Contribution of Clinically Derived Mutations inERG11to Azole Resistance in Candida albicans

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.

Download Full-text

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.

Download Full-text

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05961-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 2184-2186 ◽

Cited By ~ 87

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00995-10 ◽

2010 ◽

Vol 55 (2) ◽

pp. 940-942 ◽

Cited By ~ 63

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.

Download Full-text

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00115-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 80

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.

Download Full-text