Response regulator protein CiaR regulates the transcription of ccn-microRNAs and β-lactam antibiotic resistance conversion of Streptococcus pneumoniae.

Abstract

Introduction: Streptococcus pneumoniae does not produce β-lactamases and its reduced susceptibility to β-lactam antibiotics is predominantly caused by mutations of penicillin-binding proteins (PBPs), but mechanisms of PBP mutation-unrelated β-lactam antibiotic resistance in pneumococcal strains remain poorly characterized.

Methods: Susceptibility of S. pneumoniae ATCC49619 and its ciaR gene-knockout, -complemented, or -over-expression mutant (ΔciaR, CΔciaR, or ciaR^OE) to penicillin, cefotaxime, and imipenem was detected using E-test. Levels of pneumococcal ciaR-mRNA, five ccn-microRNAs, and six pbps-mRNAs were determined by qRT-PCR. Recombinant CiaR (rCiaR) binding to the promoters of ccn-microRNA genes was confirmed using electrophoresis mobility shift and chromatin immunoprecipitation assays. Sequence matching between the ccn-microRNAs and pbps-mRNAs were analyzed using IntaRNA software.

Results: S. pneumoniae ATCC49619 was sensitive to the three β-lactam antibiotics, but over-expression of CiaR, a response regulator protein in two-component system, caused the increase of MICs against these antibiotics. The ciaR^OE mutant exhibited the significantly increased transcription of ccn-microRNAs but notably decreased transcription of pbps-mRNAs, conversely the ΔciaR mutant displayed decreased levels of ccn-microRNAs and increasesed transcription of pbps-mRNAs. rCiaR could bind to the promoters of all ccn-microRNA genes in vitro and within cells. The three antibiotics at 1/8 minimal inhibitory concentrations caused a significant increase in the ciaR-mRNA and ccn-microRNAs. The mRNA-binding seed sequences in the five ccn-microRNAs matched all the promoter-containing sequences of pbps-mRNAs.

Conclusions: β-lactam antibiotics at low-concentrations induce PBP mutation-unrelated antibiotic resistance conversion of S. pneumoniae by decrease of PBPs through pathway of CiaR-mediated transcriptional increase of ccn-microRNAs and ccn-microRNA-dependent degradation of pbp-mRNAs.