Cloning and characterization of CYP51 from Mycobacterium avium.
Mycobacterium avium complex (MAC) causes chronic lung disease in immunocompetent people and disseminated infection in patients with AIDS. MAC is intrinsically resistant to many conventional antimycobacterial agents, it develops drug resistance rapidly to macrolide antibiotics, and patients with MAC infection experience frequent relapses or the inability to completely eradicate the infection with current treatment. Treatment regimens are prolonged and complicated by drug toxicity or intolerances. We sought to identify biochemical pathways in MAC that can serve as targets for novel antimycobacterial treatment. The cytochrome P450 enzyme, CYP51, catalyzes an essential early step in sterol metabolism, removing a methyl group from lanosterol in animals and fungi, or from obtusifoliol in plants. Azoles inhibit CYP51 function, leading to an accumulation of methylated sterol precursors. This perturbation of normal sterol metabolism compromises cell membrane integrity, resulting in growth inhibition or cell death. We have cloned and characterized a CYP51 from MAC that functions as a lanosterol 14alpha-demethylase. We show the direct interactions of azoles with purified MAC-CYP51 by absorbance and electron paramagnetic resonance spectroscopy, and determine the minimum inhibitory concentrations (MICs) of econazole, ketoconazole, itraconazole, fluconazole, and voriconazole against MAC. Furthermore, we demonstrate that econazole has a MIC of 4 mug/ml and a minimum bacteriocidal concentration of 4 mug/ml, whereas ketoconazole has a MIC of 8 mug/ml and a minimum bacteriocidal concentration of 16 mug/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.
Division of Pulmonary and Critical Care Medicine, Thoracic Diseases Research Unit, 826 Stabile Building, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Michael P
MP
Pietila
Pawan K
PK
Vohra
Bharati
B
Sanyal
Nancy L
NL
Wengenack
Sreekumar
S
Raghavakaimal
Charles F
CF
Thomas
D000890:Anti-Infective Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology; D001393:Azoles / Q000737:chemistry / Q000494:pharmacology; D002384:Catalysis; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004352:Drug Resistance, Microbial; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D009162:Mycobacterium avium / Q000201:enzymology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D058886:Sterol 14-Demethylase
D000890:Anti-Infective Agents; D000935:Antifungal Agents; D001393:Azoles; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D018942:Macrolides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase