Induction of the macrolide-resistance efflux pump Mega inhibits intoxication of Staphylococcus aureus strains by Streptococcus pneumoniae

Streptococcus pneumoniae (Spn) kills Staphylococcus aureus (Sau) through a contact-dependent mechanism that is catalyzed by cations, including iron, to convert hydrogen peroxide (H2O2) to highly toxic hydroxyl radicals (•OH). There are two well-characterized ABC transporters that contribute to the pool of iron in Spn, named Pia and Piu. Some Spn strains have acquired genes mef(E)/mel encoding another ABC trasporter (Mega) that produces an inducible efflux pump for resistance to macrolides. In macrolide-resistant Spn clinical isolates the insertion of Mega class 1. IV and 2. IVc deleted the locus piaABCD and these strains were attenuated for intoxicating Sau. The goal of this study was to investigate if the disruption of iron acquisition, or the antimicrobial-resistance activity of Mega, contributed to inhibiting the killing mechanism. Neither depletion of iron with 2,2'-dipyridyl-d8 (DP) nor incubating with a double knockout mutant SpnΔpiaAΔpiuA, inhibited killing of Sau. Clinical Spn strains carrying Mega1. IV or Mega2. IVc showed a significant delay for killing Sau. An ex vivo recombination system was used to transfer Mega1. IV or Mega2. IVc to reference Spn strains, which was confirmed by whole genome sequencing, and recombinants TIGR4Mega2. IVc, D39Mega2. IVc, and D39Mega1. IV were delayed for killing Sau. We then compared Sau killing of selected Mega-carrying Spn strains when incubated with sub-inhibitory erythromycin (Mega-induced) or sub-inhibitory cefuroxime. Remarkably, killing of Sau was completely inhibited under the Mega-induced condition whereas incubation with cefuroxime did not interfere with killing. Both mef(E) and mel were upregulated > 400-fold, and spxB (encoding an enzyme responsible for production of most H2O2) was upregulated 14.2-fold, whereas transcription of the autolysin (lytA) gene was downregulated when incubated with erythromycin. We demonstrated that erythromycin induction of Mega inhibits the •OH-mediated intoxication of Sau and that the inhibition occurred at the post-translational level suggesting that an imbalance of ions in the membrane inhibits these reactions.