Steven Park

Multilocus Sequence Typing Is a Reliable Alternative Method to DNA Fingerprinting for Discriminating among Strains of Candida albicans

ABSTRACT Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for Candida albicans have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of C. albicans, we applied it to a previously well-characterized set of 29 C. albicans isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques.

A Naturally Occurring Proline-to-Alanine Amino Acid Change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis Accounts for Reduced Echinocandin Susceptibility

ABSTRACT 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 Vmax 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.

High-frequency Triazole Resistance Found In Nonculturable Aspergillus fumigatus from Lungs of Patients with Chronic Fungal Disease

BACKGROUND Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. METHODS Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance. RESULTS Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling. CONCLUSIONS The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.

Multiple Resistance Mechanisms among Aspergillus fumigatus Mutants with High-Level Resistance to Itraconazole

ABSTRACT A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 μg ml−1 were selected in vitro (following UV irradiation as a preliminary step) to investigate mechanisms of drug resistance in this clinically important pathogen. Eight of the RIT mutants were found to have a mutation at Gly54 (G54E, -K, or -R) in the azole target gene CYP51A. Primers designed for highly conserved regions of multidrug resistance (MDR) pumps were used in reverse transcriptase PCR amplification reactions to identify novel genes encoding potential MDR efflux pumps in A. fumigatus. Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail. Analysis of the deduced amino acid sequence encoded by AfuMDR3 revealed high similarity to major facilitator superfamily transporters, while AfuMDR4 was a typical member of the ATP-binding cassette superfamily. Real-time quantitative PCR with molecular beacon probes was used to assess expression levels of AfuMDR3 and AfuMDR4. Most RIT mutants showed either constitutive high-level expression of both genes or induction of expression upon exposure to itraconazole. Our results suggest that overexpression of one or both of these newly identified drug efflux pump genes of A. fumigatus and/or selection of drug target site mutations are linked to high-level itraconazole resistance and are mechanistic considerations for the emergence of clinical resistance to itraconazole.

Effect of Candida glabrata FKS1 and FKS2 Mutations on Echinocandin Sensitivity and Kinetics of 1,3-β-d-Glucan Synthase: Implication for the Existing Susceptibility Breakpoint

ABSTRACT 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-β-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 (Vmax) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-β-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 μg/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 μg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of ≥2 μg/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.

Correlating Echinocandin MIC and Kinetic Inhibition of fks1 Mutant Glucan Synthases for Candida albicans: Implications for Interpretive Breakpoints

ABSTRACT A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-β-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 Ki 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 Km values, although some mutations decreased the Vmax. Given the association of FKS1 mutations with clinical resistance, an evaluation of the kinetic parameters for the inhibition of mutant 1,3-β-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 μg/ml for caspofungin captured nearly 100% of fks1 C. albicans strains when a kinetic inhibition rise threshold of ≤50-fold for the Ki 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 μg/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 μg/ml for the three echinocandin drugs. The 1,3-β-d-glucan synthase kinetic inhibition data support the proposed susceptibility breakpoint for caspofungin in C. albicans, but a lower susceptibility breakpoint (≤0.5 μg/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.

Assessing resistance to the echinocandin antifungal drug caspofungin in Candida albicans by profiling mutations in FKS1.

ABSTRACT Resistance of clinical isolates of Candida albicans to the echinocandin drug caspofungin is slowly emerging and is linked to mutations in short conserved regions in the FKS1 gene. The most prominent changes occurred at the serine 645 position in Fks1p with substitutions of proline, tyrosine, and phenylalanine. An allele-specific real-time PCR molecular-beacon assay was developed for rapid identification of drug resistance by targeting FKS1 mutations. Mutations altering serine 645 were reliably identified in both heterozygous and homozygous states. The molecular-beacon assay was used to evaluate two large collections of spontaneous mutants from separate strains of C. albicans with resistance (MICs, >16 μg/ml) to caspofungin with the goal of understanding the relationship between FKS1 mutations and echinocandin resistance. Of 85 resistant isolates recovered, all were identified with mutations in FKS1; 93% showed changes at Ser645, with 62% displaying a characteristic S645P substitution expressed as either a homozygous or a heterozygous mutation in FKS1. Two other prominent amino acid substitutions, S645Y and S645F, were found at frequencies of 22% and 8%, respectively. Three new mutations were also identified: T1922C, G1932T, and C1934G, encoding F641S, L644F, and S645C substitutions, respectively. One strain had the double amino acid substitution L644F and S645C. Allele-specific probes were combined in a multiplex assay for reliable screening of known FKS1 mutations. These data support the importance of FKS1p substitutions in echinocandin resistance and demonstrate the feasibility of applying molecular screening for routine resistance assessment.

Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole 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.

Calcineurin Target CrzA Regulates Conidial Germination, Hyphal Growth, and Pathogenesis of Aspergillus fumigatus†

ABSTRACT The calcineurin pathway is a critical signal transduction pathway in fungi that mediates growth, morphology, stress responses, and pathogenicity. The importance of the calcineurin pathway in fungal physiology creates an opportunity for the development of new antifungal therapies that target this critical signaling pathway. In this study, we examined the role of the zinc finger transcription factor Crz1 homolog (CrzA) in the physiology and pathogenicity of the opportunistic human fungal pathogen Aspergillus fumigatus. Genetic replacement of the crzA locus in A. fumigatus resulted in a strain with significant defects in conidial germination, polarized hyphal growth, cell wall structure, and asexual development that are similar to but with differences from defects seen in the A. fumigatus ΔcnaA (calcineurin A) strain. Like the ΔcnaA strain, the ΔcrzA strain was incapable of causing disease in an experimental persistently neutropenic inhalational murine model of invasive pulmonary aspergillosis. Our results suggest that CrzA is an important downstream effector of calcineurin that controls morphology in A. fumigatus, but additional downstream effectors that mediate calcineurin signal transduction are likely present in this opportunistic fungal pathogen. In addition, the importance of CrzA to the production of disease is critical, and thus CrzA is an attractive fungus-specific antifungal target for the treatment of invasive aspergillosis.

Acquired Echinocandin Resistance in a Candida krusei Isolate Due to Modification of Glucan Synthase

ABSTRACT A Candida krusei strain from a patient with acute myelogenous leukemia that displayed reduced susceptibility to echinocandin drugs contained a heterozygous mutation, T2080K, in FKS1. The resulting Phe655→Cys substitution altered the sensitivity of glucan synthase to echinocandin drugs, consistent with a common mechanism for echinocandin resistance in Candida spp.