Effects Of Mycobacterium Smegmatis MSMEG₆387 Gene Knockout On THP-1 Cellular Immune Function

Mycobacterium tuberculosis?MTB?,a typical intracellular pathogens,is the main pathogenic bacteria causing Tuberculosis?TB?.The treatment and prevention of TB turn more difficulty with the long-term use of anti-TB drugs and increasing the resistant strains.Due to unique virulence,the capacity of MTB to survival and replication in host macrophages is develop some schemes to escape the supervision of immune system.Lipoarabinomannan?LAM?is one of the major components of the MTBcell walls.Different structure of LAM can cause diverse host immune response.Emb C encoding of arabinosyltransferasesmediating the polymerization of arabinose into arabinan,and Emb C mutant produce diverse structure of LAM.Mycobacterium smegmatis?M.smegmatis?MSMEG₆387 is the homologous gene of emb C.This research demonstrates thatthe relationship of LAM structure and immune response in macrophages.Objectives: 1.To construct MSMEG₆387 gene knockout strain and demonstrate the change of cell wall structure and growth due to MSMEG₆387 gene knockout.2.To explore the effect of LAM structure to THP-1cell apoptosis and the immunological activity,provide clues for the relationship between LAM structure and the host innate immune response.Methods: 1.p PR27-xyl E-MSMEG₆387:kanR was constructed through genetic engineering technology,and select the recombinant which insert in specific site of genomic DNA clone using homologous recombination technology and additional antibiotics and sucrose stress screening.The insert site of MSMEG₆387:kanR DNA fragment was identified using PCR amplification and then DNA sequencing.A MSMEG₆387 gene knockout strain was selected and named M.sm-?M₆387.2.LAM was extracted using organic solvent from the M.smegmatis and M.sm-?M-₆387cell walls and identifies furtherthe structure of LAM by periodic acid-Schiff stain?PAS?method.3.In order to further understand the effect of MSEMG₆387 geneknockout on the cell wall structure and function of Mycobacterium smegmtis,we demonstrated them through four aspects :?1?To determine the effectsof MSEMG₆387 geneknockouton the growththrough the growth curve.?2?The change of cell wall structure was detected through TEM?3?The cellular morphology was observed using SEM on the M.smegmatis.?4?The cell wall character was observed through acid-fast staining method 4.In order to further understand the effect of LAM structure to host immune response.THP-1 cells was differentiated into macrophages by PMA,and then was infected by M.smegmatis and M.sm-?M₆387 strains?MOI=10:1?.we demonstrated the change of THP-1 immune response through four aspects.?1?The toxicity of M.smegmatis and M.sm-?M₆387 strain on cell was detected through MTT analysis.?2?The effect of different LAM structure on cell apoptosis was determined through the FCM.?3?TLR2 gene expression also was detected by q PCR.?4?Expression of early inflammatory response factor?IL-12 and IL-1 beta?secreted by THP-1 cells was showed through ELISA.?5?NO secreted by THP-1 was detected through the nitric oxide synthase detection kit.Results:1.MSMEG₆387 gene knockout strain was successfully constructed and named M.sm-?M₆387.LAM extract was gained and found that LAM of M.sm-?M₆387 has a smaller molecular weight.2.The results of growth curve showed that M.sm-?M₆387 strain has a slow growth.SEM results showed that the M.sm-?M₆387 turned shorter compared to wild type M.smegmatis.TEM results showed that the cell wall of M.sm-?M₆387 strains turned twisty and sunk structure.Acid-fast staining analysis found that positive cells were decreased for M.sm-?M₆387strains.3.MTT results showed that the M.sm-?M₆387strains was avirulence to THP-1 cells,and also can stimulate the proliferation of THP-1 cells.FCM results showed that the M.sm-?M₆387strains reduced cell apoptosis.q PCR results showed that gene expression of TLR2 was up-regulated.ELISA results showed that the secretion of IL-12,IL-1beta and NO of THP-1 cells was increased for MSMEG₆387 gene knockout Conclusions: The results in this study showed that MSMEG₆387 genes involved in biosynthesis of mycobacterium LAM,and MSMEG₆387 gene knockout can cause the change of LAM structural with a smaller molecular weight.Bacterial growth,cell wall structure and the change of cellular morphology were demonstrated for MSMEG₆387 gene knockout strain.So we speculated that MSMEG₆387 gene is closely related to the integrity of bacterial cell walls.Change of the LAM structure decreased macrophage apoptosis and increased the secretion of early inflammatory response factor and NO.Therefore,MSMEG₆387 gene knockout lead to the change of LAM cell wall structure,and enhance immune responses of macrophages.But the intrinsic change of LAM structure need be further studying due to MSMEG₆387 gene knockout,TLR-2 signal pathway also need be further research.