Streptococcus pneumoniae StkP kinase: binding ability with β-lactam antibiotics and correlation with drug resistance

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Objective To investigate the correlation between Streptococcus pneumoniae (S.pneumoniae) StkP kinase and drug resistance and to analyze the binding ability of StkP extracellular region (EC-StkP) to β-lactam antibiotics. Methods A stkP gene knockout (ΔstkP) mutant was constructed from S. pneumoniae strain ATCC6306 by insertional inactivation method. E-test was performed to detect the minimum inhibitory concentrations (MIC) of penicillin (PCN) and cefotaxime (CTX) against ΔstkP mutant and its wild-type strain. Bioinformatic softwares were used to predict the EC-StkP of S. pneumonia strain ATCC6306, to generate the three-dimensional structure model of EC-StkP and to analyze the correlation between the structure and functions of EC-StkP. PCR was performed to amplify the extracellular segment of stkP (EC-stkP) gene and the product of it was sequenced after T-A cloning. A prokaryotic expression system of EC-stkP gene was constructed. SDS-PAGE in combination with a gel image analysis system was used to detect the expression of the recombinant EC-StkP (EC-rStkP). The expressed EC-rStkP was extracted by Ni-NTA affinity chromatography. The binding abilities of EC-rStkP to PCN and CTX were detected by isothermal titration calorimetry (VT-ITC) and surface plasmon resonance (Biacore). Results S. pneumonia strain ATCC6306 was sensitive to PCN (MIC=0.06 μg/ml) and CTX (MIC=0.12 μg/ml), but its ΔstkP mutant was resistant to the two antibiotics (PCN MIC=16 μg/ml, CTX MIC=32 μg/ml). The 295 aa segment was predicted as the extracellular region at C-end of StkP of S. pneumoniae strain ATCC6306, containing four penicillin-binding proteins and Ser/Thr kinase-associated (PASTA) domains. The cloned EC-stkP segment and the EC-stkP segment in GenBank shared 99.6% similarity in nucleotide sequence and 100% in amino acid sequence. The constructed prokaryotic expression system for EC-stkP gene expressed EC-rStkP in soluble form. Both PCN and CTX could bind to EC-rStkP and CTX was better than PCN in term of binding ability. Conclusion The stkP gene of S. pneumonia is closely related to drug resistance and the encoded protein, Ser/Thr kinase StkP, can recognize and bind to β-lactam antibiotics. Key words: Streptococcus pneumoniae; Ser/Thr kinase; β-lactam antibiotics; Binding ability; Gene knockout/drug resistance