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

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.

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

Eukaryotic Cell ◽

10.1128/ec.00215-12 ◽

2012 ◽

Vol 11 (10) ◽

pp. 1289-1299 ◽

Cited By ~ 105

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.

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A Hyperactive Form of the Zinc Cluster Transcription Factor Stb5 CausesYOR1Overexpression and Beauvericin Resistance inCandida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01655-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 1

Author(s):

Bernardo Ramírez-Zavala ◽

Hannah Manz ◽

Frank Englert ◽

P. David Rogers ◽

Joachim Morschhäuser

Keyword(s):

Transcription Factor ◽

Target Genes ◽

Efflux Pump ◽

Hyphal Growth ◽

Pathogenic Yeast ◽

Fluconazole Resistance ◽

Content Type ◽

Activating Mutations ◽

Zinc Cluster ◽

Constitutive Overexpression

ABSTRACTGain-of-function mutations in the zinc cluster transcription factors Mrr1, Tac1, and Upc2, which result in constitutive overexpression of their target genes, are a frequent cause of fluconazole resistance in the pathogenic yeastCandida albicans. In this study, we show that an activated form of another zinc cluster transcription factor, Stb5, confers resistance to the natural compound beauvericin via the overexpression ofYOR1, encoding an efflux pump of the ATP-binding cassette transporter superfamily. Beauvericin was recently shown to potentiate the activity of azole drugs againstC. albicans. Although Yor1 did not contribute to fluconazole resistance whenC. albicanscells were treated with the drug alone, Stb5-mediatedYOR1overexpression diminished the synergistic effect of the fluconazole-beauvericin combination, thereby enhancing fluconazole resistance in beauvericin-treatedC. albicanscells. Stb5-mediatedYOR1overexpression also suppressed the inhibition of hyphal growth, an important virulence trait ofC. albicans, by beauvericin. Therefore, activating mutations in Stb5, which result in constitutiveYOR1overexpression, may enableC. albicansto acquire resistance to beauvericin and thereby overcome both the sensitization to azole drugs and the inhibition of morphogenesis caused by this compound.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02652-15 ◽

2016 ◽

Vol 60 (6) ◽

pp. 3653-3661 ◽

Cited By ~ 27

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.

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LimitedERG11Mutations Identified in Isolates ofCandida aurisDirectly Contribute to Reduced Azole Susceptibility

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01427-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 54

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.

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Constitutive Activation of MexT by Amino Acid Substitutions Results in MexEF-OprN Overproduction in Clinical Isolates ofPseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02445-17 ◽

2018 ◽

Vol 62 (5) ◽

Cited By ~ 9

Author(s):

Paulo Juarez ◽

Isabelle Broutin ◽

Christophe Bordi ◽

Patrick Plésiat ◽

Catherine Llanes

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Regulatory Gene ◽

Clinical Isolates ◽

Amino Acid Substitutions ◽

Multidrug Efflux ◽

Efflux System ◽

Content Type ◽

Constitutive Activation ◽

Multidrug Efflux System

ABSTRACTWhen overproduced, the multidrug efflux system MexEF-OprN increases the resistance ofPseudomonas aeruginosato fluoroquinolones, chloramphenicol, and trimethoprim. In this work, we demonstrate that gain-of-function mutations in the regulatory genemexTresult in oligomerization of the LysR regulator MexT, constitutive upregulation of the efflux pump, and increased resistance in clinical isolates.

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01941-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 5

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.

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Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01344-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 11

Author(s):

Zhongle Liu ◽

Lawrence C. Myers

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Transcriptional Activation ◽

Fungal Pathogens ◽

Efflux Pump ◽

Open Chromatin ◽

Drug Efflux ◽

Fluconazole Resistance ◽

Content Type ◽

Zinc Cluster

ABSTRACT Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1, a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2, the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1 GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1 GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1 GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated.

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Contribution of Clinically Derived Mutations inERG11to Azole Resistance in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03470-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 450-460 ◽

Cited By ~ 96

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.

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Ampicillin-Resistant Non-β-Lactamase-Producing Haemophilus influenzae in Spain: Recent Emergence of Clonal Isolates with Increased Resistance to Cefotaxime and Cefixime

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00354-07 ◽

2007 ◽

Vol 51 (7) ◽

pp. 2564-2573 ◽

Cited By ~ 86

Author(s):

Silvia García-Cobos ◽

José Campos ◽

Edurne Lázaro ◽

Federico Román ◽

Emilia Cercenado ◽

...

Keyword(s):

Amino Acid ◽

Haemophilus Influenzae ◽

Efflux Pump ◽

Antibiotic Use ◽

Resistance Mechanisms ◽

Amino Acid Substitutions ◽

Gene Encoding ◽

Reading Frame ◽

Recent Emergence ◽

Acrab Efflux Pump

ABSTRACT The sequence of the ftsI gene encoding the transpeptidase domain of penicillin-binding protein 3 (PBP 3) was determined for 354 nonconsecutive Haemophilus influenzae isolates from Spain; 17.8% of them were ampicillin susceptible, 56% were β-lactamase nonproducing ampicillin resistant (BLNAR), 15.8% were β-lactamase producers and ampicillin resistant, and 10.4% displayed both resistance mechanisms. The ftsI gene sequences had 28 different mutation patterns and amino acid substitutions at 23 positions. Some 93.2% of the BLNAR strains had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%). Amino acid substitutions at positions 377, 385, and 389, which conferred cefotaxime and cefixime MICs 10 to 60 times higher than those of susceptible strains, were found for the first time in Europe. In 72 isolates for which the repressor acrR gene of the AcrAB efflux pump was sequenced, numerous amino acid substitutions were found. Eight isolates with ampicillin MICs of 0.25 to 2 μg/ml showed changes that predicted the early termination of the acrR reading frame. Pulsed-field gel electrophoresis analysis demonstrated that most BLNAR strains were genetically diverse, although clonal dissemination was detected in a group of isolates presenting with increased resistance to cefotaxime and cefixime. Background antibiotic use at the community level revealed a marked trend toward increased amoxicillin-clavulanic acid consumption. BLNAR H. influenzae strains have arisen by vertical and horizontal spread and have evolved to adapt rapidly to the increased selective pressures posed by the use of oral penicillins and cephalosporins.

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A Novel VIM-Type Metallo-β-Lactamase Variant, VIM-60, with Increased Hydrolyzing Activity against Fourth-Generation Cephalosporins in Pseudomonas aeruginosa Clinical Isolates in Japan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00124-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 2

Author(s):

Tomomi Hishinuma ◽

Tatsuya Tada ◽

Hiroki Uchida ◽

Masahiro Shimojima ◽

Teruo Kirikae

Keyword(s):

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Fourth Generation ◽

Amino Acid Substitutions ◽

Content Type ◽

Catalytic Activities ◽

Genetic Context

ABSTRACT A novel VIM-type metallo-β-lactamase variant, VIM-60, was identified in multidrug-resistant Pseudomonas aeruginosa clinical isolates in Japan. Compared with VIM-2, VIM-60 had two amino acid substitutions (Arg228Leu and His252Arg) and higher catalytic activities against fourth-generation cephalosporins. The genetic context for blaVIM-60 was intI1-blaVIM-60-aadA1-aacA31-qacEdeltaI-sulI on the chromosome.

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