Correlation Of Two-component Signaling System Com/ComE And β-Lactam Antibiotics Resistance Of Streptococcus Pneumoniae

Objective:To construct prokaryotic expression systems of TCS genes comD/comE/comC which regulating competence formation of Streptococcus pneumoniae, and to determinate the alteration of drug resistance of the microbe after ComD and ComC were blocked. To generate a comD gene knock-out mutant of Streptococcus pneumoniae, and determine the correlation of comD gene and the bacterial resistance againstβ-Lactam antibiotics and understand the effect of closantel down-regulating comD, comE and comC mRNA levels. Methods:The entire comD, comE and comC genes were amplified by PCR and their prokaryotic expression systems were then established by using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rComD, rComE and rComC. Rabbits were immunized with each of the three recombinant proteins to prepare antisera. The alteration of resistance to penicillin and cefotaxime of S.pneumoniae strains were examined after ComD and ComC of the strains were blocked by antisera. 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:Compared to the reported sequences, similarities of nucleotide and amino acid sequences of the cloned comD, comE and comC genes were 98.4%-99.3% and 99.1%-100%, respectively. The constructed engineering bacteria E.coli BL21DE3 PET42a-comD),E.coli BL21DE3 pET42a-comE and E.coli BL21DE3 pET32a-comC were able to efficiently express the target recombinant proteins and the outputs of rComD, rComE and rComC were 28%, 25% and 35% of the total bacterial proteins, respectively. The double immunodiffusion titers of rabbit antisera against rComD, rComE or rComC were 1:4,1:4 and 1:8, respectively. After the ComD and / or ComC were blocked by the antisera, the cefotaxime-sensitive strains could produce resistance to the antibiotic but no alterations about the resistance of cefotaxime-resisting strains as well as the resistance to penicillin in all the tested strains could be found. The comD gene in genome DNA of the generated comD- mutant was inactivated by sequencing and immunofluorescence detection.50μmol/L or 100μmol/L closantel had a function to down-regulate the comD, comE and comC mRNA levels (P＜0.05) whereas 25μmol/L closantel did not. Both the MIC values of penicillin G and cefotaxime inhibiting comD mutant were 32μg/mL which was significantly higher than that of wild-type strain (0.06 and 1μg/mL). Conclusion:The prokaryotic expression systems of S.pneumoniae comD/come/comC genes are successfully constructed in this study, which lays a foundation for further investigating the correlation between competence formation and drug resistance. Both the ComD和ComC are involved in cefotaxime resistance of the bacterium. In this study a comD gene knock-out mutant of Streptococcus pneumoniae was successfully generated. There is a close correlation between comD gene andβ-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.