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

ObjectivesConstitutive overexpression of patAB has been observed in several unrelated fluoroquinolone-resistant laboratory mutants and clinical isolates; therefore, we sought to identify the cause of this overexpression.MethodsConstitutive 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 terminator mutations, the WT and mutated patA leader sequences were cloned upstream of a GFP reporter. Finally, mutation of the opposing base in the stem–loop structure was carried out.ResultsWe identified three novel mutations causing up-regulation of patAB. All three of these were located in the upstream region of patA and affected the same Rho-independent transcriptional terminator structure. Each mutation was predicted to destabilize the terminator stem–loop to a different degree, and there was 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.ConclusionsThis study showed that a mutation 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.

Gain- and Loss-of-Function Screens Coupled to Next-Generation Sequencing for Antibiotic Mode of Action and Resistance Studies in Streptococcus pneumoniae

“…Disruption of the stem-loop results in readthrough transcription of the downstream gene. A similar phenomenon was reported in fluoroquinolone-resistant S. pneumoniae isolates, for which SNVs disrupting the stem-loop of a Rho-independent terminator located upstream of patAB were responsible for the overexpression of this multidrug efflux pump (43). While patAB overexpression is known to be associated with resistance to a number of antibiotics (4, 32-35), here we found that the opposite is true for TMP, and it is the loss of function of PatAB that correlates with resistance, a finding also observed with (5), purH (7), and purK (11), part of the pur gene cluster (black bars) in R6spr1793 M18 and R6spr1793 M19 transformants.…”

Molecular mechanisms and epidemiology of resistance in Streptococcus pneumoniae in the Middle East region

“…The patA (spr1887) and patB (spr1885) genes belong to the same operon and work together as heterodimers to make a functional ABC transporter [56]. Even though upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are still unknown and may be due to mutations in rho-independent terminator or patAB gene duplication [57,58].…”

In Vitro Activity of Lascufloxacin against Streptococcus pneumoniae with Mutations in the Quinolone Resistance-Determining Regions

“…No additional mutations were observed in some of the strains selected by the exposure to fluoroquinolones (Tables 4 and 5). Although the gradual accumulation of GyrA and ParC mutations was the main cause of fluoroquinolone resistance, the increases in MICs in these strains were thought to be due to other mechanisms, such as GyrB and ParE mutations and an overexpression of efflux pumps, including PmrA and PatA/PatB ABC transporter (16,17). Lascufloxacin showed potent activity against first-step mutants.…”