scholarly journals Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate

2021 ◽  
Vol 7 (11) ◽  
pp. 897
Author(s):  
Danyon O. Graham ◽  
Rajni K. Wilson ◽  
Yasmeen N. Ruma ◽  
Mikhail V. Keniya ◽  
Joel D. A. Tyndall ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Crystal Structures ◽  
Differential Susceptibility ◽  
Azole Resistance ◽  
Wild Type ◽  
Gene Encoding ◽  
High Level ◽  
Structural Insights ◽  
Level Resistance

Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs.

scholarly journals Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

2000 ◽  
Vol 44 (11) ◽  
pp. 2985-2990 ◽  
Author(s):  
Hiroshi Kakeya ◽  
Yoshitsugu Miyazaki ◽  
Haruko Miyazaki ◽  
Katherine Nyswaner ◽  
Brian Grimberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Analysis ◽  
Expression System ◽  
Single Copy ◽  
Gene Replacement ◽  
Azole Resistance ◽  
High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

scholarly journals Characterization of Saccharomyces cerevisiae strains displaying high-level or low-level resistance to trichothecene antibiotics

1990 ◽  
Vol 267 (3) ◽  
pp. 709-713 ◽  
Author(s):  
M Fernandez-Lobato ◽  
M Cannon ◽  
J A Mitlin ◽  
R C Mount ◽  
A Jimenez
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosome Biogenesis ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Low Level ◽  
Ribosomal Protein L3 ◽  
High Level ◽  
Level Resistance

Biochemical and genetic analyses have been carried out on Saccharomyces cerevisiae strains characterized in vivo as sensitive, low-level-resistant or high-level-resistant to trichothecene antibiotics. Levels of drug resistance in vitro were determined for each strain and for suitable diploids derived from them. Ribosome biogenesis was also studied in selected haploids. It is suggested that resistance in all cases results from a mutation in the gene encoding ribosomal protein L3. If this is indeed the situation, then different mutations in this same gene not only can cause low-level or high-level resistance to trichothecene antibiotics but also can affect the maturation of either 40 S or 60 S ribosomal subunits.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

2004 ◽  
Vol 48 (12) ◽  
pp. 4505-4512 ◽  
Author(s):  
Chia-Geun Chen ◽  
Yun-Liang Yang ◽  
Hsin-I Shih ◽  
Chia-Li Su ◽  
Hsiu-Jung Lo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Type Strain ◽  
Antifungal Agents ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Galactosidase Activity ◽  
Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

scholarly journals Divergence of Eukaryotic Secretory Components: the Candida albicans Homolog of the Saccharomyces cerevisiae Sec20 Protein Is N Terminally Truncated, and Its Levels Determine Antifungal Drug Resistance and Growth

2001 ◽  
Vol 183 (1) ◽  
pp. 46-54 ◽  
Author(s):  
Yvonne Weber ◽  
Uwe J. Santore ◽  
Joachim F. Ernst ◽  
Rolf K. Swoboda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Transcriptional Control ◽  
Secretory Pathway ◽  
Sodium Dodecyl ◽  
Fungal Species ◽  
Gene Encoding ◽  
Vesicle Traffic ◽  
Antifungal Drug Resistance ◽  
And Function

ABSTRACT Sec20p is a component of the yeast Saccharomyces cerevisiae secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen Candida albicans. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to S. cerevisiae Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both C. albicans SEC20alleles could not be constructed, we placed SEC20 under transcriptional control of two regulatable promoters, MET3pand PCK1p. Repression of SEC20 expression in these strains prevented (MET3p-SEC20 allele) or retarded (PCK1p-SEC20 allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced SEC20 expression in the PCK1p-SEC20strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than S. cerevisiae, but the lack of the N-terminal domain and the apparent absence of a close TIP20 homolog in the C. albicans genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.

Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1

1991 ◽  
Vol 11 (5) ◽  
pp. 2593-2608 ◽  
Author(s):  
D X Tishkoff ◽  
A W Johnson ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Open Reading Frame ◽  
Strand Exchange ◽  
Wild Type ◽  
Homologous Pairing ◽  
Gene Encoding ◽  
Reading Frame ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Exchange Protein

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

scholarly journals Flavin Mononucleotide-Based Fluorescent Protein as an Oxygen-Independent Reporter in Candida albicans and Saccharomyces cerevisiae

2009 ◽  
Vol 8 (6) ◽  
pp. 913-915 ◽  
Author(s):  
D. Tielker ◽  
I. Eichhof ◽  
K.-E. Jaeger ◽  
J. F. Ernst
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Flavin Mononucleotide ◽  
Gene Encoding

ABSTRACT Hypoxia is encountered frequently by pathogenic and apathogenic fungi. A codon-adapted gene encoding flavin mononucleotide-based fluorescent protein (CaFbFP) was expressed in Candida albicans and Saccharomyces cerevisiae. Both species produced CaFbFP and fluoresced even during hypoxia, suggesting that oxygen-independent CaFbFP is a useful, novel tool for monitoring hypoxic gene expression in fungi.

scholarly journals Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

1991 ◽  
Vol 11 (8) ◽  
pp. 3868-3878 ◽  
Author(s):  
A L Munn ◽  
L Silveira ◽  
M Elgort ◽  
G S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Heavy Chain ◽  
Secretory Pathway ◽  
Genetic Locus ◽  
Wild Type ◽  
Site Mutation ◽  
Gene Encoding ◽  
Clathrin Heavy Chain ◽  
Lethal Allele

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.

scholarly journals Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

2020 ◽  
Vol 98 (5) ◽  
pp. 624-630 ◽  
Author(s):  
Yanrui Zhu ◽  
Matthew D. Berg ◽  
Phoebe Yang ◽  
Raphaël Loll-Krippleber ◽  
Grant W. Brown ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Genetic Disease ◽  
Elevated Temperatures ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Standard Genetic Code ◽  
Gene Encoding ◽  
Chromatid Cohesion ◽  
Sensitive Allele

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

scholarly journals Inactivation of KsgA, a 16S rRNA Methyltransferase, Causes Vigorous Emergence of Mutants with High-Level Kasugamycin Resistance

2008 ◽  
Vol 53 (1) ◽  
pp. 193-201 ◽  
Author(s):  
Kozo Ochi ◽  
Ji-Yun Kim ◽  
Yukinori Tanaka ◽  
Guojun Wang ◽  
Kenta Masuda ◽  
...  
Keyword(s):  
16S Rrna ◽  
High Frequency ◽  
Resistance Mutations ◽  
Gene Encoding ◽  
Adenosylmethionine Decarboxylase ◽  
Ribosomal Function ◽  
High Level ◽  
Antibiotic Resistance Mutations ◽  
Modest Level ◽  
Level Resistance

ABSTRACT The methyltransferases RsmG and KsgA methylate the nucleotides G535 (RsmG) and A1518 and A1519 (KsgA) in 16S rRNA, and inactivation of the proteins by introducing mutations results in acquisition of low-level resistance to streptomycin and kasugamycin, respectively. In a Bacillus subtilis strain harboring a single rrn operon (rrnO), we found that spontaneous ksgA mutations conferring a modest level of resistance to kasugamycin occur at a high frequency of 10−6. More importantly, we also found that once cells acquire the ksgA mutations, they produce high-level kasugamycin resistance at an extraordinarily high frequency (100-fold greater frequency than that observed in the ksgA + strain), a phenomenon previously reported for rsmG mutants. This was not the case for other antibiotic resistance mutations (Tspr and Rifr), indicating that the high frequency of emergence of a mutation for high-level kasugamycin resistance in the genetic background of ksgA is not due simply to increased persistence of the ksgA strain. Comparative genome sequencing showed that a mutation in the speD gene encoding S-adenosylmethionine decarboxylase is responsible for the observed high-level kasugamycin resistance. ksgA speD double mutants showed a markedly reduced level of intracellular spermidine, underlying the mechanism of high-level resistance. A growth competition assay indicated that, unlike rsmG mutation, the ksgA mutation is disadvantageous for overall growth fitness. This study clarified the similarities and differences between ksgA mutation and rsmG mutation, both of which share a common characteristic—failure to methylate the bases of 16S rRNA. Coexistence of the ksgA mutation and the rsmG mutation allowed cell viability. We propose that the ksgA mutation, together with the rsmG mutation, may provide a novel clue to uncover a still-unknown mechanism of mutation and ribosomal function.

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