Correlation of two-component signaling system ComD/ComE and β-lactam antibiotics resistance of Streptococcus pneumoniae
Objective To generate a comD gene knock-out mutant of Streptococcus pneumoniae,and determine the correlation of comD gene and the bacterial resistance against β-lactam antibiotics and understand the effect of closantel down-regulating comD, comE and comC mRNA levels. Methods A suicide plasmid pEVP3comD was constructed for comD gene knock-out and a comD gene knock-out mutant (comD-)was generated through homologous recombination and insertion inactivation. PCR and immunofluorescence method were used to identify the comD- mutant and real-time fluorescence quantitative PCR was applied to detect the changes of comD, comE and comC mRNA levels before and after closantel treatment in comD-mutant and wild-type strain. Double agar dilution method was performed to determine the sensitivity of comD- mutant and wild-type strain to penicillin G and cefotaxime. Results The comD gene in genome DNA of the generated comD- mutant was inactivated by sequencing and immunofluorescence detection. 50 μ mol/L or 100 μmol/L closantel had a function to down-regulate the comD, comE and comC mRNA levels ( P ＜ 0. 05) whereas 25 μmol/L closantel did not. Both the MIC values of penicillin G and cefotaxime inhibiting comD- mutant were 32 μg/ml which was significantly higher than that of wild-type strain (0.06 μg/ml and 1 μg/ml). Conclusion In this study a comD gene knock-out mutant of S. pneumoniae was successfully generated. There is a close correlation between comD gene and β-lactam antibiotics resistance of S. pneumoniae. Closantel has a function to inhibit the competence formation of S. pneumoniae through down-regulating the transcription levels of comD, comE and comC genes. 
 
Key words: 
Streptococcus pneumoniae;  Two-component signaling system ComD/ComE;  β-lactam antibiotics;  Drug resistance;  Closantel;  Expression regulation