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Populations of the causal agent of wheat tan spot, Pyrenophora tritici-repentis, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C14-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in P. tritici-repentis. Application of this compound in combination with fungicides shifted fungicide-resistant P. tritici-repentis isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.

Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14α-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMIR) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5- to 12-fold compared to that in DMI-sensitive (DMIS) isolates. Analysis of sequences upstream of CYP51 in 59 DMIR isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of CYP51 were not present in any of 22 DMIS isolates examined.

The isolation of Penicillium caseicolum mutants resistant to different fungicides which inhibit ergosterol biosynthesis is reported. Mutational frequencies for resistance were high (3 × 10−3 to 3 × 10−5). The levels of resistance toward the inhibitors of sterol C-14 demethylation were always low (<10), whereas high values were obtained with mutants resistant to inhibitors of sterol Δ14 reduction or Δ8→Δ7 isomerization, or both. Generally, there was a positive cross-resistance between fungicides showing the same biochemical mode of action but not between compounds of two different groups. Mycelial growth rate and sporulation were tested; several mutants were not affected for these characteristics. We conclude that resistance to ergosterol biosynthesis inhibitors may be used as a good marker for genetic studies through protoplast fusion.

Resistance in Monilinia fructicola to demethylation inhibitor (DMI) fungicides is beginning to emerge in North America, but its molecular basis is unknown. Two potential genetic determinants of DMI fungicide resistance including the 14α-demethylase gene (MfCYP51) and the ATP-binding cassette transporter gene MfABC1, were investigated in six resistant (DMI-R) and six sensitive (DMI-S) field isolates. No point mutations leading to an amino acid change were found in the MfCYP51 gene. The constitutive expression of the MfCYP51 gene in DMI-R isolates was significantly higher compared to DMI-S isolates. Gene expression was not induced in mycelium of DMI-R or DMI-S isolates treated with 0.3 μg of propiconazole/ml. A slightly higher average MfCYP51 copy number value was detected in DMI-R isolates (1.35) compared to DMI-S isolates (1.13); however, this difference could not be verified in Southern hybridization experiments or explain the up to 11-fold-increased MfCYP51 mRNA levels in DMI-R isolates. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp repetitive element at base pair position −117 from the translational start site in DMI-R isolates but not in DMI-S isolates. This repetitive element contained a putative promoter and was named Mona. The link between Mona and the DMI resistance phenotype became even more apparent after studying the genetic diversity between the isolates. In contrast to DMI-S isolates, DMI-R isolates contained an MfCYP51 gene of identical nucleotide sequence associated with Mona. Still, DMI-R isolates were not genetically identical as revealed by Microsatellite-PCR analysis. Also, real-time PCR analysis of genomic DNA indicated that the relative copy number of Mona among DMI-S and DMI-R isolates varied, suggesting its potential for mobility. Interestingly, constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower than that of DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. Therefore, the MfABC1 gene may play a minor role in DMI fungicide resistance in M. fructicola. Our results strongly suggest that overexpression of the MfCYP51 gene is an important mechanism in conferring DMI fungicide resistance in M. fructicola field isolates from Georgia and that this overexpression is correlated with Mona located upstream of the MfCYP51 gene.

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a “bridge” in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.

The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.

Background

Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14α-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR34/L98H). We investigated if TR34/L98H could have developed through exposure to DMIs.

Methods and Findings

Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in the Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR34/L98H isolate in 1998. Through microsatellite genotyping of TR34/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR34/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes.

Conclusions

Our findings support a fungicide-driven route of TR34/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs.

The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO7-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.

Candida albicans, the major fungal pathogen of humans, causes life-threatening infections in immunocompromised individuals. Due to limited available therapy options, this can frequently lead to therapy failure and emergence of drug resistance. To improve current treatment strategies, we have combined comprehensive chemical-genomic screening in Saccharomyces cerevisiae and validation in C. albicans with the goal of identifying compounds that can couple with the fungistatic drug fluconazole to make it fungicidal. Among the genes identified in the yeast screen, we found that only AGE3, which codes for an ADP-ribosylation factor GTPase activating effector protein, abrogates fluconazole tolerance in C. albicans. The age3 mutant was more sensitive to other sterols and cell wall inhibitors, including caspofungin. The deletion of AGE3 in drug resistant clinical isolates and in constitutively active calcineurin signaling mutants restored fluconazole sensitivity. We confirmed chemically the AGE3-dependent drug sensitivity by showing a potent fungicidal synergy between fluconazole and brefeldin A (an inhibitor of the guanine nucleotide exchange factor for ADP ribosylation factors) in wild type C. albicans as well as in drug resistant clinical isolates. Addition of calcineurin inhibitors to the fluconazole/brefeldin A combination only initially improved pathogen killing. Brefeldin A synergized with different drugs in non-albicans Candida species as well as Aspergillus fumigatus. Microarray studies showed that core transcriptional responses to two different drug classes are not significantly altered in age3 mutants. The therapeutic potential of inhibiting ARF activities was demonstrated by in vivo studies that showed age3 mutants are avirulent in wild type mice, attenuated in virulence in immunocompromised mice and that fluconazole treatment was significantly more efficacious when ARF signaling was genetically compromised. This work describes a new, widely conserved, broad-spectrum mechanism involved in fungal drug resistance and virulence and offers a potential route for single or improved combination therapies.

Author Summary

Candida albicans is a fungus that normally resides as part of the microflora in the human gut. Candida species can cause superficial infections like thrush in the healthy human population and life-threatening invasive infections in immunocompromised patients. Fungal infections are often treated with azole drugs, but due to the fungistatic nature of these agents, C. albicans can develop drug resistance, leading to therapy failure. To improve the action of azoles and convert them into fungicidal drugs, we first systematically analyzed the genetic requirements for tolerance to one such azole drug, fluconazole. We show, both genetically and pharmacologically, that components of the ARF cycling machinery are critical in mediating both azole and echinocandin tolerance in C. albicans as well as several other pathogenic Candida species and in the pathogenic mold Aspergillus fumigatus. We highlight the importance of ARF cycling in drug resistance by showing that genetic compromise of ARF functions overrides common drug resistance mechanisms in clinical samples and other key regulators of azole/echinocandin tolerance. We validated the therapeutic potential of ARF cycling in two mouse models and provide evidence that drug treatment is more efficacious when ARF activities are genetically compromised. Our study demonstrates a new mechanism involved in two important aspects of the biology of human fungal pathogens and provides a potential route for improved antifungal therapies.

Background

By disrupting the action of androgens during gestation, certain chemicals present in food, consumer products, and the environment can induce irreversible demasculinization and malformations of sex organs among male offspring. However, the consequences of simultaneous exposure to such chemicals are not well described, especially when they exert their actions by differing molecular mechanisms.

Objectives

To fill this gap, we investigated the effects of mixtures of a widely used plasticizer, di(2-ethylhexyl) phthalate (DEHP); two fungicides present in food, vinclozolin and prochloraz; and a pharmaceutical, finasteride, on landmarks of male sexual development in the rat, including changes in anogenital distance (AGD), retained nipples, sex organ weights, and malformations of genitalia. These chemicals were chosen because they disrupt androgen action with differing mechanisms of action.

Results

Strikingly, the effect of combined exposure to the selected chemicals on malformations of external sex organs was synergistic, and the observed responses were greater than would be predicted from the toxicities of the individual chemicals. In relation to other hallmarks of disrupted male sexual development, including changes in AGD, retained nipples, and sex organ weights, the combined effects were dose additive. When the four chemicals were combined at doses equal to no observed adverse effect levels estimated for nipple retention, significant reductions in AGD were observed in male offspring.

Conclusions

Because unhindered androgen action is essential for human male development in fetal life, these findings are highly relevant to human risk assessment. Evaluations that ignore the possibility of combination effects may lead to considerable underestimations of risks associated with exposures to chemicals that disrupt male sexual differentiation.

Fluconazole was observed to inhibit sterol 14 alpha-demethylase in the human pathogen Cryptococcus neoformans, and accumulation of a ketosteroid product was associated with growth arrest. A novel mechanism(s) of azole and amphotericin B cross-resistance was identified, unrelated to changes in sterol biosynthesis, as previously identified in Saccharomyces cerevisiae. Reduced cellular content of drug could account for the resistance phenotype, indicating the possible involvement of a mechanism similar to multidrug resistance observed in higher eukaryotes.

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, ≥8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.

We report a case of infection with Candida glabrata in which the organism became resistant to fluconazole and in which pre- and posttreatment isolates were available for comparison. The organism was cross-resistant to ketoconazole and itraconazole, in common with other azole-resistant yeasts. Fluconazole was a potent inhibitor of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) in lysates of cells from both susceptible and resistant cultures (50% inhibitory concentration, 0.2 microM), indicating that resistance was unrelated to changes in P-450DM. Instead, it appeared to arise from a permeability barrier to fluconazole, since resistant cells were unable to take up radiolabelled drug.

A common consequence of using agricultural fungicides is the development of resistance by fungal pathogens, which undermines reliability of fungicidal effectiveness. A potentially new strategy to aid in overcoming or minimizing this problem is enhancement of pathogen sensitivity to fungicides, or “chemosensitization.” Chemosensitization can be accomplished by combining a commercial fungicide with a certain non- or marginally fungicidal substance at levels where, alone, neither compound would be effective. Chemosensitization decreases the probability of the pathogen developing resistance, reduces the toxic impact on the environment by lowering effective dosage levels of toxic fungicides, and improves efficacy of antifungal agents. The present study shows that the antifungal activity of azole and strobilurin fungicides can be significantly enhanced through their co-application with certain natural or synthetic products against several economically important plant pathogenic fungi. Quadris (azoxystrobin) combined with thymol at a non-fungitoxic concentration produced much higher growth inhibition of Bipolaris sorokiniana, Phoma glomerata, Alternaria sp. and Stagonospora nodorum than the fungicide alone. The effect of Dividend (difenoconazole) applied with thymol significantly enhanced antifungal activity against B. sorokiniana and S. nodorum. Folicur (tebuconazole) combined with 4-hydroxybenzaldehyde (4-HBA), 2,3-dihydroxybenzaldehyde or thymol significantly inhibited growth of Alternaria alternata, at a much greater level than the fungicide alone. In addition, co-application of Folicur and 4-HBA resulted in a similar enhancement of antifungal activity against Fusarium culmorum. Lastly, we discovered that metabolites in the culture liquid of Fusarium sambucinum biocontrol isolate FS-94 also had chemosensitizing activity, increasing S. nodorum sensitivity to Folicur and Dividend.

Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14-alpha-demethylase and aromatase. Sterol 14-alpha-demethylase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14-alpha-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14-alpha-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14-alpha-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14-alpha-demethylase are inhibitors also of mammalian sterol 14-alpha-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed.

Background

Venturia inaequalis is an economically-important disease of apple causing annual epidemics of scab worldwide. The pathogen is a heterothallic ascomycete with an annual cycle of sexual reproduction on infected apple leaf litter, followed by several cycles of asexual reproduction during the apple growing season. Current disease control is achieved mainly through scheduled applications of fungicides. Genetic linkage maps are essential for studying genome structure and organisation, and are a valuable tool for identifying the location of genes controlling important traits of interest such as avirulence, host specificity and mating type in V. inaequalis. In this study, we performed a wide cross under in vitro conditions between an isolate of V. inaequalis from China and one from the UK to obtain a genetically diverse mapping population of ascospore progeny isolates and produced a map using AFLP and microsatellite (SSR) markers.

Findings

Eighty-three progeny were obtained from the cross between isolates C0154 (China) × 01/213 (UK). The progeny was screened with 18 AFLP primer combinations and 31 SSRs, and scored for the mating type locus MAT. A linkage map was constructed consisting of 294 markers (283 AFLPs, ten SSRs and the MAT locus), spanning eleven linkage groups and with a total map length of 1106 cM. The length of individual linkage groups ranged from 30.4 cM (Vi-11) to 166 cM (Vi-1). The number of molecular markers per linkage group ranged from 7 on Vi-11 to 48 on Vi-3; the average distance between two loci within each group varied from 2.4 cM (Vi-4) to 7.5 cM (Vi-9). The maximum map length between two markers within a linkage group was 15.8 cM. The MAT locus was mapped to a small linkage group and was tightly linked to two AFLP markers. The map presented is over four times longer than the previously published map of V. inaequalis which had a total genetic distance of just 270 cM.

Conclusion

A genetic linkage map is an important tool for investigating the genetics of important traits in V. inaequalis such as virulence factors, aggressiveness and mating type. The linkage map presented here represents a significant improvement over currently published maps for studying genome structure and organisation, and for mapping genes of economic importance on the V. inaequalis genome.

Azoles target the ergosterol biosynthetic enzyme lanosterol 14α-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of Candida albicans during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type C. albicans when used in conjunction with CsA and FK506. Similarly, C. albicans mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type C. albicans. These drug combinations also exert synergistic effects against two other Candida species, C. glabrata and C. krusei, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.

From each of two AIDS patients with oropharyngeal candidiasis, five Candida albicans isolates from recurrent episodes of infection which became gradually resistant against fluconazole during antimycotic treatment were analyzed for molecular changes responsible for drug resistance. In both patients, a single C. albicans strain was responsible for the recurrent infections, but the CARE-2 fingerprint pattern of the isolates exhibited minor genetic alterations, indicating that microevolution of the strains took place during fluconazole therapy. In the isolates from patient 1, enhanced mRNA levels of the MDR1 gene, encoding a multiple drug resistance protein from the superfamily of major facilitators, and constitutive high expression of the ERG11 gene, coding for the drug target enzyme sterol 14α-demethylase, correlated with a stepwise development of fluconazole resistance. The resistant strains exhibited reduced accumulation of fluconazole and, for the last in the series, a slight increase in drug needed to inhibit sterol 14α-demethylation in vitro. In the isolates from patient 2, increased MDR1 mRNA levels and the change from heterozygosity to homozygosity for a mutant form of the ERG11 gene correlated with continuously decreased drug susceptibility. In this series, reduced drug accumulation and increased resistance in the target enzyme activity, sterol 14α-demethylase, were observed. These results demonstrate that different molecular mechanisms contribute to a gradual development of fluconazole resistance in C. albicans.

We report here a biochemical study of resistance to azole antifungal agents in a field isolate (S-27) of a fungal phytopathogen. Isolates of Septoria tritici were compared in vitro, and their responses reflected that observed in the field, with S-27 exhibiting resistance relative to RL2. In untreated cultures, both RL2 and S-27 contained isomers of ergosterol and ergosta-5,7-dienol, although in differing concentrations. Under azole treatment, this phytopathogen exhibited a response similar to that of other pathogenic fungi, with a reduction in desmethyl sterols and an accumulation of 14(alpha)-methyl sterols, indicative of inhibition of the P450-mediating sterol 14(alpha)-demethylase. Growth arrest was attributed to the reduction of ergosterol combined with an accumulation of nonutilizable sterols. Strain S-27 exhibited an azole-resistant phenotype which was correlated with decreased cellular content of azole.

Background

The ability for serially homologous structures to acquire a separate identity has been primarily investigated for structures dependent on Hox gene input but is still incompletely understood in other systems. The fore and hindwings of butterflies are serially homologous structures as are the serially homologous eyespots that can decorate each of these wings. Eyespots can vary in number between fore and hindwings of the same individual and mutations of large effect can control the total number of eyespots that each of the wings displays. Here we investigate the genetics of a new spontaneous color pattern mutation, Missing, that alters eyespot number in the nymphalid butterfly, Bicyclus anynana. We further test the interaction of Missing with a previously described mutation, Spotty, describe the developmental stage affected by Missing, and test whether Missing is a mutant variant of the gene Distal-less via a linkage association study.

Results

Missing removes or greatly reduces the size of two of the hindwing eyespots from the row of seven eyespots, with no detectable effect on the rest of the wing pattern. Offspring carrying a single Missing allele display intermediate sized eyespots at these positions. Spotty has the opposite effect of Missing, i.e., it introduces two extra eyespots in homologous wing positions to those affected by Missing, but on the forewing. When Missing is combined with Spotty the size of the two forewing eyespots decreases but the size of the hindwing spots stays the same, suggesting that these two mutations have a combined effect on the forewing such that Missing reduces eyespot size when in the presence of a Spotty mutant allele, but that Spotty has no effect on the hindwing. Missing prevents the complete differentiation of two of the eyespot foci on the hindwing. We found no evidence for any linkage between the Distal-less and Missing genes.

Conclusion

The spontaneous mutation Missing controls the differentiation of the signaling centers of a subset of the serial homologous eyespots present on both the fore and the hindwing in a dose-dependent fashion. The effect of Missing on the forewing, however, is only observed when the mutation Spotty introduces additional eyespots on this wing. Spotty, on the other hand, controls the differentiation of eyespot centers only on the forewing. Spotty, unlike Missing, may be under Ubx gene regulation, since it affects a subset of eyespots on only one of the serially homologous wings.

Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14α-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.

The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.

Author Summary

Bacterial and fungal pathogens cause diseases in humans and plants alike. Antibiotics and fungicides are used for disease control, but the microbes are able to adapt quickly to these drugs by mutation. Multiple drug resistance (MDR) is well investigated in human pathogens and causes increasing problems with antibiotic therapy. Driven by the continuous use of fungicides in commercial vineyards, three types of rapidly increasing multidrug resistant populations of the grey mould fungus Botrytis cinerea have appeared in French vineyards since the mid 1990s. Using a combination of physiological, molecular and genetic techniques, we demonstrate that these MDR phenotypes are correlated with increased drug efflux activity and overexpression of two efflux transporters. Just two types of mutations, one in a regulatory protein that controls drug efflux, and the other in the gene for an efflux transporter itself, are sufficient to explain the three MDR phenotypes. We also provide evidence that a subpopulation of the French MDR strains has migrated eastward into German wine-growing regions. We anticipate that by continuous selection of multi-resistant strains, chemical control of grey mould in the field will become increasingly difficult.

Due to intrinsic resistance Candida krusei is emerging as a systemic pathogen in AIDS patients undergoing fluconazole therapy, but acquired resistance to itraconazole has not been studied biochemically. We report here studies on the basis for azole resistance and sterol composition in C. krusei. An itraconazole-resistant isolate showed reduced susceptibility to azole drugs in in vitro growth inhibition studies. Accumulation of 14 alpha-methyl-3,6-diol under azole treatment was associated with growth arrest. In vitro ergosterol biosynthesis and type II binding studies suggested no alteration in the affinity to azole drugs of the target enzyme, the cytochrome P-450 sterol 14 alpha-demethylase, in the resistant isolate. Resistance was associated with a decreased intracellular content of drug in the resistant isolate.