Effects Of CwlM On Autolysis And Biofilm Formation Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis

Tuberculosis（TB）has a high mortality and prevalence which caused by Mycobacterium tuberculosis（MTB）.In 2013,9 million people fell ill with tuberculosis and 1·5 million died.The new number of tuberculosis is 9.6 million in 2015.Compared with the data of 2013,there is an increasing trend.Many cases of tuberculosis are due to MDR-TB strains.In China,drug-resistant tuberculosis in the reported TB cases is account for a large proportion.Drug resistance of MTB has been caused TB control workers’ attention.Biofilm is considered to be one of the most important mechanisms in the bacterial resistance mechanism,MTB in vitro can form biofilm,which carries resistant cell populations that are able to survive under high concentrations of antibiotics.e DNA and autolysin genes play key roles in the biofilm formation,whereas the autolysin gene is associated with peptidoglycan hydrolase.Rv3915 gene of MTB and MSMEG₆935 of Mycobacterium smegmatis（M.smegmatis）（collectively referred to as cwl M gene）respectively encoded the CwlM protein of the respective cells.Rv3915 gene and MSMEG₆935（cwl M gene）can encode CwlM proteins,the CwlM（CwlMTB）of M.tuberculosis and CwlM（CwlMms）of M.smegmatis are amylases of peptidoglycan hydrolases that hydrolyze bacterial cell walls.Given that the hydrolysis of peptidoglycan hydrolase on the cell wall and its potential clinical value,the effects on bacterial autolysis and biofilm of CwlM as peptidoglycan hydrolase amide enzyme is worthy of our in-depth study.In this experiment,we successfully constructed the in vitro biofilm model of M.smegmatis and MTB.The expression of cwl M genes were recorded in the biofilm by fluorescence quantitative PCR（q RT-PCR）,the CwlM protein were expressed and purified,the effects of CwlMms and CwlMTB on the autolysis and biofilm were investigated.Finally,the effect of endogenous CwlMms on autolysis and biofilm was confirmed by constructing cwl M deletion of M.smegmatis.Firstly,q RT-PCR technique was used to detect the expression of Rv3915 gene and MSMEG₆935 gene in the process of biofilm formation.The results showed that the expression of MSMEG₆935 gene in the midst of the biofilm formation was 3 times（p < 0.001）higher than planktonic bacteria,the expression level of the MSMEG₆935 gene was significantly down-regulated in the late stages of biofilm formation（p < 0.05）.In the early stage of the biofilm formation,expression of Rv3915 was no significant difference（p > 0.05）than planktonic bacteria.In the mid-term of biofilm formation,the expression was significantly increased（p < 0.001）compared to the planktonic bacteria.It suggests that cwl M play an important role in the process of biofilm formation.Secondly,CwlMTB and CwlMms proteins were expressed in large quantities through prokaryotic expression systems.The pcold-6935 and pcold-3915 expression plasmids were successfully constructed in this study and the expressing strains of pcold-6935-plys and pcold-3915-plys were obtained.The soluble protein was successfully obtained by induction of 0.1 m M IPTG in low temperature.The protein was purified by affinity chromatography,ion exchange chromatography and gel filtration chromatography.SDS-PAGE and western blot results showed CwlMms and CwlMTB protein bands were single,and the purified soluble protein was target protein.The yield of CwlMms and CwlMTB was 5 mg/L approximately.thirdly,We further investigated the effects of CwlMms and CwlMTB on the autolysis and biofilm formation of M.smegmatis and MTB respectively.Mc2155 was treated with the final concentration of 200 μg/ml CwlMms protein and then we measured the OD580 nm change of bacterial suspension to to detect the autolysis capacityunder 0.1% Triton X-100.The extent of autolysis in the cultures of of H37 Rv medium in 200 μg / ml CwlMTB treated group and control group was monitored by the appearance of ICD activity.The effect of CwlMms on the biofilm and the effect of CwlMTB on the biofilm of tuberculosis were determined by crystal violet quantification.The results showed that protein treatment of Mc2155 showed stronger autolytic ability than the control group in 0.1% Triton X-100 solution,CwlMTB treated H37 Rv released more isocitrate dehydrogenase than the control group;CwlMms significantly reduced the production of the biofilm（p < 0.001）,CwlMTB protein had a tendency to inhibit tuberculosis biofilm（p <0.05）.Finally,we investigated the effect of intracellular CwlMms on the autolysis and biofilm formation of M.smegmatis by constructing a cwl M gene deletion strain.We constructed the cwl M gene deletion strain（MSΔ6935）by homologous recombination with p PR27 thermo-sensitive shuttle plasmid.The autolysis ability of Mc2155 and MSΔ6935 induced by 0.1% Triton X-100 was determined.The effects of cwl M gene deletion on the e DNA and e RNA of the two markers of biofilm were observed by scanning electron microscopy and fluorescence microscopy.The effects of cwl M gene deletion on biofilm yield were observed.The results showed that the colonies with Kana gene resistance were screened,indicating that the cwl M gene deletion strain（MSΔ6935）was successfully constructed.The results showed that the autolysis ability of MSΔ6935 strain was lower than Mc2155 strain.Compare with the Mc2155,the content of e DNA and e RNA of MSΔ6935 was decreased,indicating that the deletion of MSMEG₆935,a coding gene of CwlMms,significantly reduced the yield of the biofilm.In conclusion,the cwl M gene was differentially expressed during the process of M.smegmatis and MTB biofilm formation.The recombinant CwlMms and CwlMTB promote the autolysis of the respective cells,recombinant CwlMTB and CwlMms had an inhibitory effect on the biofilm formation of MTB and M.smegmatis.The lack of cwl M resulted in the decrease of the autolysis ability and biofilm formation of M.smegmatis,cwl M gene deletion confirmed that M.smegmatis autolytic ability and biofilm formation ability were weakened.This study provide a theoretical basis for peptidoglycan hydrolase as a new drug target screening and the use of peptidoglycan hydrolase and nucleic acid inhibitors to jointly develop anti-bacterial biofilm drugs.