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 ciaR^OE) 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 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 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.