Y132H substitution in Candida albicans sterol 14alpha-demethylase confers fluconazole resistance by preventing binding to haem

Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) is required for ergosterol biosynthesis and is the target for azole antifungal compounds. The amino acid substitution Y132H in CYP51 from clinical isolates of Candida albicans can cause fluconazole resistance by a novel change in the protein. Fluconazole binding to the mutant protein did not involve normal interaction with haem as shown by inducing a Type I spectral change. This contrasted to the wild-type protein where fluconazole inhibition was reflected in coordination to haem as a sixth ligand and where the typical Type II spectrum was obtained. The Y132H substitution occurred without drastic perturbation of the haem environment or activity allowing resistant mutants to produce ergosterol and retain fitness, an efficient strategy for resistance in nature.     

The G464S amino acid substitution in Candida albicans sterol 14alpha-demethylase causes fluconazole resistance in the clinic through reduced affinity.

Fluconazole selectively inhibits fungal sterol 14alpha-demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of sterol 14alpha-demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal sterol biosynthesis, ergosterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced sterol demethylase catalytic activity by changing the location of the 14alpha-methyl group in relation to oxygen-bound heme during the catalytic cycle.     

Amino acid variations of cytochrome P-450 lanosterol 14 alpha-demethylase (CYP51A1) from fluconazoleresistant clinical isolates of Candida albicans

We studied six clinical isolates of Candida albicans. All six isolates showed high level resistance to fluconazole (minimum inhibitory concentrations 64 microg/ml) with varying degrees of cross-resistance to other azoles but not to amphotericin B. Neither higher dosage nor upregulation of the gene encoding the cytochrome P- 450 lanosterol 14 alpha-demethylase (CYP51A1 or P-450LDM) was responsible for fluconazole resistance. The resistant and the susceptible isolates accumulated similar amounts of azoles. To examine whether resistance to fluconazole in these clinical isolates of C. albicans is mediated by an altered target of azole action, we cloned the structural gene encoding P-450LDM from the fluconazole resistant isolates. The amino acid sequences of the P-450LDMs from the isolates were deduced from the gene sequences and compared to the P-450LDM sequence of the fluconazole-susceptible C. albicans B311. The enzymes from the clinical isolates showed 2 to 7 amino acid variations scattered across the molecules encompassing 10 different loci. One-half of the amino acid changes obtained were conserved substitutions (E116D, K143R, E266D, D278E, R287K) compared to the susceptible strain. Non-conserved substitutions were T128K, R267H, S405F, G450E and G464S, three of which are in and around the hemebinding region of the molecule. R287K is the only amino acid change that was found in all six clinical isolates. One or more of these mutational alterations may lead to the expression of an azole-resistant enzyme.     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.     

白色念珠菌体外诱导氟康唑耐药性的实验研究（简报）

随着广谱抗生素的普遍应用以及免疫缺陷人群的增加,机会性致病菌念珠菌感染日益增多。深部念珠菌感染已经成为重症患者死亡的重要原因,白色念珠菌(C．albicans)是其中的主要致病菌。低毒广谱的唑类抗真菌药氟康唑是既能治疗严重真菌感染又不会产生明显副作用的少数抗真菌药之一,它的广泛使用取得良好治疗效果,但也导致菌株耐药率增加而使临床治疗失败。近10年来,在治疗     

Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles

Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways.     

Homology modeling of lanosterol 14alpha-demethylase of Candida albicans and Aspergillus fumigatus and insights into the enzyme-substrate Interactions

The crystal structure of 14alpha-sterol demethylase from Mycobacterium tuberculosis (MT_14DM) provides a good template for modeling the three dimensional structure of lanosterol 14alpha-demethylase, which is the target of azole antifungal agents. Homologous 3D models of lanosterol 14alpha-demethylase from Candida albicans (CA_14DM) and Aspergillus fumigatus (AF_14DM) were built on the basis of the crystal coordinates of MT_14DM in complex with 4-phenylimidazole and fluconazole. The reliability of the two models was assessed by Ramachandran plots, Profile-3D analysis, and by analyzing the consistency of the two models with the experimental data on the P450(14DM). The overall structures of the resulting CA_14DM model and AF_14DM model are similar to those of the template structures. The two models remain the core structure characteristic for cytochrome P450s and most of the insertions and deletions expose the molecular surface. The structurally and functionally important residues such as the heme binding residues, the residues lining the substrate access channel, and residues in active site were identified from the model. To explore the binding mode of the substrate with the two models, 24(28)-methylene-24,25-dihydrolanosterol was docked into the active site of the two models and hydrophobic interaction and hydrogen-bonding were found to play an important role in substrate recognition and orientation. These results provided a basis for experiments to probe structure-function relationships in the P450(14DM). Although CA_14DM and AF_14DM shared similar core structural character, the active site of the two models were quite different, thus allowing the rational design of specific inhibitors to the target enzyme and the discovery of novel antifungal agents with broad spectrum.     

Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.     

A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 X g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P-450.     

Genetic control of resistance to the sterol 14alpha-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae

Sexual crosses were used to determine the genetic basis of resistance to the sterol 14 alpha-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae. Three different crosses between sensitive parental strains (22-432 and 22-433 [the concentration required to inhibit growth by 50% (IG(50)) for each was 相似文献   

Anidulafungin versus fluconazole in the treatment of Candida albicans chorioretinitis

BackgroundCandida albicans chorioretinitis is the most common cause of endogenous fungal endophthalmitis. Echinocandins are recommended as first-line therapy in the treatment of invasive candidiasis (IC), but in clinically stable patients with IC and endophthalmitis caused by Candida species susceptible to azole compounds these are the first-line treatment due to their better intraocular penetration.Case reportA 42-year-old woman admitted to hospital for duodenal perforation after gastrointestinal surgery and treated with broad-spectrum antibiotics developed C. albicans candidemia. According to protocol, an antifungal treatment with anidulafungin was given. The patient presented no visual symptoms but on routinary ophthalmoscopic examination multiple bilateral chorioretinal lesions were observed. Systemic therapy was changed to fluconazole, with good systemic and ocular results.ConclusionsAzole compounds are the first-line therapy for endophthalmitis associated with candidemia. However, clinical guidelines often propose echinocandins as the first option for IC. In some cases, C. albicans chorioretinitis will require a change in the systemic treatment to assure better intraocular penetration. According to the current evidence and our own experience, routine funduscopy is not necessary in all IC patients. However, we do recommend fundus examination in patients with visual symptoms or those unable to report them (paediatric patients and patients with an altered level of consciousness), and in those who are being treated with echinocandins in monotherapy.     

Effect of oxiconazole and Ro 14-4767/002 on sterol pattern in Candida albicans

The effect of the imidazole oxiconazole and the morpholine derivative Ro 14-4767/002 on the sterol metabolism of Candida albicans was investigated at different periods of growth. Ergosterol, representing the main sterol component of control cells, was markedly reduced in oxiconazole-treated and Ro 14-4767/002-treated cells. However, the total sterol content of the cells treated with both drugs was increased due to accumulation of other sterols not present in control cells: in oxiconazole-treated cells 24-methenedihydrolanosterol, 4,14-dimethylfecosterol and 14-methylfecosterol accumulated, indicating an inhibition of C14-demethylation. This is in agreement with the mode of action described for other azoles in various pathogen fungi. In Ro 14-4767/002-treated cells the main sterol accumulated was ignosterol, indicating an inhibition of delta 14-sterol reductase and delta 8-delta 7-isomerase. This inhibition has not been described before in human pathogens although it has been previously found in plant pathogenic fungi treated with fenpropimorph.     

Isolation of the gene for cytochrome P450L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans

The Saccharomyces cerevisiae cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase)-coding gene was used as a hybridization probe to isolate two HindIII fragments of 2.5 kb and 6.85 kb from a phage lambda library of Candida albicans nucleotide sequences. Restriction endonuclease mapping and Southern blot hybridization experiments indicated that these fragments represent two allelic forms of the same gene. This cloned sequence, when introduced into S. cerevisiae or C. albicans on a multiple copy vector, produced an increase in cytochrome P450 content and resistance to imidazole antifungal agents which are inhibitors of cytochrome P450 L1A1. In addition, the cloned sequence was able to complement a cytochrome P450 L1A1 gene disruption when introduced into S. cerevisiae. These data indicate that the cloned sequence codes for the lanosterol 14 alpha-demethylase cytochrome P450 L1A1 from C. albicans.     

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor.

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.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

X-ray structure of 4,4'-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha-demethylase (CYP51)

A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14alpha-methyl group from sterol precursors by sterol 14alpha-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95A) the structure of CYP51 from Mycobacterium tuberculosis (CYP51(Mt)) co-crystallized with 4,4'-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51(Mt). The newly determined CYP51(Mt)-DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51(Mt)-estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.