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

Mutations in DNA gyrase and/or topoisomerase IV genes are frequently encoun tered in quinolone-resistant mutants of Streptococcus pneumoniae. To invest igate 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 Ser7 9Phe mutations, two changes widely associated with quinolone resistance. Ni ckel chelate chromatography yielded highly purified mutant His-tagged prote ins that, in the presence of the corresponding GyrB and ParE subunits, reco nstituted gyrase and topoisomerase IV complexes with wild-type specific act ivities. In enzyme inhibition or DNA cleavage assays, these mutant enzyme c omplexes were at least 8- to 16-fold less responsive to both sparfloxacin a nd 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 si lent in a Cipr parC (Ser79Phe) strain. These epistatic effects provide stro ng support for a model in which quinolones kill S. pneumoniae by acting not as enzyme inhibitors but as cellular poisons, with sparfloxacin killing pr eferentially through gyrase and ciprofloxacin through topoisomerase IV. By immunoblotting using subunit-specific antisera, intracellular GyrA/GyrB lev els were a modest threefold higher than those of ParC/ParE, most likely ins ufficient to allow selective drug action by counterbalancing the 20- to 40- fold preference for cleavable-complex formation through topoisomerase W obs erved in vitro. To reconcile these results, we suggest that drug-dependent differences in the efficiency by which ternary complexes are formed, proces sed, or repaired in S. pneumoniae may be key factors determining the killin g pathway.