The expression of genes involved in the ergosterol biosynthesis pathway in Candida albicans and Candida dubliniensis biofilms exposed to fluconazole.

The expression of the ERG1, ERG3, ERG7, ERG9, ERG11 and ERG25 genes in response to incubation with fluconazole and biofilm formation was investigated using reverse-transcription PCR and real-time PCR in Candida albicans and Candida dubliniensis clinical isolates. The viability of biofilm was measured using an 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and confocal scanning laser microscopy (CSLM). Expression of the ERG11 gene was found to be low or moderate and it was regulated by fluconazole addition more so than by biofilm formation. Very low or non-detectable expression of ERG1, ERG7 and ERG25 genes was detected in C. albicans. The expression of the ERG9 increased in the presence of fluconazole in some isolates. Following incubation with fluconazole, formation of biofilm by C. dubliniensis was coupled with up-regulation of the ERG3 and ERG25 genes as have been observed previously in C. albicans. Planktonic cells of both Candida species released from biofilm displayed similar resistance mechanisms to fluconazole like attached cells. The XTT reduction assay and CSLM revealed that although incubation with fluconazole decreased the biofilm thickness, these were still comprised metabolically active cells able to disseminate and produce biofilm. Our data indicate that biofilm represents a highly adapted community reflecting the individuality of clinical isolates.

Multilocus sequence typing reveals that the population structure of Candida dubliniensis is significantly less divergent than that of Candida albicans.

The pathogenic yeast Candida dubliniensis is phylogenetically very closely related to Candida albicans, and both species share many phenotypic and genetic characteristics. DNA fingerprinting using the species-specific probe Cd25 and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal gene cluster previously showed that C. dubliniensis is comprised of three major clades comprising four distinct ITS genotypes. Multilocus sequence typing (MLST) has been shown to be very useful for investigating the epidemiology and population biology of C. albicans and has identified many distinct major and minor clades. In the present study, we used MLST to investigate the population structure of C. dubliniensis for the first time. Combinations of 10 loci previously tested for MLST analysis of C. albicans were assessed for their discriminatory ability with 50 epidemiologically unrelated C. dubliniensis isolates from diverse geographic locations, including representative isolates from the previously identified three Cd25-defined major clades and the four ITS genotypes. Dendrograms created by using the unweighted pair group method with arithmetic averages that were generated using the data from all 10 loci revealed a population structure which supports that previously suggested by DNA fingerprinting and ITS genotyping. The MLST data revealed significantly less divergence within the C. dubliniensis population examined than within the C. albicans population. These findings show that MLST can be used as an informative alternative strategy for investigating the population structure of C. dubliniensis. On the basis of the highest number of genotypes per variable base, we recommend the following eight loci for MLST analysis of C. dubliniensis: CdAAT1b, CdACC1, CdADP1, CdMPIb, CdRPN2, CdSYA1, exCdVPS13, and exCdZWF1b, where "Cd" indicates C. dubliniensis and "ex" indicates extended sequence.

Antibody response to the 45 kDa Candida albicans antigen in an animal model and potential role of the antigen in adherence.

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a 'mimicry' protein because of its ability to bind antibody directed against the alpha subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C. albicans peptide DINGGGATLPQ corresponding to 11 amino-acids of the CR3-RP sequence DINGGGATLPQALXQITGVIT, determined by N-terminal sequencing, was used for immunization of rabbits to obtain polyclonal anti-CR3-PR serum and for subsequent characterization of the humoral isotypic response of rabbits. A significant increase of IgG, IgA and IgM anti-CR3-RP specific antibodies was observed after the third (P<0.01) and the fourth (P<0.001) immunization doses. The elevation of IgA levels suggested peptide immunomodulation of the IgA1 subclass, presumably in coincidence with Candida epithelial adherence. Blocking CR3-RP with polyclonal anti-CR3-RP serum reduced the ability of Candida to adhere to BECs, in comparison with the control, by up to 35 % (P<0.001), and reduced biofilm formation by 28 % (P<0.001), including changes in biofilm thickness and integrity detected by confocal laser scanning microscopy. These properties of CR3-RP suggest that it has potential for future vaccine development.

Variation of cell surface hydrophobicity and biofilm formation among genotypes of Candida albicans and Candida dubliniensis under antifungal treatment.

Candida infections are frequently associated with formation of biofilms on artificial medical devices. This work studied variation of cell surface hydrophobicity (CSH) and formation of biofilm in relation to Candida albicans and Candida dubliniensis genotypes and an effect of some conventional antifungal agents on both CSH and biofilm. The 50 isolates of C. albicans and C. dubliniensis were classified into genotypes A, B, C, and D, genotype D being exclusively represented by C. dubliniensis. No significant differences between CSH of genotypes A and B and B and C were observed with respect to cultivation temperature 25 or 37 degrees C. Candida dubliniensis showed increased CSH in comparison with other C. albicans genotypes (p < 0.001) regardless of temperature used. Using XTT reduction assay and dry masses, genotypes B and C showed reduced ability to form biofilm in comparison with genotype A (p < 0.05) and C. dubliniensis (p < 0.001). Fluconazole reduced biofilm in C. albicans genotypes A, B, and C (p < 0.05) but not CSH. The opposite effect was observed in C. dubliniensis. Voriconazole effectively reduced both biofilm formation and CSH in all tested genotypes of C. albicans and C. dubliniensis (p < 0.05).

RPD3 and ROM2 are required for multidrug resistance in Saccharomyces cerevisiae.

The PDR5 gene encodes the major multidrug resistance efflux pump in Saccharomyces cerevisiae. In drug-resistant cells, the hyperactive Pdr1p or Pdr3p transcriptional activators are responsible for the PDR5 upregulation. In this work, it is shown that the RPD3 gene encoding the histone deacetylase that functions as a transcriptional corepressor at many promoters and the ROM2 gene coding for the GDP/GTP exchange protein for Rho1p and Rho2p participating in signal transduction pathways are required for PDR5 transcription under cycloheximide-induced and noninduced conditions. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3 Delta and rom2 Delta mutants, the level of PDR5 mRNA and the rate of rhodamine 6G efflux were reduced. Unlike rpd3 Delta, in rom2 Delta mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by PDR1 or PDR3 overexpressed from heterologous promoters and by the hyperactive pdr3-9 mutant allele. The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p participating in the cell integrity pathway are involved in the control of PDR5 expression and modulation of multidrug resistance in yeast.

Functional characterization of the CgPGS1 gene reveals a link between mitochondrial phospholipid homeostasis and drug resistance in Candida glabrata.

Cardiolipin and its precursor phosphatidylglycerol are two anionic phospholipids that are essential for the biogenesis of functional mitochondria. To assess their role in mitochondrial and cellular functions in the pathogenic yeast Candida glabrata, a functional characterization of the CgPGS1 gene encoding the phosphatidylglycerolphosphate synthase has been carried out. Transposon insertion mutation in CgPGS1 resulted in the loss of phosphatidylglycerolphosphate synthase activity and in deficiency of both phosphatidylglycerol and cardiolipin. The Cgpgs1 Delta mutant cells displayed reduced amounts of cytochrome b and cytochrome a, and had impaired growth on minimal media containing non-fermentable carbon and energy sources. They did not grow at elevated temperatures and failed to form colonies after induction of mitochondrial DNA deletions. The mutant cells also displayed a decreased susceptibility to fluconazole, ketoconazole, clotrimazole, voriconazole and posaconazole. In the Cgpgs1 Delta mutant, a quantitative real time PCR revealed enhanced mRNA levels for multidrug resistance associated genes such as CgPDR1 encoding transcriptional activator and CgCDR1, CgPDH1 and CgSNQ2 coding for drug efflux transporters. These results indicate that CgPGS1 and anionic phospholipids are required for optimal mitochondrial functions and maintenance of yeast susceptibility to azole antifungals.