Molecular aspects of azole antifungal action and resistance

Lamb, David C.; Kelly, Diane; Kelly, Steven L.

doi:10.1054/drup.1999.0112

Cited by 92 publications

(85 citation statements)

References 77 publications

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“…Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 g ml ؊1 ). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.Several mechanisms can contribute to azole resistance in pathogenic fungi, such as Candida albicans (4,11,14,32,33). There has been an increase in research surrounding the potential importance of drug efflux transporters (26, 30) and changes in sterol 14␣-demethylase (ERG11 [CYP51]), the target of azole antifungals (10,12,13,14,25).…”

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confidence: 99%

A Clinical Isolate of Candida albicans with Mutations in ERG11 (Encoding Sterol 14α-Demethylase) and ERG5 (Encoding C22 Desaturase) Is Cross Resistant to Azoles and Amphotericin B

Martel

Parker

Bader

et al. 2010

Antimicrob Agents Chemother

146

102

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A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 g ml ؊1 , respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 g ml ؊1 for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14␣-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 g ml ؊1 ). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.Several mechanisms can contribute to azole resistance in pathogenic fungi, such as Candida albicans (4,11,14,32,33). There has been an increase in research surrounding the potential importance of drug efflux transporters (26, 30) and changes in sterol 14␣-demethylase (ERG11 [CYP51]), the target of azole antifungals (10,12,13,14,25). Biofilm formation (2) and the possibility for azole sequestration mechanisms (15) have also attracted attention. Following the identification of defective sterol ⌬ 5,6 -desaturase (encoded by ERG3) as a mechanism of azole resistance in Saccharomyces cerevisiae some 20 years ago (31), this mechanism has also been reported in clinical C. albicans isolates (3,8,9,19,21). There remains sustained interest in the regulation of the ERG genes and proteins that mediate fungal sterol (specifically ergosterol) biosynthesis ( Fig. 1) in azole-resistant isolates. Many antifungal compounds that are currently available to clinicians target either ergosterol (e.g., polyene antifungals) or the enzymes central to its synthesis (e.g., azole inhibitors of sterol 14␣-demethylase), and hence, the potential for the emergence of cross-resistant strains exists.Data suggest that azole inhibitors of sterol 14␣-demethylase (here called Erg11p) also bind to C22 desaturase (here called Erg5p) (6); however, the possibility that the latter could constitute a target for new antifungal compounds (6, 7) remains understudied. Here, we present information on the phenotypic and genotypic characteristics of a novel azole-resistant strain (CA108) that was initially identified as an erg5 mutant completely lacking ergosterol, using gas chromatography and mass spectrometry (GC/MS) (24). Given the importance of ergosterol for maintaining fungal cell membrane integrity (1), we were keen to investigate how CA108 was able to...

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confidence: 99%

A Clinical Isolate of Candida albicans with Mutations in ERG11 (Encoding Sterol 14α-Demethylase) and ERG5 (Encoding C22 Desaturase) Is Cross Resistant to Azoles and Amphotericin B

Martel

Parker

Bader

et al. 2010

Antimicrob Agents Chemother

146

102

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“…To test if the apoptotic-like phenotype was induced by agents that inhibited growth, without being fungicidal, we used the fungistatic azole itraconazole, which in the short term inhibits growth, without killing the cell (Lamb et al, 1999). Treatment of protoplasts with 10 mg itraconazole ml…”

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confidence: 99%

Oxidative and amphotericin B-mediated cell death in the opportunistic pathogen Aspergillus fumigatus is associated with an apoptotic-like phenotype

Mousavi

Robson

2004

Microbiology

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When protoplasts of the opportunistic fungal pathogen Aspergillus fumigatus were treated with low but toxic levels of hydrogen peroxide (0?1 mM) or amphotericin B (0?5 mg ml "1 ), loss of cell viability and death were associated with a number of phenotypic changes characteristic of apoptosis. The percentage of protoplasts staining positive with annexin V-FITC, an indicator of the externalization of phosphatidylserine and an early marker of apoptosis, rose to~55 % within 1 h. This was followed by a similar increase in apoptotic DNA fragmentation detected by the TUNEL assay, and led to a loss of cell permeability and death in~90 % of protoplasts, as indicated by the uptake of propidium iodide. The development of an apoptotic phenotype was blocked when protoplasts were pre-treated with the protein synthesis inhibitor cycloheximide, indicating active participation of the cell in the process. However, no significant activity against synthetic caspase substrates was detected, and the inclusion of the cell-permeant broad-spectrum caspase inhibitor Z-VAD-fmk did not block the development of the apoptotic-like phenotype. Higher concentrations of H 2 O 2 (1?8 mM) and amphotericin B (1 mg ml "1 ) caused protoplasts to die without inducing an apoptotic phenotype. As predicted, the fungistatic antifungal agent itraconazole, which inhibits growth without causing immediate cell death, did not induce an apoptotic-like phenotype. INTRODUCTIONThe incidence of life-threatening invasive aspergillosis in immunocompromised hosts has increased dramatically over the last two decades (Groll et al., 1996;Vogeser et al., 1997;Latgé, 1999;Denning et al., 2002). Aspergillus fumigatus is the most common aetiological agent of invasive aspergillosis, being responsible for approximately 90 % of human infections (Derouin, 1994). The incidence of invasive aspergillosis varies between 1 and 19 % for patients who have undergone solid organ transplantation (Patel & Paya, 1997;Verweij & Denning, 1997), and patients with leukaemia, AIDS and granulomatous disease are also at risk (Brown et al., 1998;Denning, 1998;Kaizer et al., 1998). In contrast, the disease is rarely found in immunocompetent hosts (Karim et al., 1997). A. fumigatus is a common, widespread saprophytic fungus, and environmental surveys indicate that humans inhale several hundred A. fumigatus conidia per day (Hospenthal et al., 1998). Ingestion and killing of spores by alveolar macrophages represent the first line of defence against infection, mainly by non-oxidative mechanisms, whilst neutrophils employ an oxidative respiratory burst to kill germinating conidia that have escaped macrophage engulfment (Schaffner et al., 1986;Levitz et al., 1986; Morgenstern et al., 1997;Roilides et al., 1998; Latgé, 2001). Invasive aspergillosis has a high mortality and morbidity rate, with only 34 % of patients showing a favourable response to antifungal therapy (Denning, 1996). The fungicidal agent amphotericin B is widely used to treat invasive aspergillosis, although it can have serious side-effect...

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“…We also found that econazole and ketoconazole inhibit MAC growth in culture, whereas the other tested azoles had no effect. As a key enzyme in sterol biosynthesis, CYP51 has been a target for antifungal drug design (24,33). Azole compounds have proven efficacy for treating localized and systemic fungal infections; however, not all fungi respond to individual azole agents.…”

Section: Discussionmentioning

confidence: 99%

“…The difference in the molecular structure of azole compounds affects their solubility and their ability to bind and inhibit CYP51 enzymes (33). Econazole and ketoconazole are imidazoles, which have five-membered ring structures containing two nitrogen atoms.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Characterization of CYP51 from Mycobacterium avium

Pietila

Vohra

Sanyal

et al. 2006

Am J Respir Cell Mol Biol

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Mycobacterium avium complex (MAC) causes chronic lung disease in immunocompetent people and disseminated infection in patients with AIDS. MAC is intrinsically resistant to many conventional antimycobacterial agents, it develops drug resistance rapidly to macrolide antibiotics, and patients with MAC infection experience frequent relapses or the inability to completely eradicate the infection with current treatment. Treatment regimens are prolonged and complicated by drug toxicity or intolerances. We sought to identify biochemical pathways in MAC that can serve as targets for novel antimycobacterial treatment. The cytochrome P450 enzyme, CYP51, catalyzes an essential early step in sterol metabolism, removing a methyl group from lanosterol in animals and fungi, or from obtusifoliol in plants. Azoles inhibit CYP51 function, leading to an accumulation of methylated sterol precursors. This perturbation of normal sterol metabolism compromises cell membrane integrity, resulting in growth inhibition or cell death. We have cloned and characterized a CYP51 from MAC that functions as a lanosterol 14␣-demethylase. We show the direct interactions of azoles with purified MAC-CYP51 by absorbance and electron paramagnetic resonance spectroscopy, and determine the minimum inhibitory concentrations (MICs) of econazole, ketoconazole, itraconazole, fluconazole, and voriconazole against MAC. Furthermore, we demonstrate that econazole has a MIC of 4 g/ml and a minimum bacteriocidal concentration of 4 g/ml, whereas ketoconazole has a MIC of 8 g/ml and a minimum bacteriocidal concentration of 16 g/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.

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Molecular aspects of azole antifungal action and resistance

Cited by 92 publications

References 77 publications

A Clinical Isolate of Candida albicans with Mutations in ERG11 (Encoding Sterol 14α-Demethylase) and ERG5 (Encoding C22 Desaturase) Is Cross Resistant to Azoles and Amphotericin B

A Clinical Isolate of Candida albicans with Mutations in ERG11 (Encoding Sterol 14α-Demethylase) and ERG5 (Encoding C22 Desaturase) Is Cross Resistant to Azoles and Amphotericin B

Oxidative and amphotericin B-mediated cell death in the opportunistic pathogen Aspergillus fumigatus is associated with an apoptotic-like phenotype

Cloning and Characterization of CYP51 from Mycobacterium avium

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