scholarly journals Gain-of-Function Mutations inUPC2Are a Frequent Cause ofERG11Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

2012 ◽  
Vol 11 (10) ◽  
pp. 1289-1299 ◽  
Author(s):  
Stephanie A. Flowers ◽  
Katherine S. Barker ◽  
Elizabeth L. Berkow ◽  
Geoffrey Toner ◽  
Sean G. Chadwick ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Clinical Isolates ◽  
Transcriptional Analysis ◽  
Single Amino Acid ◽  
Ergosterol Biosynthesis ◽  
Amino Acid Substitutions ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster

ABSTRACTInCandida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documentedUPC2gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase inERG11expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressedERG11by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation inUPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations inUPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increasedERG11expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by theMDR1gene, and the uncharacterized ATP binding cassette transporterCDR11. These findings demonstrate that gain-of-function mutations inUPC2are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account forERG11overexpression in all such isolates ofC. albicans.

scholarly journals Contribution of Clinically Derived Mutations inERG11to Azole Resistance in Candida albicans

2014 ◽  
Vol 59 (1) ◽  
pp. 450-460 ◽  
Author(s):  
Stephanie A. Flowers ◽  
Brendan Colón ◽  
Sarah G. Whaley ◽  
Mary A. Schuler ◽  
P. David Rogers
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Single Amino Acid Substitution ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Azole Resistance ◽  
Amino Acid Substitutions ◽  
Content Type ◽  
Proximal Surface

ABSTRACTInCandida albicans, theERG11gene encodes lanosterol demethylase, the target of the azole antifungals. Mutations inERG11that result in an amino acid substitution alter the abilities of the azoles to bind to and inhibit Erg11, resulting in resistance. AlthoughERG11mutations have been observed in clinical isolates, the specific contributions of individualERG11mutations to azole resistance inC. albicanshave not been widely explored. We sequencedERG11in 63 fluconazole (FLC)-resistant clinical isolates. Fifty-five isolates carried at least one mutation inERG11, and we observed 26 distinct positions in which amino acid substitutions occurred. We mapped the 26 distinct variant positions in these alleles to four regions in the predicted structure for Erg11, including its predicted catalytic site, extended fungus-specific external loop, proximal surface, and proximal surface-to-heme region. In total, 31 distinctERG11alleles were recovered, with 10ERG11alleles containing a single amino acid substitution. We then characterized 19 distinctERG11alleles by introducing them into the wild-type azole-susceptibleC. albicansSC5314 strain and testing them for susceptibilities to FLC, itraconazole (ITC), and voriconazole (VRC). The strains that were homozygous for the single amino acid substitutions Y132F, K143R, F145L, S405F, D446E, G448E, F449V, G450E, and G464S had a ≥4-fold increase in FLC MIC. The strains that were homozygous for several double amino acid substitutions had decreased azole susceptibilities beyond those conferred by any single amino acid substitution. These findings indicate that mutations inERG11are prevalent among azole-resistant clinical isolates and that most mutations result in appreciable changes in FLC and VRC susceptibilities.

scholarly journals A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate

2008 ◽  
Vol 7 (7) ◽  
pp. 1180-1190 ◽  
Author(s):  
Nico Dunkel ◽  
Teresa T. Liu ◽  
Katherine S. Barker ◽  
Ramin Homayouni ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Ergosterol Biosynthesis ◽  
Gain Of Function ◽  
Zinc Cluster ◽  
Fluconazole Resistant ◽  
Lanosterol Demethylase

ABSTRACT In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Overexpression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Overexpression of ERG11 leads to the increased production of lanosterol demethylase, which contributes to azole resistance in clinical isolates of C. albicans, but the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately upregulated with ERG11 in a fluconazole-resistant clinical isolate compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single-nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug-susceptible strain resulted in constitutive upregulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to the increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.

scholarly journals Exploring the Role of the Ω-Loop in the Evolution of Ceftazidime Resistance in the PenA β-Lactamase from Burkholderia multivorans, an Important Cystic Fibrosis Pathogen

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Scott A. Becka ◽  
Magdalena A. Taracila ◽  
Elise T. Zeiser ◽  
Julian A. Gatta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Burkholderia Cepacia ◽  
Clinical Isolates ◽  
Dynamics Simulation ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Biochemical Basis ◽  
Content Type ◽  
Burkholderia Multivorans

ABSTRACT The unwelcome evolution of resistance to the advanced generation cephalosporin antibiotic, ceftazidime is hindering the effective therapy of Burkholderia cepacia complex (BCC) infections. Regrettably, BCC organisms are highly resistant to most antibiotics, including polymyxins; ceftazidime and trimethoprim-sulfamethoxazole are the most effective treatment options. Unfortunately, resistance to ceftazidime is increasing and posing a health threat to populations susceptible to BCC infection. We found that up to 36% of 146 tested BCC clinical isolates were nonsusceptible to ceftazidime (MICs ≥ 8 μg/ml). To date, the biochemical basis for ceftazidime resistance in BCC is largely undefined. In this study, we investigated the role of the Ω-loop in mediating ceftazidime resistance in the PenA β-lactamase from Burkholderia multivorans, a species within the BCC. Single amino acid substitutions were engineered at selected positions (R164, T167, L169, and D179) in the PenA β-lactamase. Cell-based susceptibility testing revealed that 21 of 75 PenA variants engineered in this study were resistant to ceftazidime, with MICs of >8 μg/ml. Under steady-state conditions, each of the selected variants (R164S, T167G, L169A, and D179N) demonstrated a substrate preference for ceftazidime compared to wild-type PenA (32- to 320-fold difference). Notably, the L169A variant hydrolyzed ceftazidime significantly faster than PenA and possessed an ∼65-fold-lower apparent Ki (Ki app) than that of PenA. To understand why these amino acid substitutions result in enhanced ceftazidime binding and/or turnover, we employed molecular dynamics simulation (MDS). The MDS suggested that the L169A variant starts with the most energetically favorable conformation (−28.1 kcal/mol), whereas PenA possessed the most unfavorable initial conformation (136.07 kcal/mol). In addition, we observed that the spatial arrangement of E166, N170, and the hydrolytic water molecules may be critical for enhanced ceftazidime hydrolysis by the L169A variant. Importantly, we found that two clinical isolates of B. multivorans possessed L169 amino acid substitutions (L169F and L169P) in PenA and were highly resistant to ceftazidime (MICs ≥ 512 μg/ml). In conclusion, substitutions in the Ω-loop alter the positioning of the hydrolytic machinery as well as allow for a larger opening of the active site to accommodate the bulky R1 and R2 side chains of ceftazidime, resulting in resistance. This analysis provides insights into the emerging phenotype of ceftazidime-resistant BCC and explains the evolution of amino acid substitutions in the Ω-loop of PenA of this significant clinical pathogen.

scholarly journals Contribution of Clinically Derived Mutations in the Gene Encoding the Zinc Cluster Transcription Factor Mrr2 to Fluconazole Antifungal Resistance andCDR1Expression inCandida albicans

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Andrew T. Nishimoto ◽  
Qing Zhang ◽  
Brandon Hazlett ◽  
Joachim Morschhäuser ◽  
P. David Rogers
Keyword(s):  
Amino Acid ◽  
Efflux Pump ◽  
Antifungal Resistance ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Gene Encoding ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Zinc Cluster ◽  
Artificial Activation

ABSTRACTMutations in genes encoding zinc cluster transcription factors (ZCFs) such asTAC1,MRR1, andUPC2play a key role inCandida albicansazole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole. Amino acid substitutions in Mrr2 have recently been reported to contribute to fluconazole resistance in clinical isolates. In the present study, 57 C. albicansclinical isolates with elevated fluconazole MICs were examined for mutations inMRR2and expression ofCDR1. Mutations inMRR2resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 substitutions (S466L, A468G, S469T, T470N) previously reported to reduce fluconazole susceptibility. Three additional, novel amino acid substitutions (R45Q, A459T, V486M) were also discovered in fluconazole-resistant isolates. When introduced into a fluconazole-susceptible background, no change in fluconazole MIC orCDR1expression was observed for any of the mutations found in this collection. However, introduction of an allele leading to artificial activation of Mrr2 increased resistance to fluconazole as well asCDR1expression. Moreover, Mrr2 amino acid changes reported previously to have the strongest effect on fluconazole susceptibility andCDR1expression also exhibited no differences in fluconazole susceptibility orCDR1expression relative to the parent strain. While all known fluconazole resistance mechanisms are represented within this collection of clinical isolates and contribute to fluconazole resistance to different extents, mutations inMRR2do not appear to alterCDR1expression or contribute to resistance in any of these isolates.

scholarly journals Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Tsuyoshi Yamada ◽  
Mari Maeda ◽  
Mohamed Mahdi Alshahni ◽  
Reiko Tanaka ◽  
Takashi Yaguchi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Point ◽  
Point Mutations ◽  
Trichophyton Mentagrophytes ◽  
Antifungal Drugs ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Squalene Epoxidase ◽  
Amino Acid Substitutions ◽  
Content Type

ABSTRACT Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

scholarly journals SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

2014 ◽  
Vol 58 (9) ◽  
pp. 5102-5110 ◽  
Author(s):  
Bernardo Ramírez-Zavala ◽  
Selene Mogavero ◽  
Eva Schöller ◽  
Christoph Sasse ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster ◽  
Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

scholarly journals Resistance Mechanisms and Clinical Features of Fluconazole-Nonsusceptible Candida tropicalis Isolates Compared with Fluconazole-Less-Susceptible Isolates

2016 ◽  
Vol 60 (6) ◽  
pp. 3653-3661 ◽  
Author(s):  
Min Ji Choi ◽  
Eun Jeong Won ◽  
Jong Hee Shin ◽  
Soo Hyun Kim ◽  
Wee-Gyo Lee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Clinical Features ◽  
Candida Tropicalis ◽  
Resistance Mechanisms ◽  
Therapeutic Failure ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Amino Acid Substitutions ◽  
Content Type ◽  
Expression Levels

We investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates ofCandida tropicalisrecovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirty-five clinical isolates ofC. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 μg/ml), 12 FLS (MIC, 1 to 2 μg/ml), and 14 control (MIC, 0.125 to 0.5 μg/ml) isolates, were assessed.CDR1,MDR1, andERG11expression was quantified, and theERG11andUPC2genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels ofCDR1,MDR1, andERG11genes than control isolates (Pvalues of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher meanERG11expression levels than FLS isolates (P= 0.004), but there were no differences in those ofCDR1orMDR1. Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10;P= 0.011) and immunosuppression (6/6 versus 3/10;P= 0.011). These results reveal that the majority of FNSC. tropicalisisolates show overexpression ofCDR1,MDR1, andERG11genes, and fungemia develops after azole exposure in patients with immunosuppression.

scholarly journals LimitedERG11Mutations Identified in Isolates ofCandida aurisDirectly Contribute to Reduced Azole Susceptibility

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Kelley R. Healey ◽  
Milena Kordalewska ◽  
Cristina Jiménez Ortigosa ◽  
Ashutosh Singh ◽  
Indira Berrío ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Candida Auris ◽  
Content Type ◽  
Species Specific

ABSTRACTMultiple Erg11 amino acid substitutions were identified in clinical isolates ofCandida aurisoriginating from India and Colombia. Elevated azole MICs were detected inSaccharomyces cerevisiaeupon heterologous expression ofC. aurisERG11alleles that encoded for Y132F or K143R substitutions; however, expression of alleles encoding I466M, Y501H, or other clade-defined amino acid differences yielded susceptible MICs. Similar to otherCandidaspecies, specificC. aurisERG11mutations resulted directly in reduced azole susceptibility.

scholarly journals NDM-4 Metallo-β-Lactamase with Increased Carbapenemase Activity from Escherichia coli

2012 ◽  
Vol 56 (4) ◽  
pp. 2184-2186 ◽  
Author(s):  
Patrice Nordmann ◽  
Anne E. Boulanger ◽  
Laurent Poirel
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Active Sites ◽  
Hydrolytic Activity ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Content Type

ABSTRACTA clinicalEscherichia coliisolate resistant to all β-lactams, including carbapenems, expressed a novel metallo-β-lactamase (MBL), NDM-4, differing from NDM-1 by a single amino acid substitution (Met154Leu). NDM-4 possessed increased hydrolytic activity toward carbapenems and several cephalosporins compared to that of NDM-1. This amino acid substitution was not located in the known active sites of NDM-1, indicating that remote amino acid substitutions might also play a role in the extended activity of this MBL.

scholarly journals An A643V Amino Acid Substitution in Upc2p Contributes to Azole Resistance in Well-Characterized Clinical Isolates ofCandida albicans

2010 ◽  
Vol 55 (2) ◽  
pp. 940-942 ◽  
Author(s):  
Samantha J. Hoot ◽  
Adam R. Smith ◽  
Ryan P. Brown ◽  
Theodore C. White
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Gain Of Function ◽  
Ofcandida Albicans

ABSTRACTTheCandida albicansUpc2p transcription factor regulatesERG11, encoding the target of azole drugs. Gain-of-function mutations that contribute to resistance were recently identified in a series of sequential clinical isolates (N. Dunkel, T. T. Liu, K. S. Barker, R. Homayouni, J. Morschhauser, and P. D. Rogers, Eukaryot. Cell 7:1180-1190, 2008). In the present study,UPC2was sequenced from a matched set of 17 isolates. An A643V substitution was present in all of the isolates in the series that overexpressedERG11. Azole susceptibility, ergosterol levels, and expression ofERGgenes were elevated in the A643V clinical isolates and in reconstructed strains.

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