Targeted Gene Disruption of the 14- Sterol Demethylase (cyp51A) in Aspergillus fumigatus and Its Role in Azole Drug Susceptibility (original) (raw)
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Antimicrobial Agents and Chemotherapy, 2004
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.
Genes, 2020
Infections caused by Aspergillus species are being increasingly reported. Aspergillus flavus is the second most common species within this genus causing invasive infections in humans, and isolates showing azole resistance have been recently described. A. flavus has three cyp51-related genes (cyp51A, cyp51B, and cyp51C) encoding 14-α sterol demethylase-like enzymes which are the target of azole drugs. In order to study triazole drug resistance in A. flavus, three strains showing reduced azole susceptibility and 17 azole susceptible isolates were compared. The three cyp51-related genes were amplified and sequenced. A comparison of the deduced Cyp51A, Cyp51B, and Cyp51C protein sequences with other protein sequences from orthologous genes in different filamentous fungi led to a protein identity that ranged from 50% to 80%. Cyp51A and Cyp51C presented several synonymous and non-synonymous point mutations among both susceptible and non-susceptible strains. However, two amino acid mutatio...
Identification of novel genes conferring altered azole susceptibility in Aspergillus fumigatus
FEMS Microbiology Letters, 2012
Azoles are currently the mainstay of antifungal treatment both in agricultural and in clinical settings. Although the target site of azole action is well studied, the basis of azole resistance and the ultimate mode of action of the drug in fungi are poorly understood. To gain a deeper insight into these aspects of azole action, restriction-mediated plasmid integration (REMI) was used to create azole sensitive and resistant strains of the clinically important fungus Aspergillus fumigatus. Four azole sensitive insertions and four azole-resistant insertions were characterized. Three phenotypes could be recreated in wildtype AF210 by reintegration of rescued plasmid and a further four could be confirmed by complementation of the mutant phenotype with a copy of the wild-type gene predicted to be disrupted by the original insertional event. Six insertions were in genes not previously associated with azole sensitivity or resistance. Two insertions occur in transporter genes that may affect drug efflux, whereas others may affect transcriptional regulation of sterol biosynthesis genes and NADH metabolism in the mitochondrion. Two insertions are in genes of unknown function.
A Cyp51B Mutation Contributes to Azole Resistance in Aspergillus fumigatus
Journal of Fungi
The emergence and spread of Aspergillus fumigatus azole resistance has been acknowledged worldwide. The main problem of azole resistance is the limited therapeutic options for patients suffering aspergillosis. Azole resistance mechanisms have been mostly linked to the enzyme Cyp51A, a target of azole drugs, with a wide variety of modifications responsible for the different resistance mechanisms described to date. However, there are increasing reports of A. fumigatus strains showing azole resistance without Cyp51A modifications, and thus, novel resistance mechanisms are being explored. Here, we characterized two isogenic A. fumigatus clinical strains isolated two years apart from the same patient. Both strains were resistant to clinical azoles but showed different azole resistance mechanisms. One strain (CM8940) harbored a previously described G54A mutation in Cyp51A while the other strain (CM9640) had a novel G457S mutation in Cyp51B, the other target of azoles. In addition, this se...
Antimicrobial Agents and Chemotherapy, 2010
Aspergillus fumigatus sterol 14-␣ demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (K s , 8.6 M) and eburicol (K s , 22.6 M). Membrane suspensions of AF51A bound to both lanosterol (K s , 3.1 M) and eburicol (K s , 4.1 M). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the K d (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 M, respectively, in comparison with a K d value of 4 M for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with K d values ranging from 1 M for itraconazole to 11.9 M for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.
Multi-azole resistance in Aspergillus fumigatus
International Journal of Antimicrobial Agents, 2006
Azole resistance in Aspergillus spp. is unusual. We report a patient who received long-term treatment with itraconazole and voriconazole for bilateral chronic cavitary aspergillosis with aspergillomas whose isolates of Aspergillus fumigatus developed simultaneous resistance to itraconazole and voriconazole. A novel mutation (G138C) in the target gene (cyp51A) encoding 14␣-demethylase was detected. The patient had some response to intravenous caspofungin, which he received six times weekly, without the development of resistance over 9 months.
Understanding the environmental drivers of clinical azole resistance in Aspergillus species
Drug Target Insights
Aspergilli are ubiquitous fungal pathogens associated with severe life-threatening infections, especially in immunocompromised patients. Azoles are the first line of defence in the fight against most Aspergillus -related infections. However, resistance to these therapeutic compounds has developed, which is mainly due to the existence of mutations in lanosterol 14 alpha-demethylase (Cyp51A), a crucial enzyme in the pathway that produces ergosterol and is the target of azole antifungals. Azole-based antifungal medications are ineffective because of infections brought on by azole-resistant Aspergillus species, leading to a high fatality rate. However, resistant Aspergillus isolates have also been isolated from azole-naïve patients. Global agricultural practices promote the use of azole fungicides to protect crops from phytopathogens. Usage of azole fungicides on a large scale has been linked to the development of resistance among Aspergillus species prevalent in the environment. Th...