Proteomic analysis of cytoplasmic and surface proteins from yeast cells, hyphae, and biofilms of Candida albicans.

Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non-covalently attached cell-surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (>or=1.5-fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi-organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.

Farnesol-mediated inhibition of Candida albicans yeast growth and rescue by a diacylglycerol analogue.

During Candida albicans yeast cell growth to early stationary phase, metabolites accumulate in the medium, including the quorum-sensing molecule farnesol. We found that besides germ tube inhibition, 40 microM farnesol also inhibited C. albicans yeast growth under yeast growth permissive conditions. Consistent with this observation, transcriptional analysis of yeast cells resuspended in fresh medium with 40 microM farnesol revealed that genes involved in hyphal formation, GTPase activation, mitosis and DNA replication were downregulated many-fold. Farnesol-mediated inhibition of yeast growth was dependent on the growth phase of the C. albicans cells. The growth defect was relieved by addition of a diacylglycerol analogue, implicating phosphatidylinositol signalling in the delay. Although diacylglycerol is an activator of protein kinase C (PKC) in mammalian cells, there is some question about activation of fungal PKCs. A mutant strain deleted for PKC1 responded to farnesol and the diacylglycerol analogue similar to wild-type, suggesting that PKC is not the target of the diacylglycerol analogue.

Role for cell density in antifungal drug resistance in Candida albicans biofilms.

Biofilms of Candida albicans are less susceptible to many antifungal drugs than are planktonic yeast cells. We investigated the contribution of cell density to biofilm phenotypic resistance. Planktonic yeast cells in RPMI 1640 were susceptible to azole-class drugs, amphotericin B, and caspofungin at 1 x 10(3) cells/ml (standard conditions) using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay. As reported by others, as the cell concentration increased to 1 x 10(8) cells/ml, resistance was observed with 10- to 20-fold-greater MICs. Biofilms that formed in microtiter plate wells, like high-density planktonic organisms, were resistant to drugs. When biofilms were resuspended before testing, phenotypic resistance remained, but organisms, when diluted to 1 x 10(3) cells/ml, were susceptible. Drug-containing medium recovered from high-cell-density tests inhibited low-cell-density organisms. A fluconazole-resistant strain showed greater resistance at high planktonic cell density, in biofilm, and in resuspended biofilm than did low-density planktonic or biofilm organisms. A strain lacking drug efflux pumps CDR1, CDR2, and MDR1, while susceptible at a low azole concentration, was resistant at high cell density and in biofilm. A strain lacking CHK1 that fails to respond to the quorum-sensing molecule farnesol had the same response as did the wild type. FK506, reported to abrogate tolerance to azole drugs at low cell density, had no effect on tolerance at high cell density and in biofilm. These observations suggested that cell density has a role in the phenotypic resistance of biofilm, that neither the drug efflux pumps tested nor quorum sensing through Chk1p contributes to resistance, and that azole drug tolerance at high cell density differs mechanistically from tolerance at low cell density.