Fourteen Aspergillus fumigatus clinical isolates that exhibited a pattern of reduced susceptibility to triazole drugs were analyzed. The sequences of the cyp51A gene from all isolates showed the presence of a point mutation at t364a, which led to the substitution of leucine 98 for histidine (L98H), together with the presence of two copies of a 34-bp sequence in tandem in the promoter of the cyp51A gene. Quantitative expression analysis (real-time PCR) showed up to an eightfold increase in the level of expression of the cyp51A gene compared to that by the susceptible strain. Three PCR fragments of one azole-resistant strain (strain CM2627) that included the promoter with the tandem repeat and part of cyp51A with the t364a mutation or PCR fragments with only one of the modifications were used to replace the cyp51A gene of an azole drug-susceptible A. fumigatus wild-type strain (strain CM237). Only transformants which had incorporated the tandem repeat in the promoter of the cyp51A gene and the L98H amino acid substitution exhibited similarly reduced patterns of susceptibility to all triazole agents and similarly increased levels of cyp51A expression, confirming that the combination of both alterations was responsible for the azoleresistant phenotype.

Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-␤-D-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (V max ) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-␤-D-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 g/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 g/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of >2 g/ml for the three echinocandin drugs. As has been observed with Candida albicans, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS-mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.

Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced V max values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.

A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-␤-D-glucan synthase enzymes from clinical isolates of Candida albicans with nine different FKS1 mutations resulting in high MICs. Among 14 mutant Fks1p enzymes studied, the kinetic parameters 50% inhibitory concentration and K i increased 50-fold to several thousandfold relative to those for the wild type. Enzymes with mutations at Ser645 (S645P, S645Y, and S645F) within hot spot 1 showed the most prominent decrease in sensitivity, while those with mutations at the N-and C-terminal ends of hot spot 1 generally retained greater sensitivity to all three drugs. Kinetic inhibitions by caspofungin, micafungin, and anidulafungin were comparable among the fks1 mutant enzymes, although absolute values did vary with specific mutations. Amino acid substitutions in Fks1p did not alter K m values, although some mutations decreased the V max . Given the association of FKS1 mutations with clinical resistance, an evaluation of the kinetic parameters for the inhibition of mutant 1,3-␤-D-glucan synthase as a function of the MIC enabled an independent evaluation of the recently adopted susceptibility breakpoint for echinocandin drugs. Overall, a breakpoint MIC of >2 g/ml for caspofungin captured nearly 100% of fks1 C. albicans strains when a kinetic inhibition rise threshold of <50-fold for the K i was used as a measure of susceptibility. A similar MIC breakpoint for micafungin and anidulafungin was less inclusive, and a projected MIC of >0.5 g/ml was required for >95% coverage of clinical isolates. However, when MIC determinations were performed in the presence of 50% serum, all fks1 mutants showed MIC values of >2 g/ml for the three echinocandin drugs. The 1,3-␤-D-glucan synthase kinetic inhibition data support the proposed susceptibility breakpoint for caspofungin in C. albicans, but a lower susceptibility breakpoint (<0.5 g/ml) may be more appropriate for anidulafungin and micafungin. Overall, the data indicate that MIC testing with caspofungin may serve as a surrogate marker for resistance among the class of echinocandin drugs.

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma − phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

Five clinical isolates of Aspergillus fumigatus that exhibited similar patterns of reduced susceptibility to itraconazole and other triazole drugs were analyzed. Sequence analysis of genes (cyp51A and cyp51B) encoding the 14␣-sterol demethylases revealed that all five strains harbored mutations in cyp51A resulting in the replacement of methionine at residue 220 by valine, lysine, or threonine. When the mutated cyp51A genes were introduced into an A. fumigatus wild-type strain, the transformants exhibited reduced susceptibility to all triazole agents, confirming that the mutations were responsible for the resistance phenotype.

For Candida species, a bimodal wild-type MIC distribution for echinocandins exists, but resistance to echinocandins is rare. We characterized isolates from patients with invasive candidiasis (IC) breaking through >3 doses of micafungin therapy during the first 28 months of its use at our center: MICs were determined and hot-spot regions within FKS genes were sequenced. Eleven of 12 breakthrough IC cases identified were in transplant recipients. The median duration of micafungin exposure prior to breakthrough was 33 days (range, 5 to 165). Seventeen breakthrough isolates were recovered: FKS hot-spot mutations were found in 5 C. glabrata and 2 C. tropicalis isolates; of these, 5 (including all C. glabrata isolates) had micafungin MICs of >2 g/ml, but all demonstrated caspofungin MICs of >2 g/ml. Five C. parapsilosis isolates had wild-type FKS sequences and caspofungin MICs of 0.5 to 1 g/ml, but 4/5 had micafungin MICs of >2 g/ml. The remaining isolates retained echinocandin MICs of <2 g/ml and wild-type FKS gene sequences. Breakthrough IC on micafungin treatment occurred predominantly in severely immunosuppressed patients with heavy prior micafungin exposure. The majority of cases were due to C. glabrata with an FKS mutation or wild-type C. parapsilosis with elevated micafungin MICs. MIC testing with caspofungin identified all mutant strains. Whether the naturally occurring polymorphism within the C. parapsilosis FKS1 gene responsible for the bimodal wild-type MIC distribution is also responsible for micafungin MICs of >2 g/ml and clinical breakthrough or an alternative mechanism contributes to the nonsusceptible echinocandin MICs in C. parapsilosis requires further study.Invasive candidiasis (IC) is an important, life-threatening infection in hospitalized patients. The echinocandins (micafungin, caspofungin, and anidulafungin) are the newest class of medications approved for the prophylaxis and treatment of IC. They act via noncompetitive inhibition of ␤-1,3-glucan synthase, the enzyme responsible for producing ␤-1,3-D-glucan in the fungal cell wall (41). These drugs have low toxicity and few drug-drug interactions and possess a broad spectrum of antifungal activity against Candida species, including those resistant to fluconazole. In clinical trials, the echinocandins have demonstrated noninferiority for the treatment of IC versus amphotericin B deoxycholate, liposomal amphotericin B, and fluconazole (25,32,44). The echinocandins are considered interchangeable for clinical use, and a recent study comparing micafungin to caspofungin for IC supports this notion (38). Based on the accumulated experience, echinocandins are now considered a first-line therapeutic choice for IC (37).The echinocandins exhibit a bimodal MIC distribution among Candida species. MICs of C. parapsilosis, C. guilliermondii, and C. famata MICs (MIC 90 , 0.25 to 2 g/ml) are up to 133 times higher than those of C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr (MIC 90 , 0.015 to 0.25 g/ml) (42). However, this difference has not...

The role of Aspergillus fumigatus 14␣-sterol demethylase (Cyp51A) in azole drug susceptibility was assessed. Targeted disruption of cyp51A in azole-susceptible and -resistant strains decreased MICs from 2-to 40-fold. The cyp51A mutants were morphologically indistinguishable from the wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice.In Aspergillus fumigatus, there are two distinct but related Cyp51 proteins encoded by cyp51A and cyp51B (7). Erg11 activity has been shown not to be essential in yeast (6, 16) but to date, there are not reports on Cyp51 functional studies in any filamentous fungi. Functional analysis of A. fumigatus Cyp51A by targeted disruption of the cyp51A gene in three clinical strains was performed.Strains. The strains used in this study were A. fumigatus strain CM-237, which was used for describing the sequence of cyp51A and cyp51B (7), and two clinical A. fumigatus strains, CNM-CM-1252 (AF-90) and CNM-CM-796 (filamentous fungus collection of the Spanish National Center for Microbiology), with elevated MICs to azole drugs (Table 1) and different Cyp51A amino acid substitutions (5, 8).Molecular cloning and DNA sequencing. The full coding sequence of cyp51A of A. fumigatus was PCR amplified as previously described (7) and cloned into the pGEM-T vector system (Promega, Madrid, Spain) to obtain plasmid pUM100. Restriction digestion of plasmid pID621 (kindly provided by D. W. Holden) was used to obtain the 1.4-kb SalI fragment of a hygromycin B (hph) resistance cassette (4) for construction of the disruption vector. The 1.4-kb hph cassette was inserted into the XhoI restriction site of pUM100 to create pUM102. A linear 3.0-kb DNA fragment obtained by SacI/SacII double digestion of pUM102 was used for A. fumigatus strain transformations (Fig. 1A).Aspergillus transformations. A. fumigatus transformation experiments were achieved by electroporation using a protocol previously described (15) with subsequent modifications (5, 18). Hygromycin B (130 g/ml; Sigma) was used for transformants selection. Mutants were named by a letter (e.g., A) followed by a number. Genomic DNAs from hygromycin-resistant transformants and the parental strain were digested with two different restriction enzymes (SalI and EcoRV; Amersham Biosciences, Madrid, Spain). Southern analysis was performed as previously described (7,14).Antifungal susceptibility testing. Broth microdilution susceptibility testing was performed as described in NCCLS document M38-A (10), with modifications (3, 11, 13). Itraconazole (ITC), ketoconazole (KTC) (both from Janssen Pharmaceutical S.A., Madrid, Spain), voriconazole, fluconazole (FLC) (both from Pfizer S.A., Madrid, Spain), ravuconazole (BristolMyers Squibb, Madrid, Spain), and amphotericin B (AmB; Sigma Aldrich Quimica, S.A., Madrid, Spain) were tested. Susceptibility tests were performed at least three times with each strain on different days.RNA extraction and LightCycler PCR. RNA extraction from the A. fumigatus CM-237 strain and the derived CM-A8 mutant st...