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1.  Impact of Recently Emerged Sterol 14α-Demethylase (CYP51) Variants of Mycosphaerella graminicola on Azole Fungicide Sensitivity▿ 
Applied and Environmental Microbiology  2011;77(11):3830-3837.
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.
doi:10.1128/AEM.00027-11
PMCID: PMC3127603  PMID: 21478305
2.  Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function▿  
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.
doi:10.1128/AEM.02158-09
PMCID: PMC2863451  PMID: 20305029
3.  Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals ▿  
Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml−1 resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.
doi:10.1128/AEM.01332-10
PMCID: PMC3067226  PMID: 21169436
4.  ABC Transporters and Azole Susceptibility in Laboratory Strains of the Wheat Pathogen Mycosphaerella graminicola 
Antimicrobial Agents and Chemotherapy  2002;46(12):3900-3906.
Laboratory strains of Mycosphaerella graminicola with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both. Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole. No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes MgAtr1 to MgAtr5 and the sterol P450 14α-demethylase gene, CYP51. ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of Saccharomyces cerevisiae and disruption of MgAtr1 in one specific M. graminicola laboratory strain with constitutive MgAtr1 overexpression restored the level of susceptibility to cyproconazole to wild-type levels. However, the level of accumulation in the mutant with an MgAtr1 disruption did not revert to the wild-type level. We propose that variations in azole susceptibility in laboratory strains of M. graminicola are mediated by multiple mechanisms.
doi:10.1128/AAC.46.12.3900-3906.2002
PMCID: PMC132773  PMID: 12435694
5.  Triazole Fungicides Can Induce Cross-Resistance to Medical Triazoles in Aspergillus fumigatus 
PLoS ONE  2012;7(3):e31801.
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.
doi:10.1371/journal.pone.0031801
PMCID: PMC3291550  PMID: 22396740
6.  Combination Effects of (Tri)Azole Fungicides on Hormone Production and Xenobiotic Metabolism in a Human Placental Cell Line 
Consumers are exposed to multiple residues of different pesticides via the diet. Therefore, EU legislation for pesticides requires the evaluation of single active substances as well as the consideration of combination effects. Hence the analysis of combined effects of substances in a broad dose range represents a key challenge to current experimental and regulatory toxicology. Here we report evidence for additive effects for (tri)azole fungicides, a widely used group of antifungal agents, in the human placental cell line Jeg-3. In addition to the triazoles cyproconazole, epoxiconazole, flusilazole and tebuconazole and the azole fungicide prochloraz also pesticides from other chemical classes assumed to act via different modes of action (i.e., the organophosphate chlorpyrifos and the triazinylsulfonylurea herbicide triflusulfuron-methyl) were investigated. Endpoints analysed include synthesis of steroid hormone production (progesterone and estradiol) and gene expression of steroidogenic and non-steroidogenic cytochrome-P-450 (CYP) enzymes. For the triazoles and prochloraz, a dose dependent inhibition of progesterone production was observed and additive effects could be confirmed for several combinations of these substances in vitro. The non-triazoles chlorpyrifos and triflusulfuron-methyl did not affect this endpoint and, in line with this finding, no additivity was observed when these substances were applied in mixtures with prochloraz. While prochloraz slightly increased aromatase expression and estradiol production and triflusulfuron-methyl decreased estradiol production, none of the other substances had effects on the expression levels of steroidogenic CYP-enzymes in Jeg-3 cells. For some triazoles, prochloraz and chlorpyrifos a significant induction of CYP1A1 mRNA expression and potential combination effects for this endpoint were observed. Inhibition of CYP1A1 mRNA induction by the AhR inhibitor CH223191 indicated AhR receptor dependence of this effect.
doi:10.3390/ijerph110909660
PMCID: PMC4199042  PMID: 25233012
mixture toxicity; endocrine disruption; placenta; triazoles
7.  Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism 
PLoS Medicine  2008;5(11):e219.
Background
Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007.
Methods and Findings
We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries.
Conclusions
Azole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.
Editors' Summary
Background.
Aspergillosis is a group of lung diseases caused by infection with Aspergillus, a mold (fungus) that grows on decaying plant matter. Because Aspergillus is widespread in the environment, people often breathe in its spores. For most people, this is not a problem—their immune system rapidly kills the fungal spores. However, people with asthma or cystic fibrosis sometimes develop allergic bronchopulmonary aspergillosis, a condition in which the spores trigger an allergic reaction in the lungs that causes coughing, wheezing. and breathlessness. Other people can develop an aspergilloma—a fungus ball that grows in cavities in the lung caused by other illnesses such as tuberculosis. However, the most serious form of aspergillosis is invasive aspergillosis. This pneumonia-like infection, which is fatal if left untreated, affects people who have a weakened immune system (for example, people with leukemia) and can spread from the lungs into the heart, brain, and other parts of the body. Aspergillosis is usually treated with triazole drugs, which inhibit an enzyme that the fungus needs to make its cell membranes; this enzyme is encoded by a gene called cyp51A. Voriconazole is the first-line therapy for aspergillosis but itraconazole and posaconazole are also sometimes used and ravuconazole is in clinical development.
Why Was This Study Done?
About half of patients with invasive aspergillosis recover if they are given triazoles. Worryingly, however, strains of Aspergillus fumigatus (the type of Aspergillus usually involved in invasive aspergillosis) with resistance to several triazoles have recently been isolated from some patients in The Netherlands. If multi-azole resistant strains of A. fumigatus become common, they could have a serious impact on the management of invasive aspergillosis. However, noone knows what proportion of A. fumigatus strains isolated from patients with aspergillosis are resistant to several azole drugs. That is, noone knows the “prevalence” of multi-azole resistance. In this study, the researchers investigate the prevalence and development of azole resistance in A. fumigatus.
What Did the Researchers Do and Find?
Since 1994, all fungal isolates from patients at the Radboud University Nijmegen Medical Center in the Netherlands have been stored. The researchers' search of this collection yielded 1,908 A. fumigatus isolates that had been collected from 1,219 patients over a 14-year period. Of these, the isolates from 32 patients grew in the presence of itraconazole. All the itraconazole-resistant isolates (which also had increased resistance to voriconazole, ravuconazole, and posaconazole) were collected after 1999; the annual prevalence of itraconazole-resistant isolates ranged from 1.7% to 6%. The researchers then sequenced the cyp51A gene in each resistant isolate. Thirty had a genetic alteration represented as TR/L98H. This “dominant resistance mechanism” consisted of a single amino acid change in the cyp51A gene and an alteration in the gene's promoter region (the region that controls how much protein is made from a gene). The researchers also analyzed A. fumigatus isolates from patients admitted to 28 other hospitals in the Netherlands. Itraconazole resistance was present in isolates from 13 patients (out of 101 patients) from nine hospitals; the TR/L98H genetic alteration was present in 69% of the itraconazole-resistant isolates. Finally, itraconazole resistance was present in six isolates from four other countries (out of 317 isolates from six countries); only one Norwegian isolate had the TR/L98H genetic alteration.
What Do These Findings Mean?
These findings indicate that azole resistance is emerging in A. fumigatus and may already be more prevalent than generally thought. Given the dominance of the TR/L98H genetic alteration in the azole-resistant clinical isolates, the researchers suggest that A. fumigatus isolates harboring this alteration might be present and spreading in the environment rather than being selected for during azole treatment of patients. Why azole resistance should develop in A. fumigatus in the environment is unclear but might be caused by the use of azole-containing fungicides. Further studies are now urgently needed to find out if this is the case, to measure the international prevalence and spread of A. fumigatus isolates harboring the TR/L98H genetic alteration, and, most importantly, to develop alternative treatments for patients with azole-resistant aspergillosis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050219.
The MedlinePlus Medical Encyclopedia has a page on aspergillosis (in English and Spanish)
The UK National Health Service Direct health encyclopedia has detailed information about all aspects of aspergillosis
The US Centers for Disease Control and Prevention also has information about aspergillosis
Paul Verweij and colleagues show that azole resistance has emerged inAspergillus fumigatus in The Netherlands and that a dominant resistance mechanism is present in clinical isolates.
doi:10.1371/journal.pmed.0050219
PMCID: PMC2581623  PMID: 18998768
8.  Amino Acid Substitutions at the Major Insertion Loop of Candida albicans Sterol 14alpha-Demethylase Are Involved in Fluconazole Resistance 
PLoS ONE  2011;6(6):e21239.
Background
In the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. The impact of amino acid substitutions on azole interaction has been postulated by homology modeling in comparison to the crystal structure of Mycobacterium tuberculosis (MT-CYP51). Modeling of amino acid residues situated between positions 428 to 459 remains difficult to explain to date, because they are in a major insertion loop specifically present in fungal species.
Methodology/Principal Finding
Fluconazole resistance of clinical isolates displaying Y447H and V456I novel CaErg11p substitutions confirmed in vivo in a murine model of disseminated candidiasis. Y447H and V456I implication into fluconazole resistance was then studied by site-directed mutagenesis of wild-type CaErg11p and by heterogeneously expression into the Pichia pastoris model. CLSI modified tests showed that V447H and V456I are responsible for an 8-fold increase in fluconazole MICs of P. pastoris mutants compared to the wild-type controls. Moreover, mutants showed a sustained capacity for producing ergosterol, even in the presence of fluconazole. Based on these biological results, we are the first to propose a hybrid homology structure-function model of Ca-CYP51 using 3 different homology modeling programs. The variable position of the protein insertion loop, using different liganded or non-liganded templates of recently solved CYP51 structures, suggests its inherent flexibility. Mapping of recognized azole-resistant substitutions indicated that the flexibility of this region is probably enhanced by the relatively high glycine content of the consensus.
Conclusions/Significance
The results highlight the potential role of the insertion loop in azole resistance in the human pathogen C. albicans. This new data should be taken into consideration for future studies aimed at designing new antifungal agents, which circumvent azole resistance.
doi:10.1371/journal.pone.0021239
PMCID: PMC3116904  PMID: 21698128
9.  Gain of Function Mutations in CgPDR1 of Candida glabrata Not Only Mediate Antifungal Resistance but Also Enhance Virulence 
PLoS Pathogens  2009;5(1):e1000268.
CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different “hot spots,” were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.
Author Summary
Candida glabrata is a yeast causing several diseases in humans and especially in immuno-compromised people. C. glabrata infections are treated with antifungal agents, however the use of some agents, for example azoles, is associated with the development of resistance. In this yeast species, azole resistance is mediated almost exclusively by ATP Binding Cassette (ABC) multidrug transporters. Their overexpression results in enhanced efflux of azoles and thus generates resistance. Regulation of ABC transporters is therefore of pivotal importance to understanding azole resistance. In C. glabrata, the expression of ABC transporters is mediated by a zinc finger transcription factor called CgPDR1. Gain of function (GOF) mutations in CgPDR1 result in high ABC transporter expression. In this study, we investigated the occurrence of GOF mutations in a large collection of azole-resistant isolates and found a high variety of mutations localized in three distinct domains of CgPDR1. We found that these mutations are not only associated with resistance but also enhanced virulence and fitness of C. glabrata in animal models. Our study provides for the first time evidence that mutations causing antifungal resistance can also provide a selective advantage under host conditions and thus highlights the need of carefully monitoring resistance in this pathogen.
doi:10.1371/journal.ppat.1000268
PMCID: PMC2607542  PMID: 19148266
10.  Possible Environmental Origin of Resistance of Aspergillus fumigatus to Medical Triazoles▿  
Applied and Environmental Microbiology  2009;75(12):4053-4057.
We reported the emergence of resistance to medical triazoles of Aspergillus fumigatus isolates from patients with invasive aspergillosis. A dominant resistance mechanism was found, and we hypothesized that azole resistance might develop through azole exposure in the environment rather than in azole-treated patients. We investigated if A. fumigatus isolates resistant to medical triazoles are present in our environment by sampling the hospital indoor environment and soil from the outdoor environment. Antifungal susceptibility, resistance mechanisms, and genetic relatedness were compared with those of azole-resistant clinical isolates collected in a previous study. Itraconazole-resistant A. fumigatus (five isolates) was cultured from the indoor hospital environment as well as from soil obtained from flower beds in proximity to the hospital (six isolates) but never from natural soil. Additional samples of commercial compost, leaves, and seeds obtained from a garden center and a plant nursery were also positive (four isolates). Cross-resistance was observed for voriconazole, posaconazole, and the azole fungicides metconazole and tebuconazole. Molecular analysis showed the presence of the dominant resistance mechanism, which was identical to that found in clinical isolates, in 13 of 15 environmental isolates, and it showed that environmental and clinical isolates were genetically clustered apart from nonresistant isolates. Patients with azole-resistant aspergillosis might have been colonized with azole-resistant isolates from the environment.
doi:10.1128/AEM.00231-09
PMCID: PMC2698372  PMID: 19376899
11.  Clonal Expansion and Emergence of Environmental Multiple-Triazole-Resistant Aspergillus fumigatus Strains Carrying the TR34/L98H Mutations in the cyp51A Gene in India 
PLoS ONE  2012;7(12):e52871.
Azole resistance is an emerging problem in Aspergillus which impacts the management of aspergillosis. Here in we report the emergence and clonal spread of resistance to triazoles in environmental Aspergillus fumigatus isolates in India. A total of 44 (7%) A. fumigatus isolates from 24 environmental samples were found to be triazole resistant. The isolation rate of resistant A. fumigatus was highest (33%) from soil of tea gardens followed by soil from flower pots of the hospital garden (20%), soil beneath cotton trees (20%), rice paddy fields (12.3%), air samples of hospital wards (7.6%) and from soil admixed with bird droppings (3.8%). These strains showed cross-resistance to voriconazole, posaconazole, itraconazole and to six triazole fungicides used extensively in agriculture. Our analyses identified that all triazole-resistant strains from India shared the same TR34/L98H mutation in the cyp51 gene. In contrast to the genetic uniformity of azole-resistant strains the azole-susceptible isolates from patients and environments in India were genetically very diverse. All nine loci were highly polymorphic in populations of azole-susceptible isolates from both clinical and environmental samples. Furthermore, all Indian environmental and clinical azole resistant isolates shared the same multilocus microsatellite genotype not found in any other analyzed samples, either from within India or from the Netherlands, France, Germany or China. Our population genetic analyses suggest that the Indian azole-resistant A. fumigatus genotype was likely an extremely adaptive recombinant progeny derived from a cross between an azole-resistant strain migrated from outside of India and a native azole-susceptible strain from within India, followed by mutation and then rapid dispersal through many parts of India. Our results are consistent with the hypothesis that exposure of A. fumigatus to azole fungicides in the environment causes cross-resistance to medical triazoles. The study emphasises the need of continued surveillance of resistance in environmental and clinical A. fumigatus strains.
doi:10.1371/journal.pone.0052871
PMCID: PMC3532406  PMID: 23285210
12.  Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole 
Background
Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14α-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding.
Methodology/Principal Findings
We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 Å and 2.27 Å), and from the related pathogen T. brucei (resolutions of 2.7 Å and 2.6 Å), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180° depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target.
Conclusions/Significance
The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti-Chagasic drugs with improved efficacy and reduced toxicity.
Author Summary
Chagas Disease is caused by kinetoplastid protozoa Trypanosoma cruzi, whose sterols resemble those of fungi, in both composition and biosynthetic pathway. Azole inhibitors of sterol 14α-demethylase (CYP51), such as fluconazole, itraconazole, voriconazole, and posaconazole, successfully treat fungal infections in humans. Efforts have been made to translate anti-fungal azoles into a second-use application for Chagas Disease. Ravuconazole and posaconazole have been recently proposed as candidates for clinical trials with Chagas Disease patients. However, the widespread use of posaconazole for long-term treatment of chronic infections may be limited by hepatic and renal toxicity, a requirement for simultaneous intake of a fatty meal or nutritional supplement to enhance absorption, and cost. To aid our search for structurally and synthetically simple CYP51 inhibitors, we have determined the crystal structures of the CYP51 targets in T. cruzi and T. brucei, both bound to the anti-fungal drugs fluconazole or posaconazole. The structures provide a basis for a design of new drugs targeting Chagas Disease, and also make it possible to model the active site characteristics of the highly homologous Leishmania CYP51. This work provides a foundation for rational synthesis of new therapeutic agents targeting the three kinetoplastid parasites.
doi:10.1371/journal.pntd.0000651
PMCID: PMC2850312  PMID: 20386598
13.  Whole-Genome and Chromosome Evolution Associated with Host Adaptation and Speciation of the Wheat Pathogen Mycosphaerella graminicola 
PLoS Genetics  2010;6(12):e1001189.
The fungus Mycosphaerella graminicola has been a pathogen of wheat since host domestication 10,000–12,000 years ago in the Fertile Crescent. The wheat-infecting lineage emerged from closely related Mycosphaerella pathogens infecting wild grasses. We use a comparative genomics approach to assess how the process of host specialization affected the genome structure of M. graminicola since divergence from the closest known progenitor species named M. graminicola S1. The genome of S1 was obtained by Illumina sequencing resulting in a 35 Mb draft genome sequence of 32X. Assembled contigs were aligned to the previously sequenced M. graminicola genome. The alignment covered >90% of the non-repetitive portion of the M. graminicola genome with an average divergence of 7%. The sequenced M. graminicola strain is known to harbor thirteen essential chromosomes plus eight dispensable chromosomes. We found evidence that structural rearrangements significantly affected the dispensable chromosomes while the essential chromosomes were syntenic. At the nucleotide level, the essential and dispensable chromosomes have evolved differently. The average synonymous substitution rate in dispensable chromosomes is considerably lower than in essential chromosomes, whereas the average non-synonymous substitution rate is three times higher. Differences in molecular evolution can be related to different transmission and recombination patterns, as well as to differences in effective population sizes of essential and dispensable chromosomes. In order to identify genes potentially involved in host specialization or speciation, we calculated ratios of synonymous and non-synonymous substitution rates in the >9,500 aligned protein coding genes. The genes are generally under strong purifying selection. We identified 43 candidate genes showing evidence of positive selection, one encoding a potential pathogen effector protein. We conclude that divergence of these pathogens was accompanied by structural rearrangements in the small dispensable chromosomes, while footprints of positive selection were present in only a small number of protein coding genes.
Author Summary
The fungal wheat pathogen Mycosphaerella graminicola emerged in the Middle East 11,000 years ago, coinciding with host domestication. We sequenced the genome of the closest known endemic relative of M. graminicola infecting wild grass hosts. A comparative genome analysis allowed us to infer how speciation and host specialization processes have influenced pathogen evolution. The wild grass-adapted pathogen can infect wheat, but M. graminicola shows a significantly higher degree of host specificity and virulence in a detached leaf assay. The genomes of the pathogens are 7% divergent with a high degree of synteny in the 13 essential core chromosomes. However, structural rearrangements have strongly affected eight small dispensable chromosomes. These chromosomes also show altered rates of non-synonymous and synonymous substitutions. We found 43 genes showing evidence of positive selection. As the divergence of species occurred very recently, these genes are likely involved in host specialization or speciation. None of the genes have a known function, although one encodes a signal peptide and is a potential pathogen effector. We conclude that the genomic basis of the rapid emergence of the wheat-specialized pathogen M. graminicola has involved structural changes in the eight dispensable chromosomes and positive selection in a small number of genes.
doi:10.1371/journal.pgen.1001189
PMCID: PMC3009667  PMID: 21203495
14.  Three-Dimensional Models of Wild-Type and Mutated Forms of Cytochrome P450 14α-Sterol Demethylases from Aspergillus fumigatus and Candida albicans Provide Insights into Posaconazole Binding 
The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from Aspergillus fumigatus and Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in A. fumigatus and C. albicans act by interfering with the binding of the long side chain.
doi:10.1128/AAC.48.2.568-574.2004
PMCID: PMC321559  PMID: 14742211
15.  Azole Resistance Profile of Amino Acid Changes in Aspergillus fumigatus CYP51A Based on Protein Homology Modeling▿  
Molecular studies have shown that the majority of azole resistance in Aspergillus fumigatus is associated with amino acid substitutions in the cyp51A gene. To obtain insight into azole resistance mutations, the cyp51A gene of 130 resistant and 76 susceptible A. fumigatus isolates was sequenced. Out of 130 azole-resistant isolates, 105 contained a tandem repeat of 34 bp in the promoter region and a leucine-to-histidine substitution in codon 98 (designated TR/L98H). Additionally, in 12 of these TR/L98H resistant isolates, the mutations S297T and F495I were found, and in 1 isolate, the mutation F495I was found. In eight azole-resistant isolates, known azole resistance mutations were detected in codon G54, G138, or M220. In three azole-susceptible isolates, the mutation E130D, L252L, or S400I was found and in 13 azole-susceptible isolates but also in 1 azole-resistant isolate, the mutations F46Y, G98G, M172V, N248T, D255E, L358L, E427K, and C454C were found. All of the nonsynonymous mutations, apart from the mutations in codons G54, G138, and M220 and L98H, were located at the periphery of the protein, as determined by a structural model of the A. fumigatus Cyp51A protein, and were predicted neither to interact with azole compounds nor to affect structural integrity. Therefore, this wide diversity of mutations in the cyp51A gene in azole-susceptible A. fumigatus isolates is not correlated with azole resistance. Based on the Cyp51A protein homology model, the potential correlation of a mutation to azole resistance can be predicted.
doi:10.1128/AAC.01599-09
PMCID: PMC2876375  PMID: 20385860
16.  Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses 
BMC Genomics  2011;12:52.
Background
The toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.
Results
We took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with published microarray experiments obtained from two different nutritional stress conditions identified subsets of genes responding to different types of stress. Some of the genes that responded only to tebuconazole treatment appeared to be unique to the F. graminearum genome.
Conclusions
The novel F. graminearum 8 × 15 k microarray is a reliable and efficient high-throughput tool for genome-wide expression profiling experiments in fungicide research, and beyond, as shown by our data obtained for azole responses. The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.
doi:10.1186/1471-2164-12-52
PMCID: PMC3037902  PMID: 21255412
17.  Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents 
The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.
PMCID: PMC105395  PMID: 9527767
18.  Reverse Genetics in Candida albicans Predicts ARF Cycling Is Essential for Drug Resistance and Virulence 
PLoS Pathogens  2010;6(2):e1000753.
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.
doi:10.1371/journal.ppat.1000753
PMCID: PMC2816695  PMID: 20140196
19.  Rapid Induction of Multiple Resistance Mechanisms in Aspergillus fumigatus during Azole Therapy: a Case Study and Review of the Literature 
Nine consecutive isogenic Aspergillus fumigatus isolates cultured from a patient with aspergilloma were investigated for azole resistance. The first cultured isolate showed a wild-type phenotype, but four azole-resistant phenotypes were observed in the subsequent eight isolates. Four mutations were found in the cyp51A gene of these isolates, leading to the substitutions A9T, G54E, P216L, and F219I. Only G54 substitutions were previously proved to be associated with azole resistance. Using a Cyp51A homology model and recombination experiments in which the mutations were introduced into a susceptible isolate, we show that the substitutions at codons P216 and F219 were both associated with resistance to itraconazole and posaconazole. A9T was also present in the wild-type isolate and thus considered a Cyp51A polymorphism. Isolates harboring F219I evolved further into a pan-azole-resistant phenotype, indicating an additional acquisition of a non-Cyp51A-mediated resistance mechanism. Review of the literature showed that in patients who develop azole resistance during therapy, multiple resistance mechanisms commonly emerge. Furthermore, the median time between the last cultured wild-type isolate and the first azole-resistant isolate was 4 months (range, 3 weeks to 23 months), indicating a rapid induction of resistance.
doi:10.1128/AAC.05088-11
PMCID: PMC3256077  PMID: 22005994
20.  Pharmacodynamics and Dose-Response Relationships of Liposomal Amphotericin B against Different Azole-Resistant Aspergillus fumigatus Isolates in a Murine Model of Disseminated Aspergillosis 
The management of invasive aspergillosis (IA) has become more complicated due to the emergence of acquired azole resistance in Aspergillus fumigatus, which is associated with treatment failure and a mortality rate of 88%. Treatment with liposomal amphotericin B (L-AmB) may be a useful alternative to improve therapeutic outcome in azole-resistant IA. Four clinical A. fumigatus isolates obtained from patients with proven IA were studied in a nonneutropenic murine model of infection: a wild-type isolate without mutations in the cyp51A gene and three azole-resistant isolates harboring a single mutation at codon 220 (M220I) and tandem repeat mutations (a 34-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with substitutions at codon L98 [TR34/L98H] and a 46-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with mutation at codons Y121 and T289 [TR46/Y121F/T289A]), respectively. Female CD-1 mice were infected intravenously 24 h prior to the start of therapy. Groups of 11 mice were treated at days 1, 2, and 5 postchallenge with increasing 4-fold doses of L-AmB ranging from 0.004 to 16 mg/kg/day and observed for 14 days. Survival for all 4 isolates at day 14 was significantly better than that of controls. A dose-response relationship was observed independent of the azole resistance mechanism. The Hill-type model with a variable slope fitted the relationship between the dose and 14-day survival well for all isolates, with R2 values of 0.95 (wild-type), 0.97 (M220I), 0.85 (TR34/L98H), and 0.94 (TR46/Y121F/T289A), respectively. Multiple logistic regression analysis confirmed that there was no significant difference between groups. The results of these experiments indicate that L-AmB was able to prolong survival in vivo in disseminated IA independent of the presence of an azole resistance mechanism in a dose-dependent manner, and therefore, they support a role for L-AmB in the treatment of azole-resistant IA.
doi:10.1128/AAC.02226-12
PMCID: PMC3623333  PMID: 23380732
21.  Wild-Type MIC Distribution and Epidemiological Cutoff Values for Aspergillus fumigatus and Three Triazoles as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Methods▿  
Journal of Clinical Microbiology  2009;47(10):3142-3146.
Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were ≤2 μg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of ≤1 μg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of ≤0.25 μg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.
doi:10.1128/JCM.00940-09
PMCID: PMC2756953  PMID: 19692559
22.  Ergosterol Biosynthesis Inhibitors Become Fungicidal when Combined with Calcineurin Inhibitors against Candida albicans, Candida glabrata, and Candida krusei 
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.
doi:10.1128/AAC.47.3.956-964.2003
PMCID: PMC149324  PMID: 12604527
23.  The Emergence of Resistance to Fungicides 
PLoS ONE  2014;9(3):e91910.
Many studies exist about the selection phase of fungicide resistance evolution, where a resistant strain is present in a pathogen population and is differentially selected for by the application of fungicides. The emergence phase of the evolution of fungicide resistance - where the resistant strain is not present in the population and has to arise through mutation and subsequently invade the population - has not been studied to date. Here, we derive a model which describes the emergence of resistance in pathogen populations of crops. There are several important examples where a single mutation, affecting binding of a fungicide with the target protein, shifts the sensitivity phenotype of the resistant strain to such an extent that it cannot be controlled effectively (‘qualitative’ or ‘single-step’ resistance). The model was parameterized for this scenario for Mycosphaerella graminicola on winter wheat and used to evaluate the effect of fungicide dose rate on the time to emergence of resistance for a range of mutation probabilities, fitness costs of resistance and sensitivity levels of the resistant strain. We also evaluated the usefulness of mixing two fungicides of differing modes of action for delaying the emergence of resistance. The results suggest that it is unlikely that a resistant strain will already have emerged when a fungicide with a new mode of action is introduced. Hence, ‘anti-emergence’ strategies should be identified and implemented. For all simulated scenarios, the median emergence time of a resistant strain was affected little by changing the dose rate applied, within the range of doses typically used on commercial crops. Mixing a single-site acting fungicide with a multi-site acting fungicide delayed the emergence of resistance to the single-site component. Combining the findings with previous work on the selection phase will enable us to develop more efficient anti-resistance strategies.
doi:10.1371/journal.pone.0091910
PMCID: PMC3962370  PMID: 24658678
24.  Studies of the mechanism of human salivary histatin-5 candidacidal activity with histatin-5 variants and azole-sensitive and -resistant Candida species. 
Antimicrobial Agents and Chemotherapy  1997;41(10):2224-2228.
Histatins are a group of small, cationic, antifungal peptides present in human saliva. A previous molecular modeling analysis suggested structural similarity between the Phe14-His15 and His18-His19 dipeptide sequences in histatin-5 (Hsn-5; a 24-amino-acid polypeptide) and the sequence of miconazole (one of the azole-based antifungal therapeutic agents), implying that the mechanisms of killing of Candida albicans by these two molecules may be similar. To further elaborate on this observation, we have produced two variants of Hsn-5 in which Phe14-His15 or His18-His19 dipeptide sequences were replaced by Ala-Ala (F14A/H15A and H18A/H19A) to eliminate the phenyl and imidazole rings of the side chains and assessed their candidacidal activities against C. albicans. In addition, we tested azole-resistant C. albicans and Candida glabrata strains for their susceptibilities to Hsn-5. Analysis of the purified recombinant proteins for their candidacidal activities indicated that both variants were significantly less effective (the molar concentrations required to kill half of the maximum number of cells [ED50s], approximately 67 and approximately 149 microM for F14A/H15A and H18A/H19A, respectively) than the unaltered Hsn-5 (ED50, approximately 8 microM) at killing C. albicans, suggesting that the two dipeptide sequences are important for the candidacidal activity of Hsn-5. Assessment of the candidacidal activity of Hsn-5 with the well-characterized azole-resistant strains of C. albicans and C. glabrata, however, suggested that the mode of action of histatins against Candida is distinct from that of azole-based antifungal agents because Hsn-5 kills both azole-sensitive and azole-resistant strains equally well.
PMCID: PMC164097  PMID: 9333052
25.  Azole Drugs Are Imported By Facilitated Diffusion in Candida albicans and Other Pathogenic Fungi 
PLoS Pathogens  2010;6(9):e1001126.
Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a Km of 0.64 uM and Vmax of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.
Author Summary
Azole antifungals are used to treat a wide variety of fungal infections of humans, animals and plants. A great deal is known about how the azoles interact with their target enzyme within fungal cells and how the azoles are exported from the fungal cell through various efflux pumps. Altered interactions with the target enzyme and altered efflux pump expression are common mechanisms of azole resistance in fungi. However, the mechanism by which azoles enter a fungal cell is not clear—many have assumed that azoles passively diffuse into the cell. This study demonstrates that azoles are not passively diffused, or actively pumped, into the cell. Instead, azoles are imported by facilitated diffusion, mediated by a transporter. Facilitated diffusion of azoles is saturable. All clinically important azoles, and many structurally related compounds, compete for FLC import, while structurally unrelated drugs do not compete. Azole import by facilitated diffusion is shown in four species of fungi, suggesting that it is common for most if not all fungi. Altered facilitated diffusion is observed in a collection of clinical isolates, suggesting that altered import is a previously uncharacterized mechanism of resistance.
doi:10.1371/journal.ppat.1001126
PMCID: PMC2947996  PMID: 20941354

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