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

BACKGROUND

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). However, mechanisms of non-PBP mutation-related β-lactam antibiotic resistance in pneumococcal strains remain poorly characterized.

METHODS

Susceptibility of S. pneumoniae ATCC49619 and its ciaR gene knockout, complemented, or overexpression mutant (ΔciaR, CΔciaR, or ciaROE) to penicillin, cefotaxime, and imipenem was detected using an E-test. Levels of pneumococcal ciaR-mRNA, 5 ccn-microRNAs, and 6 pbps-mRNAs were determined by quantitative reverse transcription polymerase chain reaction (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 was analyzed using IntaRNA software.

RESULTS

S. pneumoniae ATCC49619 was sensitive to the 3 β-lactam antibiotics, but overexpression of CiaR, a response regulator protein in 2-component system, caused the increase of MICs against these antibiotics. The ciaROE 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 was able to bind to the promoters of all ccn-microRNA genes in vitro and within cells. The 3 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 5 ccn-microRNAs matched all the promoter-containing sequences of pbps-mRNAs.

CONCLUSIONS

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