Interrogation of related Clinical Pan-azole Resistant Aspergillus fumigatus strains - G138C, Y431C and G434C SNPs in cyp51A, Upregulation of cyp51A and Integration and Activation of Transposon Atf1 into cyp51A Promoter (original) (raw)
Related papers
Antimicrobial Agents and Chemotherapy, 2011
Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.
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...
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.
Antimicrobial agents and chemotherapy, 2013
Updated information and services can be found at: These include: REFERENCES http://aac.asm.org/content/57/8/3513#ref-list-1 at: This article cites 23 articles, 11 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on June 10, 2014 by guest http://aac.asm.org/ Downloaded from on June 10, 2014 by guest
Epidemiological and Genomic Landscape of Azole Resistance Mechanisms in Aspergillus Fungi
Frontiers in Microbiology, 2016
Invasive aspergillosis is a life-threatening mycosis caused by the pathogenic fungus Aspergillus. The predominant causal species is Aspergillus fumigatus, and azole drugs are the treatment of choice. Azole drugs approved for clinical use include itraconazole, voriconazole, posaconazole, and the recently added isavuconazole. However, epidemiological research has indicated that the prevalence of azole-resistant A. fumigatus isolates has increased significantly over the last decade. What is worse is that azole-resistant strains are likely to have emerged not only in response to long-term drug treatment but also because of exposure to azole fungicides in the environment. Resistance mechanisms include amino acid substitutions in the target Cyp51A protein, tandem repeat sequence insertions at the cyp51A promoter, and overexpression of the ABC transporter Cdr1B. Environmental azole-resistant strains harboring the association of a tandem repeat sequence and punctual mutation of the Cyp51A gene (TR34/L98H and TR46/Y121F/T289A) have become widely disseminated across the world within a short time period. The epidemiological data also suggests that the number of Aspergillus spp. other than A. fumigatus isolated has risen. Some non-fumigatus species intrinsically show low susceptibility to azole drugs, imposing the need for accurate identification, and drug susceptibility testing in most clinical cases. Currently, our knowledge of azole resistance mechanisms in non-fumigatus Aspergillus species such as A. flavus, A. niger, A. tubingensis, A. terreus, A. fischeri, A. lentulus, A. udagawae, and A. calidoustus is limited. In this review, we present recent advances in our understanding of azole resistance mechanisms particularly in A. fumigatus. We then provide an overview of the genome sequences of non-fumigatus species, focusing on the proteins related to azole resistance mechanisms.
BMC Genomics
Background: The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results: After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions: Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any "hard-wired" genetic mutations arise.
Emerging Infectious Diseases, 2009
Azoles are the mainstay of oral therapy for aspergillosis. Azole resistance in Aspergillus has been reported infrequently. The first resistant isolate in Manchester, UK, was detected in 1999. In a clinical collection of 519 A. fumigatus isolates, the frequency of itraconazole resistance was 5%, a significant increase since 2004 (p<0.001). Of the 34 itraconazole-resistant isolates we studied, 65% were cross-resistant to voriconazole and 74% (25) were crossresistant to posaconazole. Thirteen of 14 evaluable patients in our study had prior azole exposure; 8 infections failed therapy (progressed), and 5 failed to improve (remained stable). Eighteen amino acid alterations were found in the target enzyme, Cyp51A, 4 of which were novel. A population genetic analysis of microsatellites showed the existence of resistant mutants that evolved from originally susceptible strains, different cyp51A mutations in the same strain, and microalterations in microsatellite repeat number. Azole resistance in A. fumigatus is an emerging problem and may develop during azole therapy.