Mechanism of Fluconazole Resistance inCandida krusei

1998 ◽  
Vol 42 (10) ◽  
pp. 2645-2649 ◽  
Author(s):  
Alison S. Orozco ◽  
Lindsey M. Higginbotham ◽  
Christopher A. Hitchcock ◽  
Tanya Parkinson ◽  
Derek Falconer ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Candida Albicans ◽  
Absorption Maximum ◽  
Candida Krusei ◽  
Difference Spectra ◽  
Fluconazole Resistance ◽  
Cell Extracts ◽  
Demethylase Activity ◽  
Cytochrome P 450 ◽  
Soret Absorption

ABSTRACT The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14α-demethylase in this organism. The 14α-demethylase activity in cell extracts of C. kruseiwas 16- to 46-fold more resistant to inhibition by fluconazole than was 14α-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes fromC. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content ofC. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm forC. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that ofC. albicans. Thus, there are significant qualitative differences between the 14α-demethylase of C. albicansand C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14α-demethylase to inhibition by this drug.

scholarly journals On the occurrence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in rat brain.

1977 ◽  
Vol 145 (6) ◽  
pp. 1607-1611 ◽  
Author(s):  
J A Cohn ◽  
A P Alvares ◽  
A Kappas
Keyword(s):  
Carbon Monoxide ◽  
Rat Brain ◽  
Specific Activity ◽  
Aryl Hydrocarbon Hydroxylase ◽  
Hydroxylase Activity ◽  
Difference Spectra ◽  
Aryl Hydrocarbon ◽  
Brain Microsomes ◽  
Aryl Hydrocarbon Hydroxylase Activity ◽  
Cytochrome P 450

The difference spectra of the carbon monoxide-complex of dithionite-reduced rat brain microsomes, compared with both reduced microsomes, alone, and the carbon monoxide-complex of oxidized microsomes, indicate the presence of small amounts of cytochrome P-450 in brain. As in liver, cytochrome P-450 in brain is degraded in vitro to its inactive form, cytochrome P-420 by methylmercury chloride. Aryl hydrocarbon hydroxylase activity is also present in rat brain microsomes and, at lower specific activity, in brain homogenates. This carcinogen metabolizing activity is increased four-fold in rats pretreated with 3-methylcholanthrene.

scholarly journals Comparison of D0870, a new triazole antifungal agent, to fluconazole for inhibition of Candida albicans cytochrome P-450 by using in vitro assays.

1996 ◽  
Vol 40 (6) ◽  
pp. 1382-1386 ◽  
Author(s):  
K Venkateswarlu ◽  
D W Denning ◽  
N J Manning ◽  
S L Kelly
Keyword(s):  
Candida Albicans ◽  
Spectrophotometric Assay ◽  
In Vitro Assays ◽  
Type Ii ◽  
Intracellular Accumulation ◽  
Cell Extracts ◽  
Biochemical Comparison ◽  
Cytochrome P 450 ◽  
Differential Affinity

D0870 was 12 to 15 times more active than fluconazole in experiments to determine the MIC for growth arrest for two isolates of Candida albicans. A biochemical comparison of in vitro sterol biosynthesis in cell extracts showed only a twofold superiority of D0870 over fluconazole. A large differentiation (10-fold) in 50% saturating concentrations obtained by examining the binding of the azoles to microsomal P-450 was observed in a type II binding spectrophotometric assay, possibly reflecting the differential affinity for more than one P-450 enzyme. Additional mechanisms besides affinity for the target enzyme sterol 14 alpha-demethylase, such as differential intracellular accumulation of drug, may contribute to the differences in antifungal activity.

scholarly journals Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14α-sterol demethylase purified from Candida albicans

1990 ◽  
Vol 266 (2) ◽  
pp. 475-480 ◽  
Author(s):  
C A Hitchcock ◽  
K Dickinson ◽  
S B Brown ◽  
E G V Evans ◽  
D J Adams
Keyword(s):  
Enzyme Activity ◽  
Candida Albicans ◽  
Sodium Dithionite ◽  
Ergosterol Biosynthesis ◽  
Type Ii ◽  
Difference Spectra ◽  
Triazole Derivative ◽  
Antifungal Antibiotics ◽  
Total Inhibition ◽  
Cytochrome P 450

The interaction of azole antifungal antibiotics with purified Candida albicans cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) was measured spectrophotometrically and by inhibition of enzyme activity. Ketoconazole and ICI 153066 (a triazole derivative) formed low-spin complexes with the ferric cytochrome and induced type II difference spectra. These spectra are indicative of an interaction between the azole moiety and the sixth co-ordination position of P-450DM haem. Both azoles inhibited the binding of CO to the sodium dithionite-reduced ferrous cytochrome, and inhibited reconstituted P-450DM activity by binding to the cytochrome with a one-to-one stoichiometry. Similarly, total inhibition of enzyme activity occurred when equimolar amounts of clotrimazole, miconazole or fluconazole were added to reconstituted P-450DM. These results correlated with the inhibition of P-450DM in broken cell preparations, confirming that all five azoles are potent inhibitors of ergosterol biosynthesis in C. albicans.

scholarly journals Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)

2010 ◽  
Vol 54 (10) ◽  
pp. 4235-4245 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Claire M. Martel ◽  
Josie E. Parker ◽  
Nadja Melo ◽  
David C. Lamb ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Candida Albicans ◽  
Inhibitory Concentration ◽  
Tight Binding ◽  
Type I ◽  
Wild Type ◽  
Binding Properties ◽  
Fluconazole Resistance ◽  
Binding Spectra

ABSTRACT Purified Candida albicans sterol 14-α demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with Ks values of 11 and 25 μM, respectively, and a Km value of 6 μM for lanosterol. Azole binding to CaCYP51 was “tight” with both the type II spectral intensity (ΔA max) and the azole concentration required to obtain a half-ΔA max being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with Kd values of 10 to 26 μM under oxidative conditions, compared with 47 μM for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min−1), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.

scholarly journals Genetic Basis for Differential Activities of Fluconazole and Voriconazole against Candida krusei

2003 ◽  
Vol 47 (4) ◽  
pp. 1213-1219 ◽  
Author(s):  
Takashi Fukuoka ◽  
Douglas A. Johnston ◽  
Carol A. Winslow ◽  
Marcel J. de Groot ◽  
Catherine Burt ◽  
...  
Keyword(s):  
Genetic Basis ◽  
Potent Inhibitor ◽  
Aromatic Amino Acids ◽  
Candida Krusei ◽  
Predicted Amino Acid Sequence ◽  
Relative Resistance ◽  
Fluconazole Resistance ◽  
Cell Extracts ◽  
Invasive Infections

ABSTRACT 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.

scholarly journals The cytochromes of Acanthamoeba castellanii

1977 ◽  
Vol 168 (1) ◽  
pp. 113-121 ◽  
Author(s):  
S W Edwards ◽  
A H Chagla ◽  
A J Griffiths ◽  
D Lloyd
Keyword(s):  
Low Temperature ◽  
Acanthamoeba Castellanii ◽  
Difference Spectrum ◽  
Cell Respiration ◽  
Difference Spectra ◽  
Whole Cells ◽  
Microsomal Membranes ◽  
Cytochrome P 450 ◽  
Photochemical Action ◽  
Soret Absorption

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and ‘microsomal’ membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

scholarly journals Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

2021 ◽  
Vol 22 (4) ◽  
pp. 2127
Author(s):  
Jakub Suchodolski ◽  
Anna Krasowska
Keyword(s):  
Candida Albicans ◽  
Multidrug Resistance ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Pathogenic Fungus ◽  
Serum Levels ◽  
Efflux Transporters ◽  
Fluconazole Resistance ◽  
Green Fluorescent

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.

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