The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate-grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose-grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate-grown cells. We identified mating and pheromone-regulated proteins that were exclusive to lactate-grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa-specific and other niche-specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.

The pathogenic fungus Candida albicans secretes a considerable number of hydrolases and other proteins. In-depth studies of the C. albicans secretome could thus provide new candidates for diagnostic markers and vaccine development. We compared various growth conditions differing in pH, temperature and the presence of the hyphal inducer N -acetylglucosamine. The polypeptide content of the growth media was ca. 0.1-0.2% of the total biomass. Using LC-tandem mass spectrometry, we identified 44 secretory proteins, the transmembrane protein Msb2, six secretory pathwayassociated proteins and 28 predicted cytosolic proteins. Many secretory proteins are wall-related, suggesting that their presence in the growth medium is at least partially due to accidental release from the walls. Als3, Csa2, Rbt4, Sap4 and Sap6 were enriched in the medium of hyphal cultures; Bgl2, Cht3, Dag7, Eng1, Pir1, Rbe1, Scw11, Sim1/Sun42, Xog1 and Ywp1 in the medium of yeast cells; and Plb4.5 in pH 4 medium. Seven proteins (Cht3, Mp65, Orf19.5063/Coi1, Scw11, Sim1, Sun41 and Tos1) were consistently present under all conditions tested. These observations indicate that C. albicans tightly regulates its secretome. Mp65, Sun41, and Tos1 were each predicted to contain at least one highly immunogenic peptide. In total, we identified 29 highly immunogenic peptides originating from 18 proteins, including two members of the family of secreted aspartyl proteases. Fifty-six peptides were identified as proteotypic and will be useful for quantification purposes. In summary, the number of identified secretory proteins in the growth medium has been substantially extended, and growth conditions strongly affect the composition of the secretome.

The zinc cluster transcription factor Upc2p mediates upregulation of ergosterol biosynthesis genes in response to ergosterol depletion in the fungal pathogen Candida albicans. One mechanism of acquired resistance to the antifungal drug fluconazole, which inhibits ergosterol biosynthesis, is constitutively increased expression of the ERG11 gene encoding the drug target enzyme. A G648D mutation in Upc2p has recently been shown to cause hyperactivity of the transcription factor, resulting in overexpression of ergosterol biosynthesis genes and increased fluconazole resistance. In order to investigate if gain-of-function mutations in Upc2p are a common mechanism of ERG11 upregulation and fluconazole resistance, we sequenced the UPC2 alleles of four ERG11-overexpressing, fluconazole-resistant C. albicans isolates and matched susceptible isolates from the same patients. In three of the isolate pairs, no differences in the UPC2 alleles were found, suggesting that mechanisms other than Upc2p mutations can cause ERG11 overexpression. One resistant isolate had become homozygous for a UPC2 allele containing a G1927A substitution that caused an alanine-to-threonine exchange at amino acid position 643 of Upc2p. Replacement of one of the endogenous UPC2 alleles in a fluconazolesusceptible strain by the UPC2 A643T allele resulted in ERG11 overexpression and increased fluconazole resistance, which was further elevated when the A643T mutation was also introduced into the second UPC2 allele. These results further establish gain-of-function mutations in UPC2, which can be followed by loss of heterozygosity for the mutated allele, as a mechanism of ERG11 overexpression and increased fluconazole resistance in C. albicans, but other mechanisms of ERG11 upregulation also exist.

Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14␣-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14␣-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of 14 N-labeled query walls relative to a reference standard mixture of 15 N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.