scholarly journals The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

2000 ◽  
Vol 44 (1) ◽  
pp. 63-67 ◽  
Author(s):  
David C. Lamb ◽  
Diane E. Kelly ◽  
Theodore C. White ◽  
Steven L. Kelly
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Specific Activity ◽  
Difference Spectrum ◽  
Microsomal Protein ◽  
Ergosterol Biosynthesis ◽  
Wild Type ◽  
Binding Studies ◽  
Directed Mutation

ABSTRACT The cytochrome P450 sterol 14α-demethylase (CYP51) ofCandida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3β-[32-3H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.

scholarly journals Targeted Recombination within the Spike Gene of Murine Coronavirus Mouse Hepatitis Virus-A59: Q159 Is a Determinant of Hepatotropism

1998 ◽  
Vol 72 (12) ◽  
pp. 9628-9636 ◽  
Author(s):  
Isabelle Leparc-Goffart ◽  
Susan T. Hingley ◽  
Ming Ming Chua ◽  
Joanna Phillips ◽  
Ehud Lavi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Spike Gene ◽  
Recombinant Viruses ◽  
Directed Mutation ◽  
Murine Coronavirus

ABSTRACT Previous studies of a group of mutants of the murine coronavirus mouse hepatitis virus (MHV)-A59, isolated from persistently infected glial cells, have shown a strong correlation between a Q159L amino acid substitution in the S1 subunit of the spike gene and a loss in the ability to induce hepatitis and demyelination. To determine if Q159L alone is sufficient to cause these altered pathogenic properties, targeted RNA recombination was used to introduce a Q159L amino acid substitution into the spike gene of MHV-A59. Recombination was carried out between the genome of a temperature-sensitive mutant of MHV-A59 (Alb4) and RNA transcribed from a plasmid (pFV1) containing the spike gene as well as downstream regions, through the 3′ end, of the MHV-A59 genome. We have selected and characterized two recombinant viruses containing Q159L. These recombinant viruses (159R36 and 159R40) replicate in the brains of C57BL/6 mice and induce encephalitis to a similar extent as wild-type MHV-A59. However, they exhibit a markedly reduced ability to replicate in the liver or produce hepatitis compared to wild-type MHV-A59. These viruses also exhibit reduced virulence and reduced demyelination. A recombinant virus containing the wild-type MHV-A59 spike gene, wtR10, behaved essentially like wild-type MHV-A59. This is the first report of the isolation of recombinant viruses containing a site-directed mutation, encoding an amino acid substitution, within the spike gene of any coronavirus. This technology will allow us to begin to map the molecular determinants of pathogenesis within the spike glycoprotein.

scholarly journals A Single Amino Acid Substitution in the West Nile Virus Nonstructural Protein NS2A Disables Its Ability To Inhibit Alpha/Beta Interferon Induction and Attenuates Virus Virulence in Mice

2006 ◽  
Vol 80 (5) ◽  
pp. 2396-2404 ◽  
Author(s):  
Wen Jun Liu ◽  
Xiang Ju Wang ◽  
David C. Clark ◽  
Mario Lobigs ◽  
Roy A. Hall ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Nonstructural Protein ◽  
Mutant Virus ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Wild Type ◽  
Alpha Beta ◽  
A30p Mutation

ABSTRACT Alpha/beta interferons (IFN-α/β) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-α/β responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNVKUN), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-β promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNVKUN genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-α/β in infected human A549 cells than that detected following wild-type WNVKUN infection. Consequently, replication of the WNVKUNNS2A/A30P mutant virus in these cells known to be high producers of IFN-α/β was abortive. In contrast, both the mutant and the wild-type WNVKUN produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-α/β production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-α/βγ receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNVKUN and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4178-4187 ◽  
Author(s):  
O. Peyruchaud ◽  
A.T. Nurden ◽  
S. Milet ◽  
L. Macchi ◽  
A. Pannochia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

scholarly journals An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety

2001 ◽  
Vol 75 (14) ◽  
pp. 6303-6309 ◽  
Author(s):  
Marintha L. Heil ◽  
Alison Albee ◽  
James H. Strauss ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acid ◽  
Heparan Sulfate ◽  
Amino Acid Substitution ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Ross River Virus ◽  
Wild Type ◽  
Coding Region ◽  
Sulfate Production ◽  
E2 Glycoprotein

ABSTRACT Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.

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.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4178-4187 ◽  
Author(s):  
O. Peyruchaud ◽  
A.T. Nurden ◽  
S. Milet ◽  
L. Macchi ◽  
A. Pannochia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

scholarly journals Pyridines and Pyrimidines Mediating Activity against an Efflux-Negative Strain of Candida albicans through Putative Inhibition of Lanosterol Demethylase

2004 ◽  
Vol 48 (1) ◽  
pp. 313-318 ◽  
Author(s):  
Ed T. Buurman ◽  
April E. Blodgett ◽  
Kenneth G. Hull ◽  
Daniel Carcanague
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Ergosterol Biosynthesis ◽  
Clear Correlation ◽  
Wild Type ◽  
Inhibition Of Growth ◽  
Ergosterol Synthesis ◽  
A Cell ◽  
Sterol Profile ◽  
Lanosterol Demethylase

ABSTRACT The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased ERG10 transcription results in elevated specific β-galactosidase activity, was used to find novel inhibitors of ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major fungal pathogen Candida albicans (MICs > 64 μg · ml−1). However, a strain of C. albicans lacking the Cdr1p and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 μg · ml−1), suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol. Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively mediate their antifungal activity against C. albicans via lanosterol demethylase.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

scholarly journals Overproduction of l-Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

2001 ◽  
Vol 67 (7) ◽  
pp. 3064-3070 ◽  
Author(s):  
Hiroaki Motoyama ◽  
Hiroshi Yano ◽  
Yoko Terasaki ◽  
Hideharu Anazawa
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Test Tube ◽  
Amino Acid Sequences ◽  
Nutritional Requirement ◽  
Wild Type ◽  
Gene Encoding ◽  
Lysine Production ◽  
Obligate Methylotroph ◽  
In The Wild

ABSTRACT The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by l-lysine, was cloned from an l-threonine- andl-lysine-coproducing mutant of the obligate methylotrophMethylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapAmutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an l-threonine producing mutant, elevated the specific activity of DDPS 20-fold andl-lysine production 2- to 3-fold with concomitant reduction of l-threonine in test tube cultures. AL119 containing thedapA gene produced 8 g of l-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M r of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition byl-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector,l-lysine.

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