Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae

Abstract

Reversible or phenotypic tolerance to antibiotics within microbial populations has been implicated in treatment failure of chronic infections and development of persister cells. However, the molecular mechanisms regulating phenotypic drug tolerance are largely unknown. In this study, we identified a four-gene operon in Streptococcus pneumoniae that contributes to phenotypic tolerance to vancomycin (ptv). RNA sequencing, quantiative reverse transcriptase PCR, and transcriptional luciferase reporter experiments revealed that transcription of the ptv operon (consisting of ptvR, ptvA, ptvB, and ptvC) is induced by exposure to vancomycin. Further investigation showed that transcription of the ptv operon is repressed by PtvR, a PadR family repressor. Transcriptional induction of the ptv operon by vancomycin was achieved by transcriptional derepression of this locus, which was mediated by PtvR. Importantly, fully derepressing ptvABC by deleting ptvR or overexpressing the ptv operon with an exogenous promoter significantly enhanced vancomycin tolerance. Gene deletion analysis revealed that PtvA, PtvB, and PtvC are all required for the PtvR-regulated phenotypic tolerance to vancomycin. Finally, the results of an electrophoretic mobility shift assay with recombinant PtvR showed that PtvR represses the transcription of the ptv operon by binding to two palindromic sequences within the ptv promoter. Together, the ptv locus represents an inducible system in S. pneumoniae in response to stressful conditions, including those caused by antibiotics.IMPORTANCE Reversible or phenotypic tolerance to antibiotics within microbial populations is associated with treatment failure of bacterial diseases, but the underlying mechanisms regulating phenotypic drug tolerance remain obscure. This study reports our finding of a multigene locus that contributes to inducible tolerance to vancomycin in Streptococcus pneumoniae, an important opportunistic human pathogen. The vancomycin tolerance phenotype depends on the PtvR transcriptional repressor and three predicted membrane-associated proteins encoded by the ptv locus. This represents the first example of a gene locus in S. pneumoniae that is responsible for antibiotic tolerance and has important implications for further understanding bacterial responses and phenotypic tolerance to antibiotic treatment in this and other pathogens.KEYWORDS Streptococcus pneumoniae, phenotypic tolerance, vancomycin, PadR family regulator, gene regulation, transcriptional derepression

Molecular Mechanisms of hsdS Inversions in the cod Locus of Streptococcus pneumoniae

“…Transcriptional reporter strains were constructed by replacing the internal coding sequence of the bgaA gene as previously described (44). The necessary information on the generation and characteristics of these strains are provided in Table S5, which includes primers, DNA templates, parental strains, and the genotypes of resultant strains.…”

“…Transcriptional analysis by RT-PCR, qRT-PCR, and luciferase assay. The transcription of pneumococcal genes was assessed by RT-PCR, qRT-PCR, and luciferase assay as described (44). Briefly, pneumococcal total RNA was extracted with a PureLink RNA minikit (Ambion, Life Technologies, USA); a cDNA library was prepared with the iScript cDNA synthesis kit (Bio-Rad, Beijing, China) according to the supplier's instructions.…”

“…The lack of independent transcription for these two hsdS genes suggested that the hsdS B and hsdS C genes do not produce functional proteins but serve as the templates for DNA inversions with the upstream hsdS A and thereby change the sequence specificity of the type I RM system. The decrease in luciferase activity during bacterial growth was likely due to gradual exhaustion of the luciferase substrate instead of a relationship between gene transcription and the growth phase, because this phenomenon occurred in this type of assay with many target promoters (43,44).…”

Regulation of pneumococcal epigenetic and colony phases by multiple two-component regulatory systems

“…Pneumococcal mRNAs were quantified by quantitative real-time reverse transcriptase PCR (qRT-PCR) as described [72]. Briefly, total RNA extracts were prepared as described for RNAseq and used to prepare cDNA pools with iScript™ cDNA Synthesis Kit (Bio-Rad, USA).…”

“…The relative abundance of hsdS A1 mRNA of each strain is presented as (2 -ΔΔCT ) % given that the relative abundance of hsdS A1 mRNA in psrA Y247A is 100%. The transcriptional levels of RR11-regulated genes were detected by qRT-PCR with the era gene as an internal control and primers listed in S8 Table. The era gene was amplified with primers Pr7932/ Pr7933, which is commonly used as an internal control [72]. The relative gene expression was PLOS PATHOGENS calculated according to the comparative 2 -ΔΔCT method [73] and the ΔΔC T was calculated using the following equation: ΔΔC T = (C T gene of interest -C T era) mutant-(C T gene of interest-C T era) ST606.…”