Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator

Abstract

Objectives: Constitutive overexpression of patAB has been observed in several unrelated fluoroquinoloneresistant laboratorymutants and clinical isolates; therefore, we sought to identify the cause of this overexpression. Methods: Constitutive patAB overexpression in two clinical isolates and a laboratory-selected mutant was investigated using a whole-genome transformation approach. To determine the effect of the detected terminatormutations, theWTand mutated patA leader sequences were cloned upstreamof a GFP reporter. Finally, mutation of the opposing base in the stem-loop structure was carried out. Results:We identified three novelmutations causing up-regulation of patAB. All three of these were located in the upstreamregion of patA and affected the same Rho-independent transcriptional terminator structure. Each mutationwas predicted to destabilize the terminator stem-loop to a different degree, and therewas a strong correlation between predicted terminator stability and patAB expression level. Using a GFP reporter of patA transcription, these terminator mutations led to increased transcription of a downstream gene. For one mutant sequence, terminator stability could be restored by mutation of the opposing base in the stem-loop structure, demonstrating that transcriptional suppression of patAB is mediated by the terminator stem-loop structure. Conclusions: This study showed that amutation in a Rho-independent transcriptional terminator structure confers overexpression of patAB and fluoroquinolone resistance. Understanding how levels of the PatAB efflux pump are regulated increases our knowledge of pneumococcal biology and how the pneumococcus can respond to various stresses, including antimicrobials.