Novel angular naphthopyrone formation by Arp1p dehydratase involved in Aspergillus fumigatus melanin biosynthesis.

Conidial pigment is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. The biosynthetic gene cluster for 1,8-dihydroxynaphthalene (DHN)-melanin in A. fumigatus consists of six genes, alb1, ayg1, arp1, arp2, abr1 and abr2. In contrast to black DHN-melanin fungi such as Magnaporthe grisea, the polyketide synthase Alb1p in A. fumigatus produces naphthopyrone YWA1 instead of 1,3,6,8-THN (T4HN) and YWA1 is converted to T4HN by Ayg1p. The yeast transformant expressing Alb1p and Arp1p dehydratase produced an unknown compound which was identified to be a novel angular naphthopyrone named YWA3 formed from YWA1. In addition, the amount of YWA3 produced was much more than that of YWA2 formed by non-enzymatic dehydration from YWA1. To further analyse the reaction in vitro, Arp1p was overexpressed in E. coli and purified. Kinetic analysis revealed Km value of Arp1p for YWA1 to be 41 µM which is comparable with that of Ayg1p for YWA1 in conversion to T4HN. The complex structure modelling well explained the mechanism of YWA3 generation by the dehydration of angular YWA1 by Arp1p. These results indicated the possibility that polymerization of angular naphthopyrone YWA3 but not YWA2 could be involved in the characteristic bluish-green conidial pigmentation of A. fumigatus.

Sterol uptake and sterol biosynthesis act coordinately to mediate antifungal resistance in Candida glabrata under azole and hypoxic stress.

Pathogenic fungi, including Candida glabrata, develop strategies to grow and survive both in vitro and in vivo under azole stress. However, the mechanisms by which yeast cells counteract the inhibitory effects of azoles are not completely understood. In the current study, it was demonstrated that the expression of the ergosterol biosynthetic genes ERG2, ERG3, ERG4, ERG10, and ERG11 was significantly upregulated in C. glabrata following fluconazole treatment. Inhibiting ergosterol biosynthesis using fluconazole also increased the expression of the sterol influx transporter AUS1 and the sterol metabolism regulators SUT1 and UPC2 in fungal cells. The microarray study quantified 35 genes with elevated mRNA levels, including AUS1, TIR3, UPC2, and 8 ERG genes, in a C. glabrata mutant strain lacking ERG1, indicating that sterol importing activity is increased to compensate for defective sterol biosynthesis in cells. Bioinformatic analyses further revealed that those differentially expressed genes were involved in multiple cellular processes and biological functions, such as sterol biosynthesis, lipid localization, and sterol transport. Finally, to assess whether sterol uptake affects yeast susceptibility to azoles, we generated a C. glabrata aus1∆ mutant strain. It was shown that loss of Aus1p in C. glabrata sensitized the pathogen to azoles and enhanced the efficacy of drug exposure under low oxygen tension. In contrast, the presence of exogenous cholesterol or ergosterol in medium rendered the C. glabrata AUS1 wild­type strain highly resistant to fluconazole and voriconazole, suggesting that the sterol importing mechanism is augmented when ergosterol biosynthesis is suppressed in the cell, thus allowing C. glabrata to survive under azole pressure. On the basis of these results, it was concluded that sterol uptake and sterol biosynthesis may act coordinately and collaboratively to sustain growth and to mediate antifungal resistance in C. glabrata through dynamic gene expression in response to azole stress and environmental challenges.

Transcriptional profiling of Candida glabrata during phagocytosis by neutrophils and in the infected mouse spleen.

Expression microarray analysis of Candida glabrata following phagocytosis by human neutrophils was performed, and results were compared with those from C. glabrata incubated under conditions of carbohydrate or nitrogen deprivation. Twenty genes were selected to represent the major cell processes altered by phagocytosis or nutrient deprivation. Quantitative real-time PCR (qRT-PCR) with TaqMan chemistry was used to assess expression of the same genes in spleens of mice infected intravenously with Candida glabrata. The results in spleen closely paralleled gene expression in neutrophils or following carbohydrate deprivation. Fungal cells responded by upregulating alternative energy sources through gluconeogenesis, glyoxylate cycle, and long-chain fatty acid metabolism. Autophagy was likely employed to conserve intracellular resources. Aspartyl protease upregulation occurred and may represent defense against attacks on cell wall integrity. Downregulated genes were in the pathways of protein and ergosterol synthesis. Upregulation of the sterol transport gene AUS1 suggested that murine cholesterol may have been used to replace ergosterol, as has been reported in vitro. C. glabrata isolates in spleens of gp91(phox-/-) knockout mice with reduced oxidative phagocyte defenses were grossly similar although with a reduced level of response. These results are consistent with reported results of other fungi responding to phagocytosis, indicating that a rapid shift in metabolism is required for growth in a carbohydrate-limited intracellular environment.

STB5 is a negative regulator of azole resistance in Candida glabrata.

The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals (J. E. Bennett, K. Izumikawa, and K. A. Marr. Antimicrob. Agents Chemother. 48:1773-1777, 2004). Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown that Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata, the open reading frame (ORF) designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w) and shares an N-terminal Zn(2)Cys(6) binuclear cluster domain and a fungus-specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of a Δyhr178w (Δstb5) mutant with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide, and caffeine of the mutant, for which reason we designated CAGl0I02552g CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 caused a modest increase in resistance. Expression analysis found that CgSTB5 shares many transcriptional targets with CgPDR1 but, unlike the latter, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1, PDH1, and YOR1.

Prion-forming ability of Ure2 of yeasts is not evolutionarily conserved.

[URE3] is a prion (infectious protein) of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. We show that wild S. paradoxus can be infected with a [URE3] prion, supporting the use of S. cerevisiae as a prion test bed. We find that the Ure2p of Candida albicans and C. glabrata also regulate nitrogen catabolism. Conservation of amino acid sequence within the prion domain of Ure2p has been proposed as evidence that the [URE3] prion helps its host. We show that the C. albicans Ure2p, which does not conserve this sequence, can nonetheless form a [URE3] prion in S. cerevisiae, but the C. glabrata Ure2p, which does have the conserved sequence, cannot form [URE3] as judged by its performance in S. cerevisiae. These results suggest that the sequence is not conserved to preserve prion forming ability.

Microarray and molecular analyses of the azole resistance mechanism in Candida glabrata oropharyngeal isolates.

DNA microarrays were used to analyze Candida glabrata oropharyngeal isolates from seven hematopoietic stem cell transplant recipients whose isolates developed azole resistance while the recipients received fluconazole prophylaxis. Transcriptional profiling of the paired isolates revealed 19 genes upregulated in the majority of resistant isolates compared to their paired susceptible isolates. All seven resistant isolates had greater than 2-fold upregulation of C. glabrata PDR1 (CgPDR1), a master transcriptional regulator of the pleiotropic drug resistance (PDR) network, and all seven resistant isolates showed upregulation of known CgPDR1 target genes. The altered transcriptome can be explained in part by the observation that all seven resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 open reading frame. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, and S391L) and one in the activation domain (G943S), while two mutations (N764I and R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive isolate and five resistant isolates into a laboratory azole-hypersusceptible strain (Cgpdr1 strain) via integrative transformation. The Cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic backgrounds, upregulation of CgPDR1 and CgPDR1 target genes varied between the five transformants, independent of the domain locations in which the mutations occurred. In summary, gain-of-function mutations in CgPDR1 contributed to the clinical azole resistance, but different mutations had various degrees of impact on the CgPDR1 target genes.

The bZip transcription factor Cgap1p is involved in multidrug resistance and required for activation of multidrug transporter gene CgFLR1 in Candida glabrata.

Transcriptional regulation in response to environmental challenges is crucial for survival of many organisms. In this study, we characterized structural and functional properties of CgAP1, a Saccharomyces cerevisiae YAP1 ortholog, which encodes a transcription factor involved in various stress responses. Deletion of CgAP1 led to decreased resistance to hydrogen peroxide, 4-nitroquinoline-N-oxide (4-NQO), benomyl, and cadmium chloride, which could be fully recovered by reintroduction of an intact CgAP1. CgAP1 was shown to function in S. cerevisiae as it restored the drug resistance of the yap1 mutant. Moreover, overexpression of CgAP1 in a S. cerevisiae wild-type strain increased its resistance to cycloheximide, 1,10-phenanthroline, 4-NQO, and fluconazole. Overexpression of CgAP1 also phenotypically suppressed the drug sensitivity of two Yap1p-regulated transporter mutants, Deltaatr1 and Deltaflr1, to diamide, 4-NQO, and cadmium. Northern blot analysis indicated that Cgap1p regulates the benomyl-induced expression of CgFLR1, a homolog of S. cerevisiae FLR1, which encodes a transporter of the major facilitator superfamily. In contrast to the S. cerevisiae flr1 mutant, deletion of CgFLR1 in C. glabrata only resulted in increased sensitivity to benomyl, diamide, and menadione, but not 4-NQO, cycloheximide, or fluconazole. Taken together, this report demonstrated that CgAP1 plays a critical role in response to various stresses in C. glabrata and reduces the stress through transcriptional activation of its target genes including CgFLR1.

Kre29p is a novel nuclear protein involved in DNA repair and mitotic fidelity in Candida glabrata.

Candida glabrata KRE29 is an ortholog of Saccharomyces cerevisiae KRE29. S. cerevisiae Kre29p has been identified by affinity purification as a subunit of the Smc5-Smc6 complex, which is required for DNA repair and chromosome segregation. However, mutant phenotypes of S. cerevisiae KRE29 have not been well characterized and none of its orthologs' functions has been reported. Here we report phenotypic characteristics of a C. glabrata kre29 deletant. The absence of C. glabrata Kre29p resulted in decreased viability, exhibiting cell cycle arrest between late S-phase and metaphase even under normal growth conditions, and also caused an increase of plasmid loss rate, implying that Kre29p is required for mitotic chromosome transmission fidelity. The deletant showed increased sensitivity to high temperature as well as to DNA damaging agents including UV, gamma ray, 4-nitroquinoline-1-oxide and methyl methanesulfonate, and the phenotypes were restored in the KRE29 reintegrant. Consistent with the Deltakre29 phenotypes, a Kre29p-GFP fusion protein was located in the nucleus. Furthermore, Kre29p-GFP became concentrated and formed distinct foci after exposure to 4-nitroquinoline-1-oxide. These results suggest the involvement of C. glabrata Kre29p in DNA repair. To our knowledge, this is the first report addressing a cellular protein involved in DNA repair in C. glabrata.

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.

THTA, a thermotolerance gene of Aspergillus fumigatus.

Aspergillus fumigatus grows optimally from 37 to 42 degrees C but can grow at temperatures up to 55 degrees C. To study the genetic basis of thermotolerance and its role in virulence of A. fumigatus, temperature sensitive mutants were isolated. One of the mutants that grew at 42 degrees C but not at 48 degrees C was complemented and the gene, THTA, was identified. Deletion of THTA showed the same temperature sensitivity as the original mutant. THTA encodes a putative protein of 141 kDa with unknown function and the HA-tagged ThtAp accumulated to similar levels in cultures grown at either 37 or 48 degrees C. Southern blot analysis and database searches revealed the presence of THTA-related sequences in several other ascomycetous fungi. No difference in virulence was observed between the deltathtA and wild-type strains. Thus, THTA is essential for growth of A. fumigatus at high temperatures but does not contribute to the pathogenicity of the species.

Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis.

The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalene-melanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp352-His380-Ser257 constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser257-O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.

Candida glabrata erg1 mutant with increased sensitivity to azoles and to low oxygen tension.

A Candida glabrata erg1 (Cgerg1) mutant, CgTn201S, was identified by transposon mutagenesis and by increased fluconazole susceptibility. CgERG1 encodes a 489-amino-acid protein which, on the basis of its homology with Saccharomyces cerevisiae ERG1, is a squalene epoxidase essential for ergosterol synthesis. Interruption following codon 475 of CgErg1p decreased the ergosterol content by 50%; caused accumulation of the squalene precursor; increased the levels of susceptibility to fluconazole, itraconazole, and terbinafine; increased the level of resistance to amphotericin B; increased the levels of rhodamine 6G and [(3)H]-fluconazole uptake; reduced the level of growth; and blocked growth under conditions of low oxygen tension. In addition, CgTn201S efficiently took up exogenous cholesterol from cholesterol-containing serum. Cholesterol constituted 34% of the extractable sterols in CgTn201S when it was grown aerobically on serum-containing medium. Under the same conditions, C. albicans contained only 0.1 to 1.2% cholesterol. Exogenous sterols also restored growth under conditions of low oxygen tension. Finally, complementation of the Cgerg1 mutation restored the levels of [(3)H]fluconazole uptake and drug susceptibility to wild-type levels.

Cloning and functional characterization of the Rhizopus oryzae high affinity iron permease (rFTR1) gene.

Rhizopus oryzae is the most common etiologic agent of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. Therefore, the high affinity iron permease (rFTR1) which encodes a protein required to scavenge iron from the environment, is highly likely to be a critical determinant of virulence for R. oryzae. We have cloned rFTR1 by using a PCR approach relying on degenerate primers designed from the conserved regions of Saccharomyces cerevisiae high affinity iron permease. Sequence analysis of a 2.0 kb EcoRI genomic clone revealed a single open reading frame of 1107 bp that lacked introns. The putative rFtr1p had significant homology to known fungal high affinity iron permeases from Candida albicans (46% identity) and S. cerevisiae (44% identity). In R. oryzae, rFTR1 was expressed in iron-depleted and not in iron-rich media. Finally, rFTR1 restored the ability of an ftr1 null mutant of S. cerevisiae to grow on iron-limited medium and to take up radiolabeled iron, whereas S. cerevisiae transformed with the empty vector did not. These data demonstrate that we have cloned the gene encoding a R. oryzae high affinity iron permease and the putative rFtr1p is involved in assimilation of iron from iron-depleted environments.

The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass.

Many cool-season grasses harbor fungal endophytes in the genus Neotyphodium, which enhance host fitness, but some also produce metabolites--such as ergovaline--believed to cause livestock toxicoses. In Claviceps species the first step in ergot alkaloid biosynthesis is thought to be dimethylallyltryptophan (DMAT) synthase, encoded by dmaW, previously cloned from Claviceps fusiformis. Here we report the cloning and characterization of dmaW from Neotyphodium sp. isolate Lp1, an endophyte of perennial ryegrass (Lolium perenne). The gene was then disrupted, and the mutant failed to produce any detectable ergovaline or simpler ergot and clavine alkaloids. The disruption was complemented with the C. fusiformis gene, which restored ergovaline production. Thus, the biosynthetic role of DMAT synthase was confirmed, and a mutant was generated for future studies of the ecological and agricultural importance of ergot alkaloids in endophytes of grasses.

Function of Candida glabrata ABC transporter gene, PDH1.

The rapid increase in azole resistance during treatment of patients infected with Candida glabrata may be due to increased azole efflux mediated by ABC transporters, as occurs with increased expression of PDR5 in Saccharomyces cerevisiae. Two known C. glabrata homologues of PDR5 influencing azole susceptibility are PDH1 (CgCDR2) and CgCDR1. Disruption of PDH1 in a cgcdr1::ura3 strain increased susceptibility to rhodamine 6G, cycloheximide and chloramphenicol, and also increased rhodamine 6G accumulation, all properties of pdr5 null mutants. Overexpression of PDH1 in S. cerevisiae complemented the pdr5 mutation by reversing susceptibility to rhodamine 6G, chloramphenicol and cycloheximide, as well as by decreasing rhodamine 6G intracellular concentration. Expression of PDH1 in a C. glabrata cgcdr1::ura3 pdh1Delta::ura3 mutant using a multicopy plasmid almost completely restored the wild-type phenotype, showing that PDH1 at higher levels of expression can replace CgCDR1. Because PDH1 and CgCDR1 have both been reported to have upstream sequences similar to the Pdr1p- and Pdr3p-binding elements of PDR5, we sought similarities in regulation between the three genes. Abundance of PDH1 and CgCDR1 mRNA in C. glabrata was increased by rhodamine 6G, cycloheximide and oligomycin, properties in common with PDR5. PDH1, CgCDR1 and PDR5 have striking similarities in function and regulation.