Azole-resistant Clinical Isolates Research Articles

Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants.

Candida glabrata, a yeast with intrinsically low susceptibility to azoles, frequently develops increased azole resistance during prolonged treatment. Transposon mutagenesis revealed that disruption of CgPDR1 resulted in an 8- to 16-fold increase in fluconazole susceptibility of C. glabrata. CgPDR1 is a homolog of Saccharomyces cerevisiae PDR1, which encodes a transcriptional regulator of multidrug transporters. Northern blot analyses indicated that CgPDR1 regulated both constitutive and drug-induced expression of CgCDR1, a multidrug transporter gene. In agreement with the Northern analysis, the Cgpdr1 mutant had increased rhodamine accumulation, in contrast to the decreased accumulation in the CgPDR1-overexpressing strain. Northern analyses also indicated the importance of CgPDR1 in fluconazole resistance arising during therapy. Two clinically resistant isolates had higher expression of CgPDR1 and CgCDR1 compared to their paired susceptible isolates. Integrative transformation of CgPDR1 from the two resistant isolates converted the Cgpdr1 mutant into azole-resistant strains with upregulated CgPDR1 expression. Two different amino acid substitutions, W297S in one isolate and F575L in the other, accounted for the upregulated CgPDR1 expression and the resistance. Finally, CgPDR1 was shown to be required for the azole resistance due to mitochondrial deficiency. Thus, CgPDR1 encodes a transcriptional regulator of a pleiotropic drug resistance network and contributes to the azole resistance of clinical isolates and petite mutants.

Mechanisms of Azole Resistance in Clinical Isolates of Candida glabrata Collected during a Hospital Survey of Antifungal Resistance

The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

The ergosterol biosynthesis pathway, transporter genes, and azole resistance inAspergillus fumigatus

The continuous use of triazoles can result in the development of drug resistance. Azole-resistant clinical isolates, spontaneous and induced mutants of Aspergillus fumigatus have been documented. The azoles block the ergosterol biosynthesis pathway by inhibiting the enzyme 14-alpha-demethylase, product of the CYP51. Fungal azole resistance involves both amino acid changes in the target site that alter drug-target interactions and those that decrease net azole accumulation. The reduced intracellular accumulation has also been correlated with overexpression of multidrug resistance (MDR) efflux transporter genes of the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) classes. About 20 genes are involved in the A. fumigatus ergosterol biosynthesis pathway. There are several duplicated genes in this pathway. Interestingly, erg3 and erg11 showed two copies in A. fumigatus. In general, Aspergillus spp. have proportionally more MFS transporter encoding genes than Saccharomyces cerevisiae, S. pombe, and Neurospora crassa. The drug H+ (12 and 14 spanners) sub-families are also proportionally greater than in the other species. Although the numbers of ABC transporter encoding genes are comparable, again the Aspergillus spp. have more ABC transporters related to multidrug permease than the other fungal species.

Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor.

Candida glabrata has emerged as a common cause of fungal infection. This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented. Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target. Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C. glabrata these include CDR1 and PDH1. By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6- to 15-fold upregulation of CDR1 compared to susceptible strains. In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three- to fivefold, while ERG11 expression was minimally changed. Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants). Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2- to 20-fold. Effects of antifungal treatment on gene expression in susceptible C. glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4- to 12-fold. These findings suggest that these transporter genes are regulated by a common mechanism. In support of this, a mutation associated with laboratory resistance was identified in the C. glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae. The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation. Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1.

Functional similarities and differences between Candida albicans Cdr1p and Cdr2p transporters.

The Candida albicans CDR1 and CDR2 genes code for highly homologous ATP-binding cassette (ABC) transporters which are overexpressed in azole-resistant clinical isolates and which confer resistance to multiple drugs by actively transporting their substrates out of the cells. These transporters are formed by two homologous halves, each with an intracellular domain containing an ATP-binding site followed by a membrane-associated domain. We have expressed Cdr1p and Cdr2p in Saccharomyces cerevisiae to investigate their functions. The two proteins were properly expressed and functional, as determined by Western blotting, drug susceptibility assays, and rhodamine efflux. Using total membrane proteins from these transformants, we showed that Cdr1p and Cdr2p bind to the photoreactive analogue of rhodamine 123, [(125)I]iodoaryl azido-rhodamine 123 (IAARh123). IAARh123 photoaffinity labeling of membranes prepared from cells expressing either the N half or the C half of Cdr2p, or both, demonstrated that both halves contribute to rhodamine binding and can bind to rhodamine independently. Interestingly, Cdr1p was found to confer hypersusceptibility to FK520, an immunosuppressant and antifungal agent, whereas Cdr2p conferred resistance to this compound, uncovering a major functional difference between the two transporters. Furthermore, when administered in combination with azoles, FK520 sensitized cells expressing CDR1 but not those expressing CDR2. Finally, we showed that Cdr2p confers hypersusceptibility to hydrogen peroxide and resistance to diamide, while Cdr1p has no effect against these oxidative agents. Taken together, our results demonstrate that, despite a high level of structural conservation, Cdr1p and Cdr2p exhibit major functional differences, suggesting distinct biological functions.

Synthesis and antimycotic activity of N-azolyl-2,4-dihydroxythiobenzamides

N-pyrazole and N-1,2,4-triazole derivatives of 2,4-dihydroxythiobenzamide prepared from sulfinyl-bis-(2,4-dihydroxythiobenzoyl) and commercially available azole amines were tested for their antimycotic activity. The chemical structure of compounds was confirmed by IR, 1H NMR, MS and elemental analysis. The MIC values against the reference strain Candida albicans ATCC 10231, azole-resistant clinical isolates of Candida albicans and non- Candida albicans species were determined for their potential activity in vitro. The compounds exhibited comparable or higher activity than itraconazole and fluconazole tested under the same experimental conditions. Pyrazoline derivatives showed higher activity than other analogues. The strongest fungistatic activity for N-(2,3-dimethyl-1-phenyl-1,2-dihydro-5-oxo-5H-pyrazol-4-yl)-2,4-dihydroxythiobenzamide was found with MIC values significantly lower than those for the studied drugs.

Synthesis of some 1-(2,4-dihydroxythiobenzoyl)imidazoles, -imidazolines and -tetrazoles and their potent activity against Candida species

Various 1-(2,4-dihydroxythiobenzoyl)imidazoles, -imidazolines and -tetrazoles were synthesized and evaluated for their in vitro antifungal activity. Compounds were prepared by the reaction of sulfinyl-bis-(2,4-dihydroxythiobenzoyl) with properly substituted azoles. The MIC values against the Candida albicans ATCC 10231 strain, the azole-resistant clinical isolates of C. albicans and non- Candida species were determined. Tetrazole derivatives were the most active against C. albicans, imidazoline derivatives against non- Candida species. All compounds showed higher activity than that of comparable drugs.

CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans.

We report the isolation of a novel C. albicans gene designated CaALK8, by its ability to complement drug hypersensitivity of a pdr5 (ABC: ATP-binding cassette drug extrusion pump) null mutant of S. cerevisiae (JG436). CaALK8 in JG436 conferred resistance to drugs such as cycloheximide (CYH), fluconazole (FCZ), O-phenanthroline (PHE) and 4-nitroquinoline oxide (NQO). The gene was so designated because its sequence was identical to a partial sequence entry named as ALK8 in the Candida database (http://alces.med.umn.edu/candida.html). CaALK8 encodes for a putative 515 amino acid protein highly homologous to alkane-inducible cytochromes P450 (CYP52 gene family) of C. maltosa and C. tropicalis. The ability of CaALK8 to confer drug resistance was also established by its expression in another drug-hypersensitive strain of S. cerevisiae (AD 1234568), which was deleted in seven ABC efflux pumps. The homozygous disruption of CaALK8 in a wild-type C. albicans strain (CAI4) did not result in altered drug susceptibilities. The overexpression of CaALK8 in CAI4 resulted in only FCZ resistance. However, a distinct MDR phenotype was evident when CaALK8 was overexpressed in a drug-hypersensitive C. albicans strain disrupted in both CDR1 and CDR2 (ABC drug extrusion pumps of C. albicans). Alk8p, similar to other Alk proteins from C. maltosa and C. tropicalis, could hydroxylate alkanes and fatty acids. In this study we demonstrate that several drugs could compete with the hydroxylation activity by directly interacting with CaAlk8p. Taken together, our results suggest that a member of the CYP52 gene family could mediate MDR in C. albicans, although it does not seem to be involved in the development of azole resistance in clinical isolates. The nucleotide sequence reported in this paper has been submitted to GenBank under Accession No. Y14766.

Accumulation of 3-ketosteroids induced by itraconazole in azole-resistant clinical Candida albicans isolates.

The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378-2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14alpha-methyl-ergosta-8,24(28)-diene-3beta,6alpha-dio l (3, 6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14alpha-methyl-ergosta-5,7,22,24(28)-tetraene-3betaol, and 14alpha-methyl-fecosterol. In strains C48 and C56, no further increase of 3,6-diol was observed after exposure to itraconazole. Ergosterol synthesis was less sensitive to itraconazole inhibition, as was expected for these azole-resistant isolates which overexpress ATP-binding cassette transporter genes CDR1 and CDR2. In addition to 3,6-diol, substantial amounts of obtusifolione were found after exposure to itraconazole. This toxic 3-ketosteroid was demonstrated previously to accumulate after itraconazole treatment in Cryptococcus neoformans and Histoplasma capsulatum but has not been reported in Candida isolates. Accumulation of obtusifolione correlated with nearly complete growth inhibition in these azole-resistant strains compared to that found in the susceptible parent strain, although the onset of growth inhibition only occurred at higher concentrations of itraconazole. ERG25 and ERG26 are the only genes assigned to the 4-demethylation process, of which the 3-ketoreductase is part. To verify whether mutations in these ERG25 genes contributed to obtusifolione accumulation, their nucleotide sequences were determined in all three related isolates. No mutations in ERG25 alleles of isolates C48 and C56 were found, suggesting that this gene is not involved in obtusifolione accumulation. The molecular basis for the accumulation of this sterol in these two strains remains to be established.

Identification of polymorphic mutant alleles of CaMDR1, a major facilitator of Candida albicans which confers multidrug resistance, and its in vitro transcriptional activation.

CaMDR1 (Candida albicans Multi Drug Resistance) encodes a major facilitator whose expression in Saccharomyces cerevisiae confers resistance to several unrelated drugs. We describe here the identification and molecular characterization of seven mutant alleles of CaMDR1 (CaMDR1-1 to 1-7). The complete sequencing of CaMDR1 alleles revealed several in-frame point mutations leading to a change in amino-acid residues where insertion/replacement of an aspartate residue in a serine-asparagine-aspartate-rich domain was most noteworthy. Interestingly, these alleles showed a distinct drug resistance profile. The expression of CaMDR1, or of its alleles, in C. albicans cells was enhanced by benomyl, methotrexate and several other unrelated drugs, and was more pronounced in at least one of the azole-resistant clinical isolates.

Expression of CDR1, a multidrug resistance gene of Candida albicans: transcriptional activation by heat shock, drugs and human steroid hormones

We have examined the expression of CDR1 ( Candida drug resistance gene) in different stress conditions. There was a significant but transient enhancement of CDR1 expression associated with elevated temperatures. Most noteworthy transcriptional activation was observed with miconazole and vinblastine. Interestingly, β-estradiol and progesterone were also able to enhance CDR1 expression. Elevated levels of CDR1 and CDR2 (a homologue of CDR1) mRNA were found in some azole-resistant clinical isolates of C. albicans. CaMDR1 (benomyl-resistant) expression, however, did not differ among all the resistant isolates. Our results confirm the existence of multiple mechanisms of azole resistance in C. albicans.

Expression ofCDR1, a multidrug resistance gene ofCandida albicans: transcriptional activation by heat shock, drugs and human steroid hormones

We have examined the expression of CDR1 (Candida drug resistance gene) in different stress conditions. There was a significant but transient enhancement of CDR1 expression associated with elevated temperatures. Most noteworthy transcriptional activation was observed with miconazole and vinblastine. Interestingly, beta-estradiol and progesterone were also able to enhance CDR1 expression. Elevated levels of CDR1 and CDR2 (a homologue of CDR1) mRNA were found in some azole-resistant clinical isolates of C. albicans. CaMDR1 (benomyl-resistant) expression, however, did not differ among all the resistant isolates. Our results confirm the existence of multiple mechanisms of azole resistance in C. albicans.