scholarly journals Contributions of both ATP-Binding Cassette Transporter and Cyp51A Proteins Are Essential for Azole Resistance in Aspergillus fumigatus

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Sanjoy Paul ◽  
Daniel Diekema ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Azole Resistance ◽  
Atp Binding ◽  
Wild Type ◽  
Gene Encoding ◽  
Pathogenic Yeast ◽  
Genetic Changes ◽  
Content Type ◽  
Transporter Protein ◽  
Atp Binding Cassette

ABSTRACT While azole drugs targeting the biosynthesis of ergosterol are effective antifungal agents, their extensive use has led to the development of resistant organisms. Infections involving azole-resistant forms of the filamentous fungus Aspergillus fumigatus are often associated with genetic changes in the cyp51A gene encoding the lanosterol α14 demethylase target enzyme. Both a sequence duplication in the cyp51A promoter (TR34) and a substitution mutation in the coding sequence (L98H) are required for the full expression of azole resistance. A mechanism commonly observed in pathogenic yeast such as Candida albicans involves gain-of-function mutations in transcriptional regulatory proteins that induce expression of genes encoding ATP-binding cassette (ABC) transporters. We and others have found that an ABC transporter protein called Cdr1B (here referred to as AbcG1) is required for wild-type azole resistance in A. fumigatus. Here, we test the genetic relationship between the TR34 L98H allele of cyp51A and an abcG1 null mutation. Loss of AbcG1 from a TR34 L98H cyp51A-containing strain caused a large decrease in the azole resistance of the resulting double-mutant strain. We also generated antibodies that enabled the detection of both the wild-type and L98H forms of the Cyp51A protein. The introduction of the L98H lesion into the cyp51A gene led to a decreased production of immunoreactive enzyme, suggesting that this mutant protein is unstable. Our data confirm the importance of AbcG1 function during azole resistance even in a strongly drug-resistant background.

scholarly journals Evidence that Ergosterol Biosynthesis Modulates Activity of the Pdr1 Transcription Factor inCandida glabrata

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Bao Gia Vu ◽  
Grace Heredge Thomas ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Candida Glabrata ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Atp Binding ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Atp Binding Cassette Transporter ◽  
Encoding Gene ◽  
Atp Binding Cassette

ABSTRACTA crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of these drugs is a major risk factor in successful treatment of a variety of fungal diseases.Candida glabratais a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance.C. glabrataoften acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encodingCDR1locus. GOF alleles ofPDR1have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, anERGgene regulator. We show that Upc2A binds directly to thePDR1andCDR1promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs.IMPORTANCEA likely contributor to the increased incidence of non-albicanscandidemias involvingCandida glabratais the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding geneCDR1. Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding geneERG11. These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of theERGpathway, Upc2A, is shown to directly bind to thePDR1andCDR1promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.

scholarly journals Contributions of Aspergillus fumigatus ATP-Binding Cassette Transporter Proteins to Drug Resistance and Virulence

2013 ◽  
Vol 12 (12) ◽  
pp. 1619-1628 ◽  
Author(s):  
Sanjoy Paul ◽  
Daniel Diekema ◽  
W. Scott Moye-Rowley
Keyword(s):  
Drug Resistance ◽  
Aspergillus Fumigatus ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Sequence Similarity ◽  
Atp Binding ◽  
Content Type ◽  
Transporter Proteins ◽  
Encoding Genes ◽  
Atp Binding Cassette

ABSTRACTIn yeast cells such as those ofSaccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of twoAspergillus fumigatusproteins that share high sequence similarity withS. cerevisiaePdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The twoA. fumigatusgenes encoding the ABC transporters sharing the highest sequence similarity toS. cerevisiaePdr5 are calledabcAandabcBhere. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains ofA. fumigatus. Loss ofabcBinvariably elicited increased azole susceptibility, whileabcAdisruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from thehspApromoter inA. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane inA. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen.

scholarly journals Itraconazole, Voriconazole, and Posaconazole CLSI MIC Distributions for Wild-Type and Azole-Resistant Aspergillus fumigatus Isolates

2018 ◽  
Vol 4 (3) ◽  
pp. 103 ◽  
Author(s):  
Jochem Buil ◽  
Ferry Hagen ◽  
Anuradha Chowdhary ◽  
Paul Verweij ◽  
Jacques Meis
Keyword(s):  
Aspergillus Fumigatus ◽  
Minimal Inhibitory Concentration ◽  
Susceptibility Testing ◽  
Inhibitory Concentration ◽  
Azole Resistance ◽  
Wild Type ◽  
Gene Encoding ◽  
Environmental Resistance ◽  
Resistance Selection ◽  
Susceptibility Profiles

Azole resistance in Aspergillus fumigatus is most frequently conferred by mutations in the cyp51A gene encoding 14α-sterol demethylases. TR34/L98H and TR46/Y121F/T289A are the two most common mutations associated with environmental resistance selection. We studied the minimal inhibitory concentration (MIC) distribution of clinical A. fumigatus isolates to characterize the Clinical and Laboratory Standards Institute (CLSI) susceptibility profiles of isolates with the wild-type (WT) cyp51A genotype, and isolates with the TR34/L98H and TR46/Y121F/T289A cyp51A mutations. Susceptibility testing was performed according to CLSI M38-A2. The MICs of 363 A. fumigatus isolates were used in this study. Based on the CLSI epidemiological cut-off values (ECVs), 141 isolates were phenotypically non-WT and 222 isolates had a phenotypically WT susceptibility. All isolates with the TR34/L98H mutation had an itraconazole MIC > 1 mg/L which is above the CLSI ECV. Eighty-six of 89 (97%) isolates with the TR34/L98H mutation had voriconazole and posaconazole MICs above the CLSI ECV, i.e., MICs of 1 and 0.25 mg/L, respectively. The isolates with a TR46/Y121F/T289A mutation showed a different phenotype. All 37 isolates with a TR46/Y121F/T289A mutation had a voriconazole MIC above the CLSI ECV, while 28/37 (76%) isolates had an itraconazole MIC > 1 mg/L. Interestingly, only 13 of 37 (35%) isolates had a posaconazole MIC > 0.25 mg/L.

scholarly journals Five-Year Survey (2014 to 2018) of Azole Resistance in Environmental Aspergillus fumigatus Isolates from China

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Duantao Cao ◽  
Ruilin Wu ◽  
Suxia Dong ◽  
Feiyan Wang ◽  
Chao Ju ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Soil Samples ◽  
Azole Resistance ◽  
Clinical Settings ◽  
Wild Type ◽  
Content Type

ABSTRACT A total of 191 soil samples from Hangzhou, China, were submitted to detect non-wild-type (non-WT) Aspergillus fumigatus and its associated mechanisms. There were 2 (4.7%), 13 (12.4%), and 31 (23.1%) isolates identified as non-WT in 2014, 2016, and 2018, respectively. The resistant mutations of TR34/L98H, TR46/Y121F/T289A, and TR34/L98H/S297T/F495I were found in 3, 5, and 5 non-WT isolates. The G448S mutation, previously only found in clinical settings, was detected in A. fumigatus from soil samples.

scholarly journals In VitroBiochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

2015 ◽  
Vol 59 (12) ◽  
pp. 7771-7778 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Josie E. Parker ◽  
Claire L. Price ◽  
W. David Nes ◽  
Steven L. Kelly ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Inhibitory Concentration ◽  
Point Mutations ◽  
Residual Activity ◽  
Azole Resistance ◽  
Wild Type ◽  
Biochemical Basis ◽  
Content Type ◽  
Resistant Phenotype

ABSTRACTThe incidence of triazole-resistantAspergillusinfections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinantAspergillus fumigatusCPR1 (AfCPR1), andEscherichia colimembrane suspensions containing recombinantA. fumigatusCYP51 proteins, allowingin vitroscreening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed thatA. fumigatusCYP51B (Af51B IC50, 0.50 μM) was 34-fold more susceptible to inhibition by fluconazole thanA. fumigatusCYP51A (Af51A IC50, 17 μM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 μM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 μM triazole, which could confer azole resistance inA. fumigatusstrains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance ofA. fumigatusstrains harboring G54W, L98H, and M220K Af51A point mutations.

scholarly journals The Strength of Synergistic Interaction between Posaconazole and Caspofungin Depends on the Underlying Azole Resistance Mechanism of Aspergillus fumigatus

2015 ◽  
Vol 59 (3) ◽  
pp. 1738-1744 ◽  
Author(s):  
Eleftheria Mavridou ◽  
Joseph Meletiadis ◽  
Antony Rijs ◽  
Johan W. Mouton ◽  
Paul E. Verweij
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanism ◽  
Fungal Growth ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Synergistic Interactions ◽  
Drug Concentrations

ABSTRACTThe majority of azole resistance mechanisms inAspergillus fumigatuscorrespond to mutations in thecyp51Agene. As azoles are less effective against infections caused by multiply azole-resistantA. fumigatusisolates, new therapeutic options are warranted for treating these infections. We therefore investigated thein vitrocombination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistantA. fumigatusisolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly (ttest,P< 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates (P< 0.01). FIC-2 values were inversely correlated with POSA MICs (rs= −0.52,P= 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA (rs= 0.76,P< 0.0001) and CAS (rs= 0.52,P= 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) againstA. fumigatusisolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

scholarly journals Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of theAspergillus fumigatus cyp51AGene in Primary Clinical Samples from Neutropenic Patients

2012 ◽  
Vol 56 (7) ◽  
pp. 3905-3910 ◽  
Author(s):  
Birgit Spiess ◽  
Wolfgang Seifarth ◽  
Natalia Merker ◽  
Susan J. Howard ◽  
Mark Reinwald ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Positive Culture ◽  
Cross Reactivity ◽  
Resistant Isolate ◽  
Clinical Samples ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Pcr Assays

ABSTRACTThe increasing incidence of azole resistance inAspergillus fumigatuscausing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediatingcyp51Agene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of theAspergillus fumigatus cyp51Agene to establish PCR assays with consecutive DNA sequence analysis to detect and identify theA. fumigatus cyp51Atandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially dilutedA. fumigatusand human DNA,A. fumigatus cyp51Agene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg ofA. fumigatusDNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons forA. fumigatuswild-type DNA confirmed thecyp51Awild-type sequence, and PCR products from one azole-resistantA. fumigatusisolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

scholarly journals The Aspergillus fumigatus Mismatch Repair MSH2 Homolog Is Important for Virulence and Azole Resistance

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Thaila Fernanda dos Reis ◽  
Lilian Pereira Silva ◽  
Patrícia Alves de Castro ◽  
Rafaela Andrade do Carmo ◽  
Marjorie Mendes Marini ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Mismatch Repair ◽  
Genetic Instability ◽  
Galleria Mellonella ◽  
Fungal Infections ◽  
Invasive Pulmonary Aspergillosis ◽  
Azole Resistance ◽  
Repair System ◽  
Wild Type ◽  
Content Type

ABSTRACT The genetic stability of every living organism depends on accurate DNA replication and repair systems. Here, we investigated the Aspergillus fumigatus MSH2 mismatch repair (MMR) gene MshA and how it impacts virulence and the evolution of azole resistance. We examined mshA gene variation in 62 environmental and clinical A. fumigatus strains. We have observed 12 strains with variants (18.2%), and 8 strains among them showed missense variants. We demonstrated that A. fumigatus mshA null mutants are haploid and have conserved karyotypes with discrete gross chromosomal rearrangements. The ΔmshA strains are not sensitive to several DNA-damaging agents. The lack of mshA caused a significant reduction of virulence of A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonella. Wild-type and ΔmshA populations did not show any significant changes in drug resistance acquisition after they were transferred 10 times in minimal medium in the absence of any stress. However, these populations rapidly acquired virulence in the ΔmshA background and high levels of resistance to posaconazole in the presence of this drug (at least 200-fold-higher levels of resistance than those derived from the wild-type strain). Taken together, these results suggest that genetic instability caused by ΔmshA mutations can confer an adaptive advantage, mainly increasing posaconazole resistance and virulence acquisition. IMPORTANCE Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases in immunocompromised patients, with mortality rates as high as 90%. Systemic fungal infections such as IA are usually treated with triazoles; however, epidemiological research has shown that the prevalence of azole-resistant Aspergillus fumigatus isolates has increased significantly over the last decade. There is very little information about the importance of genomic stability for A. fumigatus population structure, azole resistance, and virulence. Here, we decided to investigate whether the mismatch repair system could influence A. fumigatus azole resistance and virulence, focusing on one of the components of this system, MSH2. Although the mutation frequency of mshA (the A. fumigatus MSH2 homologue) is low in environmental and clinical isolates, our results indicate that loss of mshA function can provide increased azole resistance and virulence when selected for. These results demonstrate the importance of genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance and establishing fitness in the host.

scholarly journals Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus fumigatus on Bronchoalveolar Lavage Fluid

2015 ◽  
Vol 53 (3) ◽  
pp. 868-874 ◽  
Author(s):  
Ga-Lai M. Chong ◽  
Wendy W. J. van de Sande ◽  
Gijs J. H. Dingemans ◽  
Giel R. Gaajetaan ◽  
Alieke G. Vonk ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Azole Resistance ◽  
Wild Type ◽  
Pcr Assay ◽  
Content Type ◽  
Resistant Strains

Azole resistance inAspergillus fumigatusis increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevantAspergillusspecies, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistantA. fumigatusstrains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence ofAspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence ofAspergilluswas <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2Aspergillusspecies and 14A. fumigatussamples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection ofAspergillusspecies directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

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