Analysis and identification of a drug resistance-associated two-component signaling system composed of Streptococcus pneumoniae StkP/CiaR proteins / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To identify a two-component signaling system (TCSS) composed of β-lactam antibiotic resistance-associated intracellular CiaR and transmembrane serine/threonine kinase StkP of Streptococcus pneumoniae ( S. pneumoniae). Methods:The intracellular segment of stkP gene (IC- stkP) was amplified by PCR and the PCR product was sequenced after T-A cloning. A prokaryotic expression system for IC- stkP segment was established. SDS-PAGE was used to detect the expression of the target recombinant proteins (rIC-StkP and rCiaR) by the established prokaryotic expression system and a previously established prokaryotic expression system for ciaR gene. Ni-NTA affinity chromatography was used to purify the recombinant proteins. The rIC-StkP-captured target proteins of S. pneumoniae were identified by LC-MS/MS after Co-IP. The ability of rCiaR to bind to rIC-StkP was detected by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Results:The established prokaryotic expression system for IC- stkP segment could effectively express rIC-StkP. Both rIC-StkP and rCiaR after purification showed a single protein band on SDS-PAGE. CiaR could be specifically co-precipitated with rIC-StkP. Three extracted cleaved peptides were found in CiaR molecule with exactly matched sequences. SPR and ITC showed that rCiaR could strongly bind to rIC-StkP with high affinity and the KD values were 1.526×10 -9 mol/L and 1.980×10 -6 mol/L, respectively. Conclusions:S. pneumoniae CiaR could act as the downstream response regulatory protein of StkP kinase to compose StkP/CiaR TCSS.