scholarly journals Disruption of the Candida albicans CYB5 Gene Results in Increased Azole Sensitivity

2004 ◽  
Vol 48 (9) ◽  
pp. 3425-3435 ◽  
Author(s):  
K. M. Rogers ◽  
C. A. Pierson ◽  
N. T. Culbertson ◽  
C. Mo ◽  
A. M. Sturm ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Sterol Synthesis ◽  
Squalene Epoxidase ◽  
Gene Products ◽  
Direct Transformation ◽  
Azole Antifungals ◽  
Increased Sensitivity ◽  
Sterol Profile ◽  
Encoding Gene ◽  
Cytochrome P 450

ABSTRACT Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme. Cytochrome b 5 is required for sterol C5-6 desaturation and the encoding gene, CYB5, is nonessential in Saccharomyces cerevisiae. Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable. The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals. Based on this phenotype, the CYB5 gene in the human pathogen Candida albicans was investigated to determine whether the cyb5 genotype was viable and would also demonstrate azole sensitivity. Sequential disruption of the CYB5 alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the CYB5 gene. Subsequent disruption procedures with a pMAL2-CYB5 rescue cassette and a CYB5-URA3 blaster cassette resulted in viable cyb5 strains with no third copy. The C. albicans CYB5 gene is concluded to be nonessential. Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol. The C. albicans cyb5 strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the S. cerevisiae cyb5. The C. albicans cyb5 shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the ERG2 and ERG24 gene products. These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment.

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 775-787 ◽  
Author(s):  
Hannes Nahrstedt ◽  
Christine Schröder ◽  
Friedhelm Meinhardt
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Mitomycin C ◽  
Bacillus Megaterium ◽  
Functional Complementation ◽  
Homologous Gene ◽  
Gene Products ◽  
Null Mutant ◽  
Promoter Regions ◽  
Increased Sensitivity

Isolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.

scholarly journals The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (6) ◽  
pp. 2719-2727 ◽  
Author(s):  
S Silve ◽  
P Leplatois ◽  
A Josse ◽  
P H Dupuy ◽  
C Lanau ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Proliferation ◽  
Gene Product ◽  
In Vitro Assays ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isomerase Activity ◽  
Encoding Gene ◽  
Resistant Mutants

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

scholarly journals Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

2010 ◽  
Vol 9 (9) ◽  
pp. 1329-1342 ◽  
Author(s):  
Claire A. Walker ◽  
Beatriz L. Gómez ◽  
Héctor M. Mora-Montes ◽  
Kevin S. Mackenzie ◽  
Carol A. Munro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Cell Walls ◽  
Chitin Synthesis ◽  
Melanin Production ◽  
Content Type ◽  
Encoding Gene ◽  
Chitinase Genes

ABSTRACT The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.

In vitro synergy of azole antifungals and methotrexate against Candida albicans

Life Sciences ◽  
2019 ◽  
Vol 235 ◽  
pp. 116827 ◽  
Author(s):  
Jianxun Yang ◽  
Lei Gao ◽  
Pei Yu ◽  
Janet Cheruiyot Kosgey ◽  
Lina Jia ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Azole Antifungals

scholarly journals Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase

2012 ◽  
Vol 79 (5) ◽  
pp. 1639-1645 ◽  
Author(s):  
Josie E. Parker ◽  
Andrew G. S. Warrilow ◽  
Hans J. Cools ◽  
Bart A. Fraaije ◽  
John A. Lucas ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Type Ii ◽  
Inhibitor Binding ◽  
Binding Properties ◽  
Content Type ◽  
Azole Antifungals ◽  
A Cell ◽  
Antifungal Action ◽  
Binding Spectra

ABSTRACTProthioconazole is a new triazolinthione fungicide used in agriculture. We have usedCandida albicansCYP51 (CaCYP51) to investigate thein vitroactivity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment ofC. albicanscells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts fromC. albicansgrown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.

scholarly journals Enhanced Production of Farnesol by Candida albicans Treated with Four Azoles

2004 ◽  
Vol 48 (6) ◽  
pp. 2305-2307 ◽  
Author(s):  
Jacob M. Hornby ◽  
Kenneth W. Nickerson
Keyword(s):  
Candida Albicans ◽  
Clinical Isolates ◽  
Farnesyl Pyrophosphate ◽  
Dimorphic Fungus ◽  
Azole Antifungals ◽  
Appl Environ ◽  
Enhanced Production

ABSTRACT The dimorphic fungus Candida albicans excretes farnesol, which is produced enzymatically from the sterol biosynthetic intermediate farnesyl pyrophosphate. Inhibition of C. albicans by four azole antifungals, fluconazole, ketoconazole, miconazole, and clotrimazole, caused elevated farnesol production (10- to 45-fold). Furthermore, farnesol production occurs in both laboratory strains and clinical isolates (J. M. Hornby et al., Appl. Environ. Microbiol. 67:2982-2992, 2001) of C. albicans.

scholarly journals Squalene epoxidase encoded by ERG1 affects morphogenesis and drug susceptibilities of Candida albicans

2005 ◽  
Vol 55 (6) ◽  
pp. 905-913 ◽  
Author(s):  
Ritu Pasrija ◽  
Shankarling Krishnamurthy ◽  
Tulika Prasad ◽  
Joachim F. Ernst ◽  
Rajendra Prasad
Keyword(s):  
Candida Albicans ◽  
Squalene Epoxidase

scholarly journals Pyridines and Pyrimidines Mediating Activity against an Efflux-Negative Strain of Candida albicans through Putative Inhibition of Lanosterol Demethylase

2004 ◽  
Vol 48 (1) ◽  
pp. 313-318 ◽  
Author(s):  
Ed T. Buurman ◽  
April E. Blodgett ◽  
Kenneth G. Hull ◽  
Daniel Carcanague
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Ergosterol Biosynthesis ◽  
Clear Correlation ◽  
Wild Type ◽  
Inhibition Of Growth ◽  
Ergosterol Synthesis ◽  
A Cell ◽  
Sterol Profile ◽  
Lanosterol Demethylase

ABSTRACT The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased ERG10 transcription results in elevated specific β-galactosidase activity, was used to find novel inhibitors of ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major fungal pathogen Candida albicans (MICs > 64 μg · ml−1). However, a strain of C. albicans lacking the Cdr1p and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 μg · ml−1), suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol. Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively mediate their antifungal activity against C. albicans via lanosterol demethylase.

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