Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator.
Bottom Line: To determine the effect of the detected terminator mutations, the WT and mutated patA leader sequences were cloned upstream of a GFP reporter.This 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.
Affiliation: Antimicrobials Research Group, School of Immunity and Infection, Institute of Microbiology and Infection and College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.Show MeSH
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Mentions: It was predicted that reduced stability of the terminator loop would increase transcription of patAB, by increasing transcriptional read-through into the patA coding sequence.11 However, this had not been experimentally demonstrated. To investigate this, we synthesized four DNA fragments corresponding to the patA upstream region (−146 to −13 bp), flanked by restriction sites. One fragment corresponded to the WT (R6) sequence, and the remaining three introduced the mutations C(−33)T (v168), A(−47)C (v101) and G(−46)A (v87). These were cloned upstream of a promoter-less GFP allele in a plasmid named pBAV1K-gfp82, derived from pBAV1K-t5-gfp (construction described in the Materials and methods section). The resulting constructs were transformed into R6 cells and transformants selected using 100 mg/L kanamycin. Fluorescence from each construct was measured during growth of the strains in BHI broth. Compared with a strain carrying pBAV1K-gfp82 empty vector, the pBAV1K-gfp82-WT construct produced consistently higher fluorescence throughout the growth cycle (Figure 4). Introduction of the terminator mutations (constructs pBAV1K-gfp82-v168, pBAV1K-gfp82-v101 and pBAV1K-gfp82-v87) resulted in a significant 1.5- to 3-fold increase in fluorescence in all three cases after 2.5, 3.5 and 5.5 h of growth, respectively, representing late-log, stationary and late-stationary phases, respectively (Figure 4; P < 0.05, two-tailed Student's t-test).Figure 4.
Affiliation: Antimicrobials Research Group, School of Immunity and Infection, Institute of Microbiology and Infection and College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.