scholarly journals Impact of Erg11 amino acid substitutions identified in Candida auris clade III isolates on triazole drug susceptibility

2021 ◽  
Author(s):  
Benjamin Williamson ◽  
Adam Wilk ◽  
Kevin D. Guerrero ◽  
Timothy D. Mikulski ◽  
Tony N. Elias ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Drug Susceptibility ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Gene Variants ◽  
Candida Auris ◽  
Single Substitution

ERG11 sequencing of 28 Candida auris clade III isolates revealed the presence of concomitant V125A and F126L substitutions. Heterologous expression of Erg11-V125A/F126L in Saccharomyces cerevisiae led to reduced fluconazole and voriconazole susceptibilities. Generation of single substitution gene variants through site-directed mutagenesis uncovered that F126L primarily contributes to the elevated triazole MICs. A similar, yet diminished pattern of reduced susceptibility was observed with long-tailed triazoles posaconazole and itraconazole for V125A/F126L, F126L, Y132F, and K143R alleles.

scholarly journals Impact of Erg11 amino acid substitutions identified in Candida auris clade III isolates on triazole drug susceptibility

2021 ◽  
Author(s):  
Benjamin Williamson ◽  
Adam Wilk ◽  
Kevin D. Guerrero ◽  
Timothy D. Mikulski ◽  
Tony N. Elias ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Drug Susceptibility ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Gene Variants ◽  
Candida Auris ◽  
Single Substitution

ERG11 sequencing of 28 Candida auris clade III isolates revealed the presence of concomitant V125A and F126L substitutions. Heterologous expression of Erg11-V125A/F126L in Saccharomyces cerevisiae led to reduced fluconazole and voriconazole susceptibilities. Generation of single substitution gene variants through site-directed mutagenesis uncovered that F126L primarily contributes to the elevated triazole MICs. A similar, yet diminished pattern of reduced susceptibility was observed with long-tailed triazoles posaconazole and itraconazole for V125A/F126L, F126L, Y132F, and K143R alleles.

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 Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

scholarly journals VIM-19, a Metallo-β-Lactamase with Increased Carbapenemase Activity from Escherichia coli and Klebsiella pneumoniae

2009 ◽  
Vol 54 (1) ◽  
pp. 471-476 ◽  
Author(s):  
Jose-Manuel Rodriguez-Martinez ◽  
Patrice Nordmann ◽  
Nicolas Fortineau ◽  
Laurent Poirel
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Class 1 Integron ◽  
Carbapenem Resistant ◽  
Class 1

ABSTRACT Two carbapenem-resistant isolates, one Escherichia coli isolate and one Klebsiella pneumoniae isolate, recovered from an Algerian patient expressed a novel VIM-type metallo-β-lactamase (MBL). The identified bla VIM-19 gene was located on a ca. 160-kb plasmid and located inside a class 1 integron in both isolates. VIM-19 differed from VIM-1 by the Asn215Lys and Ser228Arg substitutions, increasing its hydrolytic activity toward carbapenems. Site-directed mutagenesis experiments showed that both substitutions were necessary for the increased carbapenemase activity of VIM-19. This study indicates that MBLs with enhanced activity toward carbapenems may be obtained as a result of very few amino acid substitutions.

Investigating the active site of human trimethyllysine hydroxylase

2019 ◽  
Vol 476 (7) ◽  
pp. 1109-1119 ◽  
Author(s):  
Yali Wang ◽  
Y. Vijayendar Reddy ◽  
Abbas H. K. Al Temimi ◽  
Hanka Venselaar ◽  
Frank H. T. Nelissen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Biosynthesis Pathway ◽  
Recognition Sites ◽  
Aromatic Cage ◽  
Basic Insight ◽  
Insight Into

Abstract The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate and (2S)-Nε-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242–D244–H389 residues, R391–R398 involved in 2OG binding and several residues (D231, N334 and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2S)-Nε-trimethyllysine.

scholarly journals A Novel FexA Variant from a Canine Staphylococcus pseudintermedius Isolate That Does Not Confer Florfenicol Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5763-5766 ◽  
Author(s):  
Elena Gómez-Sanz ◽  
Kristina Kadlec ◽  
Andrea T. Feßler ◽  
Myriam Zarazaga ◽  
Carmen Torres ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Base Pairs ◽  
Chloramphenicol Resistance ◽  
Content Type ◽  
Resistance Phenotype ◽  
First Time

ABSTRACTTransposon Tn558integrated in the chromosomalradCgene was detected for the first time inStaphylococus pseudintermedius. It carried a novelfexAvariant (fexAv) that confers only chloramphenicol resistance. The exporter FexAv exhibited two amino acid substitutions, Gly33Ala and Ala37Val, both of which seem to be important for substrate recognition. Site-directed mutagenesis that reverted the mutated base pairs to those present in the originalfexAgene restored the chloramphenicol-plus-florfenicol resistance phenotype.

scholarly journals Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy

2016 ◽  
Vol 60 (4) ◽  
pp. 2248-2256 ◽  
Author(s):  
Jennifer Giandhari ◽  
Adriaan E. Basson ◽  
Katherine Sutherland ◽  
Chris M. Parry ◽  
Patricia A. Cane ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Pediatric Patients ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Resistance Mutations ◽  
Phenotypic Resistance ◽  
Protease Resistance ◽  
Hiv 1

ABSTRACTProtease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.

scholarly journals Alterations in PBP 1A Essential for High-Level Penicillin Resistance in Streptococcus pneumoniae

1998 ◽  
Vol 42 (6) ◽  
pp. 1329-1333 ◽  
Author(s):  
Anthony M. Smith ◽  
Keith P. Klugman
Keyword(s):  
Amino Acid ◽  
South African ◽  
Site Directed Mutagenesis ◽  
Penicillin Resistance ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Penicillin Binding Proteins ◽  
Development Of Resistance ◽  
High Level

ABSTRACT High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 μg/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 μg/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 μg/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 μg/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 μg/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 μg/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.

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