Effect Of MSMEG₆281 Knockout On Cell Wall Synthesis And Pathogenicity Of Mycobacterium Smegmatis

Mycobacterium tuberculosis?MTB?is the main pathogen of tuberculosis?TB?,and its cell wall has unique structure,including peptidoglycan?PG?,Arabinogalactan?AG?and branch mycolic acid?MA?,which play an important role in preventing MTB against the resistance of drugs and acidic environment.The mycobacterium tuberculosis Rv3717 protein is an extracellular secreted protein with a molecular weight of 24.8 KD and has recently been reported to have a N-acetyl muramyl-L-alanine amidase activity that hydrolyzes cell wall peptidoglycans[1],which is a kind of novel mycobacterial autolysin,and also play an important role on in the occurrence and development of tuberculosis.Mycobacterium smegmatis?M.smegmatis mc²155?,as a model of MTB,has a similar cell wall structure to MTB.Mycobacterium smegmatis MSMEG₆281 is a homologous protein of Rv3717.Previous studies have shown that the protein encoded by Rv3717?MSMGE₆281?gene may play an important role in mycobacterial division and pathogenicity.Therefore,this studydemonstrate the function of MSMEG₆281 through the construction of MSMEG₆281 gene knockout strain,further indicate the relationship of MSMEG₆281 and mycobacterium virulence.These results lay the foundation for the development of anti-tuberculosis drug.OBJECTIVE:To indicate the function of MSMEG₆281?Rv3717?on bacterial growth,division,cell wall biosynthesis and mycobacterial pathogenicity,further to provide some clues for the mechanism of eliciting innate immune response duringmycobacterium tuberculosis infection.Methods:MSMEG₆281 gene knockout strain was successfully constructed by twice homologous recombination using conditional replication plasmid,and the expression of MSMEG₆281 gene was identified by RT-PCR.The effect of MSMEG₆281 gene knockout on the growth of mycobacterium smegmatis was determined by growth curve,and the integrity of cell wall was detected by acid-fast staining and cell wall permeability analysis.The effect of MSMEG₆281 gene knockdown on cell wall structure and bacterial morphology was observed by transmission electron microscopy?TEM?.The expression of MSMEG₆396,MSMEG₆398,MSMEG₂078 and MSMEG₃580 were analyzed by RT-PCR analysis of MSMEG₆281 gene knockout.On the other hand,the cells were aerosolized by aerosol generating device,and the mice were infected with M.sm-?M₆281 with 10⁸CFU/ml of wild-type Mycobacterium smegmatis and the two groups of mice were subjected to aerosol attack on average The rats were sacrificed at10⁸ CFU/day for 7 days.The expression of IL-6,IL-1?and IL-10 in spleen tissue of mice were detected by RT-PCR at the same time.RESULTS:MSMEG₆281 gene knockout strain was successfully constructed and named M.sm-?M₆281.Growth curve tests showed that MSMEG₆281 was a non-essential gene for growth of M.smegmatis.Morphological analysis showed that MSMEG₆281 gene knockdown,the cell acid-resistant staining decreased,and the cell wall surface blurred,the cell boundary is not clear,and jagged,indicating that MSMEG₆281 gene knockout may affect the cell wall structure,affecting cell wall integrity.In the detection of bacterial cell wall permeability,M sm-?M₆281 in SDS and crystal violet no significant change.RT-PCR showed that the expression of MSMEG₆396 and MSMEG₆398 were down-regulated.The results showed that the expression of IL-6 and IL-1?was down-regulated in the MSMEG₆281 gene after knockout of M.sm-?M₆281,so Rv3717 protein may play an important role ininflammatory and pathological response during MTB infection.In summary,the MSMEG-6281 is non-essential gene for M.smegmatis,and MSMEG₆281 gene knockout results in the changes ofmycobacterial morphology and cell wall structure.The expression of genes involving in adhesin synthesis are down-regulated,andthe pathogenicity in mice is decreased without affecting bacterial growth and cell wall permeability.