β-lactam antibiotic binding sites in Streptococcus pneumoniae StkP kinase extracellular region
10.3760/cma.j.cn112309-20211228-00436
- VernacularTitle:肺炎链球菌StkP激酶胞外区与β-内酰胺类抗生素结合位点分析
- Author:
Yanying HUANG
1
;
Shaodong LI
;
Jie YAN
;
Aihua SUN
Author Information
1. 浙江大学医学院附属杭州市胸科医院病理科，杭州 310003
- Keywords:
Streptococcus pneumoniae;
Ser/Thr kinase;
β-lactam antibiotics;
SXXK
- From:
Chinese Journal of Microbiology and Immunology
2022;42(7):556-561
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To analyze the binding ability of motifs in the serine/threonine kinase StkP extracellular region (EC-StkP) of Streptococcus pneumoniae to β-lactam antibiotics. Methods:Three motifs (SXXK) in the EC-StkP were mutated into AXXA, respectively or simultaneously. Four mutant plasmids (EC- stkp-AXXA1, EC- stkp-AXXA2, EC- stkp-AXXA3 and EC- stkp-AXXA4) were transfected into recipient cells for cloning and expression. SDS-PAGE combined with gel image analysis was used to detect the expression of the recombinant mutant proteins (EC-rStkP-AXXA1, EC-rStkP-AXXA2, EC-rStkP-AXXA3 and EC-rStkP-AXXA4). The expressed mutated proteins were extracted and purified by Ni-NTA affinity chromatography. The binding abilities of the mutant proteins to penicillin (PCN) and cefotaxime (CTX) were detected by isothermal titration calorimetry (ITC 200) and surface plasmon resonance (Biacore t200). Results:PCN and CTX could not bind to the expressed proteins with mutations in the first or the third motif (EC-rStkP-AXXA1, EC-rStkP-AXXA3, EC-rStkP-AXXA4). EC-rStkP-AXXA2 could weakly bind to CTX, but not to PCN.Conclusions:All three motifs in the EC-StkP of Streptococcus pneumoniae could bind to β-lactam antibiotics with the first and the third motifs being more important.
