazole, consistent with a role in drug impedance and contribution of multiple matrix components. IMPORTANCE This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.

Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of Candida albicans biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by BGL2, PHR1, and XOG1, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by FKS1. Furthermore, our observations suggest that this matrix delivery pathway works independently from the C. albicans ZAP1 matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics. Biofilms are a community of microbes that grow attached to each other and adherent to a surface. One distinguishing feature of this form of growth is the presence of a surrounding extracellular matrix which is proposed to provide a structural scaffold and protection for biofilm cells. This later function contributes to the extreme resistance to anti-infective therapies, another innate characteristic of biofilms. One carbohydrate component of the matrix of Candida albicans, β-1, 3 glucan, has been linked to overall accumulation of matrix material and the antifungal drug resistance phenotype. Although the glucan synthase pathway has been implicated in glucan production, the delivery and incorporation of these carbohydrates into the matrix remains a mystery. The current investigation describes three gene products that serve a matrix delivery role. The functions of these gene products include glucanase and glucanosyltransferase activities. Mutants unable to produce these enzymes demonstrate reduced matrix glucan, decreased total matrix biomass accumulation, and enhanced susceptibility to antifungal drug therapy. The observations here offer insight into a novel pathway that contributes to biofilm maintenance. Enzymes in this biofilm-specific process may provide useful anti-biofilm drug targets.

Significance: Candida albicans is the most common fungal pathogen and frequently grows as a biofilm. These adherent communities tolerate extremely high concentrations of antifungals due in large part to the protective extracellular matrix. The present studies observe a novel reliance on multiple matrix constituents for structure and function. Furthermore, the results demonstrate how the biofilm community assembles these matrix components in the extracellular space. Our findings reveal a coordinated mechanism by which the defining trait of the biofilm lifestyle arises and identify a number of potential therapeutic targets.

The coexistence of sexual and asexual reproductive cycles within the same individual is a striking phenomenon in numerous fungi. In the fungus Aspergillus nidulans (teleomorph: Emericella nidulans) endogenous oxylipins, called psi factor, serve as hormone-like signals that modulate the timing and balance between sexual and asexual spore development. Here, we report the identification of A. nidulans ppoA, encoding a putative fatty acid dioxygenase, involved in the biosynthesis of the linoleic acid derived oxylipin psiBα. PpoA is required for balancing anamorph and teleomorph development. Deletion of ppoA significantly reduced the level of psiBα and increased the ratio of asexual to sexual spore numbers 4-fold. In contrast, forced expression of ppoA resulted in elevated levels of psiBα and decreased the ratio of asexual to sexual spore numbers 6-fold. ppoA expression is mediated by two developmental regulators, VeA and the COP9 signalosome, such that ppoA transcript levels are correlated with the initiation of asexual and sexual fruiting body formation. PpoA localizes in lipid bodies in these tissues. These data support an important role for oxylipins in integrating mitotic and meiotic spore development.

Oxylipins called psi factors have been shown to alter the ratio of asexual to sexual sporulation in the filamentous fungusAspergillus nidulans. Analysis of theA. nidulansgenome has led to the identification of three fatty acid oxygenases (PpoA, PpoB and PpoC) predicted to produce psi factors. Here, it is reported that deletion ofppoB(ΔppoB) reduced production of the oleic-acid-derived oxylipin psiBβand increased the ratio of asexual to sexual spore development. Generation of the triple mutant ΔppoAΔppoBΔppoCresulted in a strain deficient in producing oleic- and linoleic-acid-derived 8′-hydroxy psi factor and caused increased and mis-scheduled activation of sexual development. Changes in asexual to sexual spore development were positively correlated to alterations in the expression ofbrlAandveA, respectively. PpoB and/or its products antagonistically mediate the expression levels ofppoAandppoC, thus revealing regulatory feedback loops among these three genes. Phylogenetic analyses showed thatppogenes are present in both saprophytic and pathogenic Ascomycetes and Basidiomycetes, suggesting a conserved role for Ppo enzymes in the life cycle of fungi.

Candida auris is an emerging fungal pathogen responsible for health care–associated outbreaks that arise from persistent surface and skin colonization. We characterized the arsenal of adhesins used by C. auris and discovered an uncharacterized adhesin, Surface Colonization Factor (Scf1), and a conserved adhesin, Iff4109, that are essential for the colonization of inert surfaces and mammalian hosts. SCF1 is apparently specific to C. auris , and its expression mediates adhesion to inert and biological surfaces across isolates from all five clades. Unlike canonical fungal adhesins, which function through hydrophobic interactions, Scf1 relies on exposed cationic residues for surface association. SCF1 is required for C. auris biofilm formation, skin colonization, virulence in systemic infection, and colonization of inserted medical devices.