Richard D. Cannon

Overexpression of Candida albicans CDR1, CDR2, or MDR1 Does Not Produce Significant Changes in Echinocandin Susceptibility

ABSTRACT The micafungin and caspofungin susceptibilities of Candida albicans laboratory and clinical isolates and of Saccharomyces cerevisiae strains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicans gave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2 showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1 or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1 showed slightly decreased susceptibility to micafungin in these assays. A C. albicans clinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.

Chemosensitization of Fluconazole Resistance in Saccharomyces cerevisiae and Pathogenic Fungi by a d-Octapeptide Derivative

ABSTRACT Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5+) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 μg ml−1), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 μg ml−1). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer d-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted d-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC50], 4 μM) which chemosensitized AD/PDR5+ to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC50, 30 μM) and Cdr2p (IC50, 2 μM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter BenRp, or the FLC drug target lanosterol 14α-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC50, 1 μM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor.

Overexpression of the Candida albicans ATP‐binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ∼u20031.89u2003×u2003106 member d‐octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4‐methoxy‐2,3,6‐trimethylbenzenesulphonyl derivative of the d‐octapeptide d‐NH2‐FFKWQRRR‐CONH2, as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C.u2003albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C.u2003albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization‐resistant suppressor mutants of S.u2003cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug‐like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3.

Regulation of N-acetylglucosaminidase production in Candida albicans.

Abstract The N-acetylglucosaminidase of Candida albicans is a secreted hydrolytic enzyme that contributes to the yeast’s virulence. There was a significant increase in the N-acetylglucosaminidase activity of C. albicans cells released from carbon starvation in medium containing N-acetylglucosamine. The increased enzyme activity in N-acetylglucosamine-grown cells correlated with increased transcription of the HEX1 gene, which encodes C. albicans N-acetylglucosaminidase. In contrast, glucose repressed HEX1 transcription, and glucose-grown cells had on average 94-fold lower N-acetylglucosaminidase activities than did N-acetylglucosamine-grown cells. N-acetylglucosaminidase induction in cells grown on N-acetylglucosamine was also repressed by fructose, mannose or galactose, although to a lesser extent than by glucose, and sucrose repressed enzyme production by only 10%. Eighty-eight percent of the enzyme in N-acetylglucosamine-grown cells was localised in the periplasm, and after incubation for 5 h, 30 or 70% of the total enzyme activity was secreted into the medium by yeast or mycelial cells, respectively. The cellular location of the enzyme and the regulation of production by the carbon source indicate a scavenging role for C. albicans N-acetylglucosaminidase.