The gene that determines resistance to tioconazole and to acridine derivatives in Aspergillus nidulans may have a corresponding gene in Trichophyton rubrum

Understanding the genetic mechanisms involved in resistance to antifungal agents is important in the fight against pathogenic fungi. In the present investigation we studied a strain of the model fungus Aspergillus nidulans which presents resistance to tioconazole and behaves as the wild strain in the presence of other azole derivatives. Genetic analysis revealed that this resistance is due to a mutation in a single gene located on chromosome II, closely linked to the allele responsible for resistance to acriflavine and other acridine derivatives, i.e., acrA1. This result suggests that a multidrug resistance (MDR)-type mechanism may be involved. Two tioconazole-resistant strains of the pathogenic fungus Trichophyton rubrum obtained after mutagenic treatment also became simultaneously resistant to acriflavine and ethidium bromide, suggesting the existence of a resistance mechanism similar to that observed with the acrA1 mutation in A. nidulans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Papulacandin B resistance in budding and fission yeasts: isolation and characterization of a gene involved in (1,3)beta-D-glucan synthesis in Saccharomyces cerevisiae.

Papulacandin B, an antifungal agent that interferes with the synthesis of yeast cell wall (1,3)beta-D-glucan, was used to isolate resistant mutants in Schizosaccharomyces pombe and Saccharomyces cerevisiae. The resistance to papulacandin B always segregated as a recessive character that defines a single complementation group in both yeasts (pbr1+ and PBR1, respectively). Determination of several kinetic parameters of (1,3)beta-D-glucan synthase activity revealed no differences between S. pombe wild-type and pbr1 mutant strains except in the 50% inhibitory concentration for papulacandin B of the synthases (about a 50-fold increase in mutant activity). Inactivation of the synthase activity of both yeasts after in vivo treatment with the antifungal agent showed that mutant synthases were more resistant than the corresponding wild-type ones. Detergent dissociation of the S. pombe synthase into soluble and particulate fractions and subsequent reconstitution indicated that the resistance character of pbr1 mutants resides in the particulate fraction of the enzyme. Cloning and sequencing of PBR1 from S. cerevisiae revealed a gene identical to others recently reported (FKS1, ETG1, CWH53, and CND1). Its disruption leads to reduced levels of both (1,3)beta-D-glucan synthase activity and the alkali-insoluble cell wall fraction. Transformants containing the PBR1 gene reverse the defect in (1,3)beta-D-glucan synthase. It is concluded that Pbr1p is probably part of the (1,3)beta-D-glucan synthase complex.     

Isolation of dieldrin resistance from field populations of Drosophila melanogaster (Diptera: Drosophilidae)

High levels (about 4,000-fold) of resistance to dieldrin were isolated by screening field-collected populations of Drosophila melanogaster (Meigen). The resistance was made homozygous following 2-4 generations of selection. A single, major gene mapping to the left arm of chromosome III was solely responsible for resistance. The implications of the recovery of resistant mutants from field populations of D. melanogaster are discussed.     

Azaserine resistance in Escherichia coli: chromosomal location of multiple genes.

Resistance to azaserine in Escherichia coli is the result of mutations in at least three different loci. All spontaneously arising azaserine-resistant mutants harbor a lesion in the aroP gene. However, a lesion in this gene is not solely responsible for resistance. All spontaneously arising intermediate-level azaserine-resistant mutants also harbor a lesion in a gene designated azaA, which lies near min 43 on the chromosome. High-level resistant mutants harbor lesions in the aroP and azaA genes and in a third gene designated azaB, which lies near min 69 on the chromosome. Transport studies demonstrate that mutants harboring lesions in the azaA gene are not defective in the transport of the aromatic amino acids, but that mutants which harbor lesions in the azaB gene are defective in phenylalanine transport but not in tyrosine or tryptophan transport.     

A multiple resistance locus on chromosome arm 3BS in wheat confers resistance to stem rust (Sr2), leaf rust (Lr27) and powdery mildew

Sr2 is the only known durable, race non-specific adult plant stem rust resistance gene in wheat. The Sr2 gene was shown to be tightly linked to the leaf rust resistance gene Lr27 and to powdery mildew resistance. An analysis of recombinants and mutants suggests that a single gene on chromosome arm 3BS may be responsible for resistance to these three fungal pathogens. The resistance functions of the Sr2 locus are compared and contrasted with those of the adult plant resistance gene Lr34.     

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.     

Genetics of organophosphate resistance in field populations of the house fly (Diptera: Muscidae)

The genetics of resistance to the organophosphate insecticide diazinon were investigated in four populations of the house fly, Musca domestica L., collected in the southern United States. Crosses were made between individual females of lines derived from each population and males of a susceptible strain with three recessive mutants on chromosome II. Individual F1 females were crossed to mutant males, and the progenies were scored for resistance to diazinon and for the presence of mutant phenotypes. A major chromosome II gene for resistance to diazinon was present in all populations at an overall frequency of 83%. Map distances between the resistance gene and the mutant aristapedia and between the mutants aristapedia and stubby wing were highly variable in all populations. Recombination among the visible mutants was usually reduced in resistant progenies relative to susceptible progenies. The data suggest that a single major gene for resistance to diazinon was present on chromosome II in all test populations at variable map positions and is usually associated with a chromosome rearrangement, probably an inversion. The results are similar to those obtained earlier with house fly populations selected for resistance to insecticides in the laboratory; therefore, they seem to be characteristic of field and laboratory populations of the house fly. Overall, the data offer an explanation for previous results suggesting the existence of multiple, closely linked genes for metabolic resistance to insecticides on house fly chromosome II.     

A genetic analysis of cyromazine resistance in Drosophila melanogaster (Diptera: Drosophilidae)

Flies resistant to the insect growth regulator cyromazine were selected in the F1 generation from a cyromazine-susceptible strain of Drosophila melanogaster (Meigen) treated with ethyl methanesulfonate. Four resistant strains were isolated by screening with cyromazine at a concentration > LC100 of susceptibles. In each strain, resistance is conferred by a single gene mutation. Cyromazine resistance in two of the mutants (rst(1a)cyr1 and rst(1a)cyr2) localizes to map position 17 of the X chromosome. Evidence is presented that these mutations are alleles of the gene rst(1a)cyr. Cyromazine resistance in another of the mutants (Rst(1b)Cyr) is also X-linked, and localizes to map position 49 of the X chromosome. The location of the gene conferring cyromazine resistance in the other mutant (Rst(2b)Cyr) is map position 66 of chromosome II. This is possibly an allele of a previously characterized cyromazine resistance gene, Rst(2)Cyr. Dosage-mortality analyses demonstrate a low level of cyromazine resistance is conferred in all strains.     

In vitro inhibitory effects of farnesol and interactions between farnesol and antifungals against biofilms of Candida albicans resistant strains

Antifungal resistance is a serious problem in clinical infections. Farnesol, which is a potential antifungal agent against biofilms formed by Candida albicans resistant strains (a fluconazole-resistant isolate derived from SC5314 and two clinical Candida resistant isolates), was investigated in this study. The inhibitory effects of farnesol on biofilms were examined by XTT assay. The morphological changes and biofilm thicknesses were analyzed by scanning electron microscopy and confocal laser scanning microscopy, respectively. Additionally, the checkerboard microdilution method was used to investigate the interactions between farnesol and antifungals (fluconazole, amphotericin B, caspofungin, itraconazole, terbinafine and 5-flurocytosine) against biofilms. The results showed decreased SMICs of farnesol and thinner biofilms in the farnesol-treated groups, indicating that farnesol inhibited the development of biofilms formed by the resistant strain. Furthermore, there were synergistic effects between farnesol and fluconazole/5-flurocytosine, while there were antagonistic effects between farnesol and terbinafine/itraconazole, respectively, on the biofilms formed by the resistant strains.     

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.     

Modification of Ribosomes in Cryptopleurine-Resistant Mutants of Yeast

Cryptopleurine-resistant mutants of Saccharomyces cerevisiae were isolated. A single, recessive nuclear gene, very closely linked to the mating locus (2.1 centimorgans), is responsible for resistance. Ribosomes from the mutants were found to be resistant to cryptopleurine when analyzed by poly(U)-directed polyphenylalanine synthesis. Analysis of the distribution of ribosomes between monosomes and polysomes in sensitive cells exposed to cryptopleurine suggests that some step is inhibited during the elongation phase of protein synthesis.     

Identification of Saccharomyces cerevisiae ribosomal protein L3 as a target of curvularol, a G1-specific inhibitor of mammalian cells

The cellular target of curvularol, a G1-specific cell-cycle inhibitor of mammalian cells, was identified by a genetic approach in Saccharomyces cerevisiae. Since the wild-type W303 strain was highly resistant to curvularol, a drug hypersensitive parental strain was constructed in which various genes implicated in general drug resistance had been disrupted. Curvularol resistant mutants were isolated, and strains that exhibited a semi-dominant, curvularol-specific resistance phenotype were selected. All five strains examined were classified into a single genetic complementation group designated YCR1. A mutant gene responsible for curvularol resistance was identified as an allele of the RPL3 gene encoding the ribosomal protein L3. Sequence analysis of the mutant genes revealed that Trp255Cys and Trp255Leu substitutions of Rpl3p are responsible for curvularol resistance. Rpl3p mutants in which Trp255 residue was replaced by other amino acids were constructed. All of these replacements led to varying degrees of increased resistance to curvularol and growth defects.     

In Candida albicans, resistance to flucytosine and terbinafine is linked to MAT locus homozygosity and multilocus sequence typing clade 1

A panel of 637 isolates of Candida albicans that had been typed by multilocus sequence typing (MLST) and tested for susceptibility to amphotericin B, caspofungin, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, terbinafine and voriconazole was the material for a statistical analysis of possible associations between antifungal susceptibility and other properties. For terbinafine and flucytosine, the greatest proportion of low-susceptibility isolates, judged by two resistance breakpoints, was found in MLST clade 1 and among isolates homozygous at the MAT locus, although only three isolates showed cross-resistance to the two agents. Most instances of low susceptibility to azoles, flucytosine and terbinafine were among oropharyngeal isolates from HIV-positive individuals. Statistically significant correlations were found between terbinafine and azole minimal inhibitory concentrations (MICs), while correlations between flucytosine MICs and azole MICs were less strong. It is concluded that a common regulatory mechanism may operate to generate resistance to the two classes of agent that inhibit ergosterol biosynthesis, terbinafine and the azoles, but that flucytosine resistance, although still commonly associated with MAT homozygosity, is differently regulated.     

Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants.

In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of beta-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective beta-lactams.     

Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique

Identification of genetic markers involved in stress response to physical factors or chemical substances in organisms is a challenging task. Typing of upregulated gene expression due to selective antibacterial pressure is a promising approach in the search of molecular mechanisms responsible for development of resistance. cDNA-Fluorescent Amplified Fragment Length Polymorphism (cDNA-FAFLP) strategy was developed and applied in the search of antimycotic drug resistance marker(s) in medically important fungi as an alternative method to microarray analysis. We compared differential gene expression of two sensitive Candida albicans reference strains (ATCC 10231 and ATCC 60133) and two of their paired resistant to fluconazole and itraconazole mutants. Resistant mutants Candida albicans FLC-R, resistant to fluconazole (MIC > 128 μg/ml) and Candida albicans ICZ-R, resistant to itraconazole (MIC > 4 μg/ml) were obtained in subcultures with gradual increase of the antifungal in the culture medium. cDNA-AFLP profile in both itraconazole resistant mutants showed specific spectrophotometric peaks with 5–6-fold RNA overexpression product of 500 bp length compared to the sensitive strains. Fluconazole mutants do not reveal RNA level changes under tested by us typing conditions. These results indicate that the cDNA-FAFLP strategy is a relatively rapid, simple, and reliable method for simultaneous typing of both constitutive and induced differences in expression of host genes providing insight into the biological processes involved in response to drugs in bacteria and fungi. Moreover, this methodology could be tested for typing of the genome response of any organism to physical or chemical stress factors.     

Mutations in the genes for synthesis of the outer core region of the lipopolysaccharide of Yersinia enterocolitica O:3

AIMS: The aim of this study was to construct non-polar frame-shift mutations in some of the individual genes responsible for the biosynthesis of the branching outer core (OC) hexasaccharide of the lipopolysaccharide (LPS) in Yersinia enterocolitica O:3 (YeO:3). METHODS AND RESULTS: Chromosomal segments of YeO:3 containing wbcN, wbcO and wbcQ genes were cloned into a suicide vector. A frame-shift mutation was introduced into each gene by modifying a unique restriction enzyme recognition site. Each recombinant plasmid with a modified OC gene was mobilized into YeO:3 to allow for allelic exchange between the modified gene and the wild type chromosomal gene. The exchange was confirmed by demonstrating the absence of the particular restriction site in the chromosome of each mutant strain. Analysis of LPS by gel electrophoresis showed that the LPS of the mutants was lacking the OC. Therefore, the constructed wbcN, wbcO and wbcQ strains are true mutants with frame-shifts in the corresponding genes. CONCLUSIONS: The products of the wbcN, wbcO and wbcQ genes are putative glycosyltransferases and, based on the present analysis, essential for the biosynthesis of the OC hexasaccharide. The absence of OC in the LPS of these mutants further supports the hypothesis that the OC hexasaccharide is a single O-antigen O-unit that is not polymerized in YeO:3. SIGNIFICANCE AND IMPACT OF THE STUDY: These mutants provide information on the unique nature of the synthesis of OC of YeO:3 LPS. They are valuable for future biochemical studies to establish the roles of the products of individual OC genes.     

Mechanisms of antifungal resistance

The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allylamines has not been reported from human pathogens, but, resistant phenotypes have been reported for variants of Saccharomyces cerevisiae and of Ustilago maydis. Tolerance to morpholines is seldom found. Intrinsic resistance to griseofulvin is due to the absence of a prolonged energy-dependent transport system for this antibiotic. Resistance to azole antifungal agents is known to be exceptional, although it does now appear to be increasing in importance in some groups of patients infected with e.g. Candida spp., Histoplasma capsulatum or Cryptococcus neoformans. For example, resistance to fluconazole is emerging in C. albicans, the major agent of oro-pharyngeal candidosis in AIDS patients, after long-term suppressive therapy. In the majority of cases, primary and secondary resistance to fluconazole and cross-resistance to other azole antifungal agents seems to originate from decreased intracellular accumulation of the azoles, which may result from reduced uptake or increased efflux of the molecules. In most C. albicans isolates the decreased intracellular levels can be correlated with enhanced azole efflux, a phenomenon linked to an increase in the amounts of mRNA of a C. albicans ABC transporter gene CDR1 and of a gene (BEN(r) or CaMDR) coding for a transporter belonging to the class of major facilitator multidrug efflux transporters. Not only fluconazole, ketoconazole and itraconazole are substrates for CDR1, terbinafine and amorolfine have also been established as substrates, BEN(r) overexpression only accounts for fluconazole resistance. Other sources of resistance: changes in membrane sterols and phospholipids, altered or overproduced target enzyme(s) and compensatory mutations in the Delta5,6-desaturase.     

Yeast resistance to polyene antibiotics. I. Production of Saccharomyces cerevisiae mutants resistant to nystatin and their genetic analysis]

239 nistatin-resistant mutants were selected after UV-irradiation of yeasts. Phenotypical analysis has revealed two main groups of the mutants: 1) resistant to nistatin and resistant or sensitive (in different combinations) to haptaens; 2) resistant to nistatin and having an increased resistance to haptens. It is found that the sensitivity dominates over the resistance and hyper-resistance. Genetic analysis of the mutant collection has shown that the resistance to nistatin is determined by 5 nuclear genes (hysr). Hyper-resistance is controlled by mutations in other genes, which are not connected with stable phenotype. Genes of hyper-resistance can be considered as minus-modificators of pleiothrophic cross-resistance, characteristic of hysr genes. Plus-modificator genes of polyenic resistance are described. The gene hysr1 is linked with its chromosome.     

Telomeric gene deletion and intrachromosomal amplification in antimony‐resistant Leishmania

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next‐generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony‐resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate‐dependent peroxidase (APX) and glucose‐6‐phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.     

Inheritance and RAPD tagging of multiple genes for resistance to net blotch in barley.

A doubled haploid barley (Hordeum vulgare L.) population that was created from a cross between cultivars 'Léger' and 'CI 9831' was characterized by RAPD (random amplified polymorphic DNA) markers for resistance to isolate WRS857 of Pyrenophora teres Drechs. f. sp. maculata Smedeg., the causal agent of the spot form of net blotch. Resistance, which initially appeared to be conferred by a single gene from the approximate 1:1 (resistant : susceptible) segregation ratio of the doubled-haploid (DH) progeny, was found to be associated with three different genomic regions by RAPD analysis. Of 500 RAPD random primers that were screened against the parents, 195 revealed polymorphic bands, seven showed an association to the resistance in bulks, and these seven markers were mapped to three unlinked genomic regions. Two of these regions, one of which was mapped to chromosome 2, have major resistance genes. The third region has some homology to the chromosome 2 region. This study demonstrates the simultaneous location of markers for more than one gene governing a trait by using RAPD and bulked segregant analysis (BSA).