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

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Abstract
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 (Cip r ) phenotype was silent in a sparfloxacin-resistant (Spx r ) S . pneumoniae gyrA (Ser81Phe) strain expressing a demonstrably wild-type topoisomerase IV, whereas Spx r was silent in a Cip r 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.