Biofilms are a protected niche for microorganisms, where they are safe from antibiotic treatment and can create a source of persistent infection. Using two clinically relevant Candida albicans biofilm models formed on bioprosthetic materials, we demonstrated that biofilm formation proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in a polysaccharide matrix. Fluorescence and confocal scanning laser microscopy revealed that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements. In both models, antifungal resistance of biofilm-grown cells increased in conjunction with biofilm formation. The expression of agglutinin-like ( ALS ) genes, which encode a family of proteins implicated in adhesion to host surfaces, was differentially regulated between planktonic and biofilm-grown cells. The ability of C. albicans to form biofilms contrasts sharply with that of Saccharomyces cerevisiae , which adhered to bioprosthetic surfaces but failed to form a mature biofilm. The studies described here form the basis for investigations into the molecular mechanisms of Candida biofilm biology and antifungal resistance and provide the means to design novel therapies for biofilm-based infections.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

Denture biofilms represent a protective reservoir for oral microbes. The study of the biology of Candida in these biofilms requires a reliable model. A reproducible model of C. albicans denture biofilm was developed and used to determine the susceptibility of two clinically relevant C. albicans isolates against 4 antifungals. C. albicans, growing as a biofilm, exhibited resistance to amphotericin B, nystatin, chlorhexidine, and fluconazole, with 50% reduction in metabolic activity (50% RMA) at concentrations of 8, 16, 128, and > 64 μg/mL, respectively. In contrast, planktonically cultured C. albicans were susceptible (50% RMA for the same antifungals was obtained at 0.25, 1.0, 4.0, and 0.5 μg/mL, respectively). In conclusion, results obtained by means of our biofilm model show that biofilm-associated C. albicans cells, compared with cells grown in planktonic form, are resistant to antifungals used to treat denture stomatitis.

The agglutinin-like sequence (ALS) family of Candida albicans includes eight genes that encode large cell-surface glycoproteins. The high degree of sequence relatedness between the ALS genes and the tremendous allelic variability often present in the same C. albicans strain complicated definition and characterization of the gene family. The main hypothesis driving ALS family research is that the genes encode adhesins, primarily involved in host-pathogen interactions. Although adhesive function has been demonstrated for several Als proteins, the challenge of studying putative adhesins in a highly adhesive organism like C. albicans has led to varying ideas about how best to pursue such investigations, and results that are sometimes contradictory. Recent analysis of alsΔ/alsΔ strains suggested roles for Als proteins outside of adhesion to host surfaces, and a broader scope of Als protein function than commonly believed. The availability and use of experimental methodologies to study C. albicans at the genomic level, and the ALS family en masse, have advanced knowledge of these genes and emphasized their importance in C. albicans biology and pathogenesis.

In Candida albicans wild‐type cells, the β1,6‐glucanase‐extractable glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI‐CWPs, including Als1p and Als3p, are attached via β1,6‐glucan to β1,3‐glucan. The remaining GPI‐CWPs are linked through β1,6‐glucan to chitin. The β1,6‐glucanase‐resistant protein fraction is small and consists of Pir‐related CWPs, which are attached to β1,3‐glucan through an alkali‐labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Δ and pmt1Δ mutant strains, which are defective in N‐ and O‐glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI‐CWPs through β1,6‐glucan to chitin. In these cells, the level of Pir‐CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a β1,6‐glucan‐deficient hemizygous kre6Δ mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.