scholarly journals The Evolution of Azole Resistance inCandida albicansSterol 14α-Demethylase (CYP51) through Incremental Amino Acid Substitutions

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Andrew G. Warrilow ◽  
Andrew T. Nishimoto ◽  
Josie E. Parker ◽  
Claire L. Price ◽  
Stephanie A. Flowers ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fold Increase ◽  
Nitrilotriacetic Acid ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Double Amino Acid

ABSTRACTRecombinantCandida albicansCYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed inEscherichia coliand purified by Ni2+-nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC50) using CYP51 reconstitution assays. The greatest increase in the IC50compared to that of the wild-type enzyme was observed with the five double substitutions Y132F+K143R (15.3-fold), Y132H+K143R (22.1-fold), Y132F+F145L (10.1-fold), G307S+G450E (13-fold), and D278N+G464S (3.3-fold). The single substitutions K143R, D278N, S279F, S405F, G448E, and G450E conferred at least 2-fold increases in the fluconazole IC50, and the Y132F, F145L, Y257H, Y447H, V456I, G464S, R467K, and I471T substitutions conferred increased residual CYP51 activity at high fluconazole concentrations.In vitrotesting of select CaCYP51 mutations inC. albicansshowed that the Y132F, Y132H, K143R, F145L, S405F, G448E, G450E, G464S, Y132F+K143R, Y132F+F145L, and D278N+G464S substitutions conferred at least a 2-fold increase in the fluconazole MIC. The catalytic tolerance of the purified proteins to voriconazole, itraconazole, and posaconazole was far lower and limited to increased residual activities at high triazole concentrations for certain mutations rather than large increases in IC50values. Itraconazole was the most effective at inhibiting CaCYP51. However, when tested against CaCYP51 mutant strains, posaconazole seemed to be the most resistant to changes in MIC as a result of CYP51 mutation compared to itraconazole, voriconazole, or fluconazole.

scholarly journals EUCAST Determination of Olorofim (F901318) Susceptibility of Mold Species, Method Validation, and MICs

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Karin Meinike Jørgensen ◽  
Karen M. T. Astvad ◽  
Rasmus Krøger Hare ◽  
Maiken Cavling Arendrup
Keyword(s):  
Fusarium Solani ◽  
Susceptibility Testing ◽  
Wild Type ◽  
Preparation Methods ◽  
Content Type ◽  
Microtiter Plates ◽  
Upper Limits ◽  
Limited Variation

ABSTRACT Olorofim is a novel antifungal agent with in vitro activity against Aspergillus and some other molds. Here, we addressed technical aspects for EUCAST olorofim testing and generated contemporary MIC data. EUCAST E.Def 9.3.1 testing was performed comparing two plate preparation methods (serial dilution in medium [serial plates] versus predilution in DMSO [ISO plates]), two lots of olorofim, visual (visual-MIC) versus spectrophotometer (spec-MIC) reading, and four polystyrene plates using 34 to 53 Aspergillus isolates from five genera. Subsequently, olorofim MICs were compared to itraconazole, voriconazole, posaconazole, and amphotericin B MICs for 298 clinical mold isolates (2016 to 2017). Wild-type upper limits (WT-UL) were determined following EUCAST principles for epidemiologic cutoff value (ECOFF) setting. Olorofim median MICs comparing serial plates and ISO plates were identical (25/36 [69%]) or one dilution apart (11/36 [31%]). Interperson agreement for visual-MICs was 92% to 94%/100% for ≤1/≤2 dilutions, respectively. The visual-MIC values across tested microtiter plates and olorofim lots revealed only discrete differences (≤1 dilution lower for treated plates). No single spec-MIC criterion was applicable to all species. Olorofim MICs were low against 275 Aspergillus species isolates (modal MIC, 0.06 mg/liter; MIC range, < 0.004 to 0.25 mg/liter) and three dermatophytes (MICs 0.03 to 0.06 mg/liter). MICs against Fusarium were diverse, with full inhibition of F. proliferatum (MIC, 0.016), 50% growth inhibition of Fusarium solani at 1 to 2 mg/liter, and no inhibition of F. dimerum. Olorofim displayed potent in vitro activity against most mold isolates and was associated with limited variation in EUCAST susceptibility testing.

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 In vitro analysis of human immunodeficiency virus type 1 resistance to nevirapine and fitness determination of resistant variants

2002 ◽  
Vol 83 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Maria Dolores Iglesias-Ussel ◽  
Concepción Casado ◽  
Eloísa Yuste ◽  
Isabel Olivares ◽  
Cecilio López-Galíndez
Keyword(s):  
Fold Increase ◽  
Resistant Mutant ◽  
Wild Type ◽  
Resistance Mutations ◽  
Phenotypic Resistance ◽  
Drug Concentrations ◽  
Immunodeficiency Virus ◽  
Vitro Analysis

Nevirapine-resistant variants were generated by serial passages in MT-2 cells in the presence of increasing drug concentrations. In passage 5, mutations V106A, Y181C and G190A were detected in the global population, associated with a 100-fold susceptibility decrease. Sequence analysis of biological clones obtained from passage 5 and subsequent passages showed that single mutants, detected in first passages, were progressively replaced in passage 15 by double mutants, correlating with a 500-fold increase in phenotypic resistance. Fitness determination of single mutants confirmed that, in the presence of nevirapine, every variant was more fit than wild-type with a fitness order Y181C>V106A>G190A>wild-type. Unexpectedly, in the absence of the drug, the Y181C resistant mutant was more fit than wild-type, with a fitness gradient Y181C>wild-type >G106A⩾V190A. Using a molecular clone in which the Y181C mutation was introduced by in vitro mutagenesis, the greater fitness of the Y181C mutant was confirmed in new competition cultures. These data exemplify the role of resistance mutations on virus phenotype but also on virus evolution leading, occasionally, to resistant variants fitter than the wild-type in the absence of the drug.

scholarly journals Source of the Fitness Defect in Rifamycin-ResistantMycobacterium tuberculosisRNA Polymerase and the Mechanism of Compensation by Mutations in the β′ Subunit

2018 ◽  
Vol 62 (6) ◽  
pp. e00164-18 ◽  
Author(s):  
Maxwell A. Stefan ◽  
Fatima S. Ugur ◽  
George A. Garcia
Keyword(s):  
Amino Acid Substitutions ◽  
Β Subunit ◽  
Wild Type ◽  
Rna Transcription ◽  
Content Type ◽  
Rna Hydrolysis ◽  
Rifampin Resistance ◽  
Compensatory Mutations

ABSTRACTMycobacterium tuberculosisis a critical threat to human health due to the increased prevalence of rifampin resistance (RMPr). Fitness defects have been observed in RMPrmutants with amino acid substitutions in the β subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi β-barrel (DPBB) domain of the β′ subunit of RNAP. To identify factors contributing to the fitness defects observedin vivo, severalin vitroRNA transcription assays were utilized to probe initiation, elongation, termination, and 3′-RNA hydrolysis with the wild-type and RMPrM. tuberculosisRNAPs. We found that the less prevalent RMPrmutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMPrmutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the βS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the β′ subunit.

scholarly journals Impact of the E540V Amino Acid Substitution in GyrB ofMycobacterium tuberculosison Quinolone Resistance

2011 ◽  
Vol 55 (8) ◽  
pp. 3661-3667 ◽  
Author(s):  
Hyun Kim ◽  
Chie Nakajima ◽  
Kazumasa Yokoyama ◽  
Zeaur Rahim ◽  
Youn Uck Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Dna Cleavage ◽  
Recombinant Dna ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Quinolone Resistance ◽  
Wild Type ◽  
Wild Type Enzyme

ABSTRACTAmino acid substitutions conferring resistance to quinolones inMycobacterium tuberculosishave generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance inM. tuberculosis, we expressed recombinant DNA gyrases inEscherichia coliand characterized themin vitro. Wild-type and GyrB-E540V DNA gyrases were reconstitutedin vitroby mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance inM. tuberculosis.

scholarly journals Altering Catalytic Properties of 3-Chlorocatechol-Oxidizing Extradiol Dioxygenase fromSphingomonas xenophaga BN6 by Random Mutagenesis

2001 ◽  
Vol 183 (7) ◽  
pp. 2322-2330 ◽  
Author(s):  
Ulrich Riegert ◽  
Sibylle Bürger ◽  
Andreas Stolz
Keyword(s):  
Amino Acid ◽  
Random Mutagenesis ◽  
Ring Cleavage ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Extradiol Dioxygenase ◽  
Mutant Forms ◽  
Sphingomonas Xenophaga

ABSTRACT The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.

scholarly journals Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility inEnterococcus faecalis

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Louis B. Rice ◽  
Charlene Desbonnet ◽  
Amelia Tait-Kamradt ◽  
Monica Garcia-Solache ◽  
John Lonks ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enterococcus Faecalis ◽  
Active Site ◽  
Fluorescent Protein ◽  
Joint Infection ◽  
Point Mutations ◽  
Promoter Sequence ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Content Type

ABSTRACTEnterococcus faecalisstrains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report anE. faecalisstrain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yieldedE. faecaliswith MICs of penicillins and carbapenems higher than those for wild-type strainE. faecalisJH2-2. Sequence analysis of thepbp4gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predictedpbp4−35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription ofpbp4in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed inE. faecalisLS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.IMPORTANCEEnterococcus faecalisis an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report anE. faecalisstrain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to “suppress” infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.

scholarly journals Use of a Two-Color Genetic Screen To Identify a Domain of the Global Regulator Lrp That Is Specifically Required forpap Phase Variation

1998 ◽  
Vol 180 (5) ◽  
pp. 1224-1231 ◽  
Author(s):  
Linda Kaltenbach ◽  
Bruce Braaten ◽  
Julie Tucker ◽  
Margareta Krabbe ◽  
David Low
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Phase Variation ◽  
Genetic Screen ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Global Regulator ◽  
Transcription In Vitro

ABSTRACT The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as ilvIH, activation ofpapBA transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate ilvIH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on ilvIH transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for pap and ilvIH DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp’s were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity forpap Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-lockedpap derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp’s are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on pap transcription state.

scholarly journals Mutations of theompK36Porin Gene and Promoter Impact Responses of Sequence Type 258, KPC-2-Producing Klebsiella pneumoniae Strains to Doripenem and Doripenem-Colistin

2013 ◽  
Vol 57 (11) ◽  
pp. 5258-5265 ◽  
Author(s):  
Cornelius J. Clancy ◽  
Liang Chen ◽  
Jae H. Hong ◽  
Shaoji Cheng ◽  
Binghua Hao ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Independent Predictor ◽  
Gene Mutations ◽  
Sequence Type ◽  
Wild Type ◽  
Content Type ◽  
Time Kill

ABSTRACTDoripenem-colistin exerts synergy against some, but not all,Klebsiella pneumoniaecarbapenemase (KPC)-producingK. pneumoniaestrainsin vitro. We determined if doripenem MICs and/orompK36porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 μg/ml) and colistin (2 μg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 μg/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutantompK36, respectively. The most common mutations were IS5promoter insertions (n= 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD;n= 8), which were associated with higher doripenem MICs than other mutations or wild-typeompK36(allPvalues ≤ 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135 GD, IS5, and wild-type/other mutants, respectively (P= 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was ≤8 μg/ml (P= 0.0007 and 0.09, respectively), but not if the MIC was >8 μg/ml (P= 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS5mutants (P= 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5mutations was the only independent predictor of doripenem-colistin responses at 24 h (P= 0.002). In conclusion,ompK36genotypes identified ST258 KPC-K. pneumoniaestrains that were most likely to respond to doripenem-colistin.

scholarly journals Pathoadaptive Alteration of Salmonella Biofilm Formation in Response to the Gallbladder Environment

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Michael R. Neiger ◽  
Juan F. González ◽  
Geoffrey Gonzalez-Escobedo ◽  
Harkness Kuck ◽  
Peter White ◽  
...  
Keyword(s):  
Mouse Model ◽  
Typhoid Fever ◽  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Fold Increase ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Content Type ◽  
Chronic Carriage

ABSTRACT Typhoid fever, a human-specific disease, is primarily caused by the pathogen Salmonella enterica serovar Typhi. It is estimated that 3 to 5% of people infected with typhoid fever become chronic carriers. Studies have demonstrated that a mechanism of chronic carriage involves biofilm formation on gallstone surfaces. In the course of a previous study using a chronic carriage mouse model, a Salmonella enterica serovar Typhimurium isolate was recovered from a mouse gallstone that exhibited a 2-fold increase in biofilm formation over the wild type. In order to identify the gene(s) responsible for the phenotype, the genomic sequences of this isolate and others were determined and compared. These sequences identified single nucleotide polymorphisms (SNPs) in 14 genes. Mutations in the most promising candidates, envZ and rcsB, were created, but neither showed increased biofilm-forming ability separately or in combination. The hyperbiofilm isolate did, however, present variations in cellular appendages observable using different techniques and a preferential binding to cholesterol. The isolate was also examined for systemic virulence and the ability to colonize the gallbladder/gallstones in a mouse model of chronic infection, demonstrating a systemic virulence defect and decreased gallbladder/gallstone colonization. Finally, to determine if the appearance of hyperbiofilm isolates could be replicated in vitro and if this was a common event, wild-type Salmonella spp. were grown long term in vitro under gallbladder-mimicking conditions, resulting in a high proportion of isolates that replicated the hyperbiofilm phenotype of the original isolate. Thus, Salmonella spp. acquire random mutations under the gallbladder/gallbladder-simulating conditions that may aid persistence but negatively affect systemic virulence. IMPORTANCE Chronic carriers are the main reservoirs for the spread of typhoid fever in regions of endemicity. Salmonella Typhi forms biofilms on gallstones in order to persist. A strain with enhanced biofilm-forming ability was recovered after a nine-month chronic-carriage mouse study. After sequencing this strain and recreating some of the mutations, we could not duplicate the phenotype. The isolate did show a difference in flagella, a preference to bind to cholesterol, and a systemic virulence defect. Finally, gallbladder conditions were simulated in vitro. After 60 days, there was a 4.5-fold increase in hyperbiofilm isolates when a gallstone was present. These results indicate that Salmonella spp. can undergo genetic changes that improve persistence in gallbladder albeit at the cost of decreased virulence.

Sign in / Sign up

Export Citation Format

Share Document