PDR16-mediated azole resistance in Candida albicans

Many Candida albicans azole-resistant (AR) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16Delta mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an AR clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16Delta/pdr16Delta mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the AR phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (AS) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.     

Genotypic analysis of Candida albicans isolates obtained from removable prosthesis wearers

Aims: To assess of the genotypic diversity of Candida albicans isolated from removable prosthesis wearers, with and without denture‐related stomatitis (DRS). The occurrence of different genotypes in pathological and control cases was investigated. Methods and Results: One hundred and sixty‐four isolates of C. albicans obtained from different oral cavity locations were compared by randomly amplified polymorphic DNA (RAPD). The coherence of this analysis was confirmed by genotyping a selected group of isolates with pulsed field gel electrophoresis (PFGE). Among the 164 isolates, 150 were grouped into seven groups on the basis of their RAPD patterns. Three of these groups (comprising 54 isolates) had significant (α < 0·10) predominance of clinical or control cases. For the other isolates, no significant differences were observed between control and DRS cases. Occasionally, more than one genotype was found in the same person. These findings were sustained by PFGE analysis. No relevant associations between the genotypic patterns and pathology level were found. Conclusions: This study evidenced that C. albicans with similar genotypes may be found in individuals with DRS and in control cases. Significance and Impact of the Study: This conclusion hints the involvement of other aetiological factors that alone or in association with C. albicans may trigger the emergence of DRS.     

白念珠菌唑类药物耐药相关转录因子研究进展

近年来白念珠菌的感染率呈逐年上升趋势,随着唑类药物的广泛应用,耐药菌株不断增多,已成为临床治疗的一大难题.白念珠菌的耐药机制主要与ERG 11基因的突变和过表达、药物外排泵相关基因表达增多及生物膜的形成等有关,由于转录因子是耐药基因表达的关键调节因子,关于锌簇转录因子与耐药关系的研究越来越多,如TAC 1、MRR 1、MRR 2、UPC 2、NDT 80等,其点突变可引起某些耐药基因的过表达而介导耐药,该领域研究已成为热点,该文就此研究进展做一概述.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Genotypic subgrouping of clinical isolates of Candida albicans and Candida dubliniensis by 25S intron analysis

AIMS: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates. METHODS: Twenty-one invasive and 18 noninvasive isolates were examined by PCR amplification of a transposable intron region in the 25S rRNA gene. Isolates were genotyped following analysis of the size of resulting DNA amplicons. The isolates could be subdivided into four genotypes (A-D). RESULTS: There was no significant difference between the frequency and genotype distribution of the invasive and noninvasive Candida isolates. IMPACT OF THE STUDY: Therapeutic prophylaxis against candidal infections remains an area of controversy. Any diagnostic markers that reflect the potential of isolates to become invasive should be fully explored, so that more focused antifungal intervention should be targeted at these patients with these potential invasive markers. This study demonstrated that analysis of the transposable intron region in the 25S rRNA gene may be useful in helping to differentiate C. albicans from C. dubliniensis isolates, without the need for sequence analysis, which may not be readily available at primary diagnostic laboratories. However, employment of this genotypic assay is not a suitable locus to determine invasiveness and other more reliable markers of invasiveness should be sought.     

Reduced intracellular accumulation of azole antifungal results in resistance in Candida albicans isolate NCPF 3363

Candida albicans strain NCPF 3363 was isolated from a British patient with chronic mucocutaneous candidiasis (CMC) and confirmed to be resistant to azole antifungal compounds. In this study we investigate the molecular basis of resistance and show that azole tolerance in NCPF 3363 was associated with reduced intracellular accumulation of drug and not reduced affinity for the target site, as previously indicated. Relative impermeability or the presence of transporters related to those responsible for multidrug resistance are implicated in the mechanism of resistance.     

Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles

Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways.     

白念珠菌耐药机制新进展

近年来对于深部真菌感染的研究报道越来越多,其已日益成为一些重要疾病临床治疗过程中的常见并发症,其中,白念珠菌病的发病率仍居高不下.虽然目前有多种抗真菌药物应用于临床,但其耐药现象愈来愈严重,给临床治疗带来了极大的挑战.近来有关白念珠菌耐药机制的研究有了较新的进展.该文就新发现的白念珠菌的耐药机制,作一概述.     

Population dynamics and the evolution of antifungal drug resistance in Candida albicans

Candida albicans is an important human fungal pathogen. Resistance to all major antifungal agents has been observed in clinical isolates of Candida spp. and is a major clinical challenge. The rise and expansion of drug-resistant mutants during exposure to antifungal agents occurs through a process of adaptive evolution, with potentially complex population dynamics. Understanding the population dynamics during the emergence of drug resistance is important for determining the fundamental principles of how fungal pathogens evolve for resistance. While few detailed reports that focus on the population dynamics of C. albicans currently exist, several important features on the population structure and adaptive landscape can be elucidated from existing evolutionary studies in in vivo and in vitro systems.     

Differential inhibition of Candida albicans CYP51 with azole antifungal stereoisomers

Azole antifungal compounds are important in agriculture and in the treatment of mycotic infection The target enzyme, sterol 14α-demethylase (CYP51), is inhibited through binding of triazole N-4 to the haem of this P450, as a sixth ligand together with the N-1 substituent groups interacting in some way with the apoprotein. Here we use Saccharomyces cerevisiae expression systems for the target enzyme of Candida albicans to investigate binding of enantiomers of the azole antifungal compounds SCH39304 and tetraconazole. A molecular model produced previously provided qualitative explanations for these differences. Interaction of the azole antifungal aromatic group with Phe-233 or -235 may cause the higher activity for (R)-tetraconazole while inactivity of the (SS)-enantiomer of SCH39304 was predicted to result from incompatibility of the hydrophilic sulfonyl moiety when located into the hydrophobic pocket of the active site.     

Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.     

Divergence in fitness and evolution of drug resistance in experimental populations of Candida albicans

The dissemination and persistence of drug-resistant organisms in nature depends on the relative fitness of sensitive and resistant genotypes. While resistant genotypes are expected to be at an advantage compared to less resistant genotypes in the presence of drug, resistance may incur a cost; resistant genotypes may be at a disadvantage in the absence of drug. We measured the fitness of replicate experimental populations of the pathogenic yeast Candida albicans founded from a single progenitor cell in a previous study (L. E. Cowen, D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, and L. M. Kohn, J. Bacteriol. 182:1515-1522, 2000) and evolved in the presence, and in the absence, of the antifungal agent fluconazole. Fitness was measured both in the presence and in the absence of fluconazole by placing each evolved population in direct competition with the drug-sensitive ancestor and measuring the reproductive output of each competitor in the mixture. Populations evolved in the presence of drug diverged in fitness. Any significant cost of resistance, indicated by reduced fitness in the absence of drug, was eliminated with further evolution. Populations evolved in the absence of drug showed more uniform increases in fitness under both conditions. Fitness in the competition assays was not predicted by measurements of the MICs, doubling times, or stationary-phase cell densities of the competitors in isolation, suggesting the importance of interactions between mixed genotypes in competitions.     

Reduced azole susceptibility of oral isolates of Candida albicans from HIV-positive patients and a derivative exhibiting colony morphology variation.

Approximately 50% (15/28) of a selection of oral isolates of Candida albicans from separate individuals infected with the human immunodeficiency virus (HIV) exhibited low susceptibility to ketoconazole as determined by hyphal elongation assessment. Nine of these isolates exhibited colony morphology variation or switching at 37 degrees C, of which six expressed low ketoconazole susceptibility. To determine whether colony morphology variation could give rise to derivatives with reduced azole susceptibility, several high-frequency switching variants of three HIV-patient isolates were recovered and assessed. All but one of the variants expressed similar azole susceptibility profiles to their respective parental strains. However, the C. albicans derivative 132ACR expressed significantly reduced susceptibility to ketoconazole in comparison to its parental strain 132A. In whole cells, on the basis of total growth the switched derivative 132ACR was markedly less susceptible than its parental isolate 132A to ketoconazole at 10 microM. A much smaller difference was observed with fluconazole at 10 microM, with the switched derivative 132ACR exhibiting a threefold lower susceptibility compared with the parental isolate 132A. The incorporation of [14C]acetate in control and azole-treated cells of both organisms was higher for the parental strain. When cell lysates of strain 132A and its derivative 132ACR were incubated with [14C]mevalonic acid and ketoconazole, the IC50 for 14C-label incorporation into C-4 demethyl sterols was fivefold higher for lysates of the switched derivative 132ACR compared with those of the parental strain 132A. With fluconazole the IC50 value for the derivative 132ACR was 25-fold higher than for strain 132A. The 14-sterol demethylase of the switched derivative 132ACR was possibly less sensitive to azole inhibition than that of the enzyme of strain 132A. These studies indicated that colony morphology variation in vitro can generate derivatives with stable, reduced azole susceptibility without prior exposure to azoles.     

rDNA-based genotyping of clinical isolates of Candida albicans

The study presents an analysis of the restriction pattern ofrDNA fragments of 95 C. albicans isolates previously classified on the basis of the presence of the intron in rDNA into genotypes A (62 isolates), B (28), and C (5). Most isolates (61) with genotype A were classified as "subtype a" and one as "subtype d" (Karahan and Akar; 2005). No differences were observed in the restriction patterns of the tested genotype B isolates. Similarly, most genotype C strains (4/5) showed the same restriction pattern. The results indicate low subtyping variations of the analyzed isolates, which is in contrast to published data obtained from a Turkish collection of yeasts.