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Cloning and Characterization of CYP51 from Mycobacterium avium

Thoracic Diseases Research Unit, Division of Pulmonary and Critical Care Medicine; Mycobacteriology and Mycology Laboratory, Department of Laboratory Medicine and Pathology; and Division of Endocrinology, Mayo Clinic College of Medicine, Rochester, Minnesota

Correspondence and requests for reprints should be addressed to Charles F. Thomas, Jr., Thoracic Diseases Research Unit, 826 Stabile Building, Mayo Clinic College of Medicine, Rochester, MN 55905. E-mail: thomas.charles{at}mayo.edu

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 14-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 µg/ml and a minimum bacteriocidal concentration of 4 µg/ml, whereas ketoconazole has a MIC of 8 µg/ml and a minimum bacteriocidal concentration of 16 µg/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.

Key Words: azoles • CYP51 • ERG11 • lanosterol