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Antimicrobial Agents and Chemotherapy, November 2001, p. 3140-3147, Vol. 45, No. 11
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.11.3140-3147.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Quinolone Resistance Mutations in Streptococcus pneumoniae GyrA and ParC Proteins: Mechanistic Insights into Quinolone Action from Enzymatic Analysis, Intracellular Levels, and Phenotypes of Wild-Type and Mutant Proteins

Xiao-Su Pan, Genoveva Yague,dagger and L. Mark Fisher*

Molecular Genetics Group, Department of Biochemistry and Immunology, St. George's Hospital Medical School, University of London, London SW17 0RE, United Kingdom

Received 25 April 2001/Returned for modification 9 June 2001/Accepted 23 August 2001

Mutations in DNA gyrase and/or topoisomerase IV genes are frequently encountered in quinolone-resistant mutants of Streptococcus pneumoniae. To investigate the mechanism of their effects at the molecular and cellular levels, we have used an Escherichia coli system to overexpress S. pneumoniae gyrase gyrA and topoisomerase IV parC genes encoding respective Ser81Phe and Ser79Phe mutations, two changes widely associated with quinolone resistance. Nickel chelate chromatography yielded highly purified mutant His-tagged proteins that, in the presence of the corresponding GyrB and ParE subunits, reconstituted gyrase and topoisomerase IV complexes with wild-type specific activities. In enzyme inhibition or DNA cleavage assays, these mutant enzyme complexes were at least 8- to 16-fold less responsive to both sparfloxacin and ciprofloxacin. The ciprofloxacin-resistant (Cipr) phenotype was silent in a sparfloxacin-resistant (Spxr) S. pneumoniae gyrA (Ser81Phe) strain expressing a demonstrably wild-type topoisomerase IV, whereas Spxr was silent in a Cipr parC (Ser79Phe) strain. These epistatic effects provide strong support for a model in which quinolones kill S. pneumoniae by acting not as enzyme inhibitors but as cellular poisons, with sparfloxacin killing preferentially through gyrase and ciprofloxacin through topoisomerase IV. By immunoblotting using subunit-specific antisera, intracellular GyrA/GyrB levels were a modest threefold higher than those of ParC/ParE, most likely insufficient to allow selective drug action by counterbalancing the 20- to 40-fold preference for cleavable-complex formation through topoisomerase IV observed in vitro. To reconcile these results, we suggest that drug-dependent differences in the efficiency by which ternary complexes are formed, processed, or repaired in S. pneumoniae may be key factors determining the killing pathway.

* Corresponding author. Mailing address: Molecular Genetics Group, Department of Biochemistry and Immunology, St. George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, United Kingdom. Phone: 44 208 725 5782. Fax: 44 208 725 2992. E-mail: lfisher{at}sghms.ac.uk.

dagger Present address: Departamento de Genetica y Microbiologia, Facultad de Medicina, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain.

Antimicrobial Agents and Chemotherapy, November 2001, p. 3140-3147, Vol. 45, No. 11
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.11.3140-3147.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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