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Antimicrobial Agents and Chemotherapy, May 2007, p. 1643-1648, Vol. 51, No. 5
0066-4804/07/$08.00+0     doi:10.1128/AAC.01282-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Expression and Purification of an Active Form of the Mycobacterium leprae DNA Gyrase and Its Inhibition by Quinolones{triangledown}

Stéphanie Matrat,1,3,{dagger} Stéphanie Petrella,1,3,{dagger} Emmanuelle Cambau,2,3 Wladimir Sougakoff,1,3 Vincent Jarlier,1,3 and Alexandra Aubry1,3*

Laboratoire de Bactériologie, EA1541, Faculté de Médecine Pierre et Marie Curie, Site Pitié-Salpêtrière, Assistance Publique Hôpitaux de Paris, Université Paris 6, INSERM, U655-LRMA, Paris,1 Laboratoire de Bactériologie-Virologie-Hygiène, Centre Hospitalo-Universitaire Henri Mondor, Faculté de Médecine de Créteil, Université Paris 12, Créteil,2 Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Paris, France3

Received 13 October 2006/ Returned for modification 15 January 2007/ Accepted 16 February 2007

Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the mouse footpad system, which requires at least 12 months before the results become available. We have developed an in vitro assay for studying the activities of quinolones against the DNA gyrase of M. leprae. We overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits separately as His-tagged proteins by using a pET plasmid carrying the gyrA and gyrB genes. The soluble 97.5-kDa GyrA and 74.5-kDa GyrB subunits were purified by nickel chelate chromatography and were reconstituted as an enzyme with DNA supercoiling activity. Based on the drug concentrations that inhibited DNA supercoiling by 50% or that induced DNA cleavage by 25%, the 13 quinolones tested clustered into three groups. Analysis of the quinolone structure-activity relationship demonstrates that the most active quinolones against M. leprae DNA gyrase share the following structural features: a substituted carbon at position 8, a cyclopropyl substituent at N-1, a fluorine at C-6, and a substituent ring at C-7. We conclude that the assays based on DNA supercoiling inhibition and drug-induced DNA cleavage on purified M. leprae DNA gyrase are rapid, efficient, and safe methods for the screening of quinolone derivatives with potential in vivo activities against M. leprae.

* Corresponding author. Mailing address: Faculté de Médecine Pierre et Marie Curie, Site Pitié-Salpêtrière, 91, Boulevard de l'Hôpital, 75634 Paris cedex 13, France. Phone: 33 1 40 77 97 46. Fax: 33 1 45 82 75 77. E-mail: aubry{at}chups.jussieu.fr

{triangledown} Published ahead of print on 26 February 2007.

{dagger} Both of these authors equally contributed to the present study.

Antimicrobial Agents and Chemotherapy, May 2007, p. 1643-1648, Vol. 51, No. 5
0066-4804/07/$08.00+0     doi:10.1128/AAC.01282-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Matrat, S., Cambau, E., Jarlier, V., Aubry, A. (2008). Are All the DNA Gyrase Mutations Found in Mycobacterium leprae Clinical Strains Involved in Resistance to Fluoroquinolones?. Antimicrob. Agents Chemother. 52: 745-747 [Abstract] [Full Text]  


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