Search

BackgroundThe antifungal posaconazole concentrates within host cells and protects against Aspergillus fumigatus. The specific subcellular location of posaconazole and the mechanism by which cell-associated posaconazole inhibits fungal growth remain uncharacterized.MethodsPosaconazole was conjugated with the fluorophore boron-dipyrromethene (BDP-PCZ). A549 pulmonary epithelial cells and A. fumigatus were exposed to BDP-PCZ individually and in coculture. BDP-PCZ subcellular localization and trafficking were observed using confocal microscopy and flow cytometry.ResultsBDP-PCZ concentrated within A549 cell membranes, and in particular within the endoplasmic reticulum. Epithelial cell-associated BDP-PCZ rapidly transferred to and concentrated within A. fumigatus cell membranes on contact. BDP-PCZ transfer to conidia did not require phagocytosis and was markedly enhanced by the conidial hydrophobin RodA. Within AF, BDP-PCZ also concentrated in membranes including the endoplasmic reticulum and colocalized with the azole target enzyme CYP51a. Concentration of BDP-PCZ within host and fungal cell membranes persisted for >48 hours and could be competitively inhibited by posaconazole but not voriconazole.ConclusionsPosaconazole concentrates within host cell membranes and rapidly transfers to A. fumigatus, where it accumulates to high concentrations and persists at the site of its target enzyme. These intracellular and intercellular pharmacokinetic properties probably contribute to the efficacy of PCZ.

The yeast-like fungus Cryptococcus neoformans favours respiration as a mechanism of energy production, and thus depends heavily on mitochondrial function. Previous studies of a C. neoformans vad1[DELTA] mutant revealed reduced expression of the mitochondrial elongation factor TUF1 and defects in glycerol utilization, consistent with mitochondrial dysfunction. In this study, we found that in trans expression of TUF1 in the vad1[DELTA] mutant suppressed the mitochondrial defects, including growth on respiration-dependent carbon sources and fluconazole resistance, associated with VAD1 deletion. Tetracycline, an inhibitor of mitochondrial translation, was found to confer resistance to fluconazole in the wild-type and vad1[DELTA] mutant, whereas the fluconazole susceptibility of the TUFl-overexpressing strain was unaffected by tetracycline treatment. In the presence of fluconazole, the vad1[DELTA] mutant exhibited increased activation of the global transcriptional regulator Sre1. TUF1 overexpression failed to alter cleavage of Sre1 in response to fluconazole in the vad1[DELTA] mutant, suggesting that TUF1 repression in the vad1[DELTA] mutant is distal to Sre1, or that it occurs through an independent pathway. DOI 10.1099/mic.0.035923-0

Many genes in the Candida albicans ergosterol biosynthetic pathway are controlled by the transcriptional activator Upc2p, which is upregulated in the presence of azole drugs and has been suggested to regulate its own transcription by an autoregulatory mechanism. The UPC2 promoter was cloned upstream of a luciferase reporter gene (RLUC). UPC2-RLUC activity was induced in response to ergosterol biosynthesis inhibitors and in response to anaerobicity. Under both conditions, induction correlates with the magnitude of sterol depletion. Azole inducibility in the parental strain was approximately 100-fold, and in a UPC2 homozygous deletion strain was 17-fold, suggesting that, in addition to autoregulation, UPC2 transcription is controlled by a novel, Upc2p-independent mechanism(s). Curiously, basal UPC2-RLUC activity was fivefold higher in the deletion strain, which may be an indirect consequence of the lower sterol level in this strain, or a direct consequence of repression by an autoregulatory mechanism. These results suggest that transcriptional regulation of UPC2 expression is important in the response to antifungal drugs, and that this regulation occurs through Upc2p-dependent as well as novel Upc2p-independent mechanisms.

The tetracycline (TET) promoter has been used in several systems as an inducible regulator of gene expression. In control analyses, the standard Candida albicans laboratory strain SC5314 was found to have altered susceptibility to a variety of antifungal drugs in the presence of relatively high concentrations (50-200 [micro]g [ml.sup.-1]) of TET. Altered susceptibility was most notable with exposure to amphotericin B (AMB), with a 32-fold increase in susceptibility, and terbinafine (TRB), with a 32-fold decrease in susceptibility. The TET/AMB synergy was observed in several clinical isolates of C. albicans and in the distantly related species Aspergillus fumigatus and Cryptococcus neoformans. The TET/AMB synergy is not related to efflux pump activity, as determined by FACS analyses and by analysis of a strain containing efflux pump deletions. Gene expression analyses by luciferase and by quantitative real-time reverse transcriptase PCR failed to identify significant alterations in expression of any genes associated with resistance. C. albicans grown with TET for 48 h does show a reduction in total cellular ergosterol. Analysis of growth curves suggests that the TET effect is associated with lack of a diauxic shift, which is related to a loss of mitochondrial function. MitoTracker fluorescent dye was used to demonstrate that TET has a direct effect on mitochondrial function. These results demonstrate the need for careful analysis of TET effects when using a TET-inducible promoter, especially in studies that involve antifungal drugs. This study defines some limits to the use of the TET-inducible promoter, and identifies effects on cells that are the result of TET exposure alone, not the result of expression of a targeted gene.

Research has been conducted on post-translational protein modification process, prenylation which is mediated by prenyltransferases in eukaryotic cells. The analysis of the RAM2 gene of Candida albicans prenylation pathway has been carried out, and the results demonstrate that protein prenylation is indispensible cellular process in C. albicans.

Research has been conducted on the antifungal drug resistance in Candida albicans. The correlation between the heterozygosity loss at the C. albicans mating-type-like locus and resistance to the azole antifungals has been investigated and the results indicate that gene disruption of the C. albicans mating-type-like loci does not affect azole susceptibility.

The DNA fragment the exhibited a coding region for a protein with high amino acid similarity to IMP dehydrogenases was cloned and sequence from Candida albicans. The Candida albicans IMH3 gene was characterized as a 2.9-kilodalton DNA fragment that encoded an IMP dehydrogenase. The gene also conferred resistance to the specific IMP dehydrogenase inhibitor mycophenolic acid in a dose-dependent manner. Furthermore, IMH3 was expressed in equal levels in the hyphal and yeast form of Candida albicans.

Cell phenotype and environmental factors may affect the production of specific secreted aspartyl proteinase (Sap) isoenzymes in Candida albicans. Sap activity has been linked to virulence. Monitoring levels of Sap1, Sap2 and Sap3 production in different strains and different growth conditions revealed that the WO-1 strain is unique in producing Saps in certain conditions. Phenotype switching in this strain and yeast

A SAP1 gene of Candida albicans has SAP4 and CARE2 elements positioned upstream. Aspartyl proteinase is the SAP gene product. Distinctive marking of SAP1 alleles indicates the presence of SAD4 gene. Characterization of the WO-1 strain allele shows a nucleotide corresponding to the CARE2 repetitive element.

Three secreted aspartyl proteinases have been located, as characterized by molecular mass and N-terminal sequences from one strain of Candida albicans (Wo-1) in which a phenotypic switching system is expressed. The mature type of one of three particular proteinase isoenzymes, two of which correlate to SAP 1 and SAP 2 genes, is represented by these three proteins. The signals and functioning included in the change between white and opaque cells can be examined since the three proteinase isoenzymes in C. albicans have been identified and their expression has been noted.

Abstract Background The fungal pathogen Candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC), can evolve rapidly. In recent years it has become clear that plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH) at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and FLC resistance in Candida albicans. Results The data presented here support a role for homologous recombination in cell growth and DNA damage sensitivity, as Candida albicans rad54Δ/rad54Δ mutants were hypersensitive to MMS and menadione, and had an aberrant cell and nuclear morphology. The Candida albicans rad54Δ/rad54Δ mutant was defective in invasion of Spider agar, presumably due to the altered cellular morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance and cell growth, and deletion of either Candida albicans RAD54 or Candida albicans RDH54 did not alter FLC susceptibility. Conclusions Together, these results support a role for homologous recombination in genome stability under nondamaging conditions. The nuclear morphology defects in the rad54Δ/rad54Δ mutants show that Rad54 performs an essential role during mitotic growth and that in its absence, cells arrest in G2. The viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that Rdh54 can partially compensate for Rad54 during mitotic growth.

The analysis of one of the universal Leu codons, CTG, from several Candida species has shown that it is connected with strange translational events. The positions specified by the CTG codon containing a nonmodified serine (Ser) in Candida albicans are demonstrated by using mass spectrometry and Edman sequencing of peptides from the secreted aspartyl proteinase isoenzyme (Sap1). Unorthodox tRNAs that acknowledge the CTG codon and that are charged in vitro with serine (Ser) have been also identified.