The Function And Mechanism Study Of Mycobacterium Tuberculosis McelF Protein In Mycobacterial Infection

The mammalian cell entry(mce)protein encoded by Mycobacterium tuberculosis(Mtb)plays an important role in the process of bacterial invasion of host cells.Mtb contains 4 mce operons and mce lF belongs to one of the 24 family members of mce gene,the mcelF is predicted as components of mycobacteria cell wall based on its protein sequence.However,its actual function is still unknown.In order to study the specific function and mechanism of mcelF protein in the process of Mtb infection,we constructed a recombinant Mycobacterium smegmatis(MS-mcelF)in which the mcelF was up-regulated.We found that MS-mcelF could inhibit the production of pro-inflamlatory factors IL-1β,IL-6,TNF-or and iNOS in infected RAW264.7 cells.Furthermore,we found that the expression of cellular inflammatory factor and iNOS were inhibited in MS-mcelF infected Bone marrow derived macrophages(BMDMs)from WT and TLR2 knock-out mice.But the expression of cellular inflammatory factor and iNOS had no significant change in infected BMDMs from TLR4 knock-out mice.The phosphorylation of IKKa,IκB,p65,JNK in the NF-κB Signaling pathway was significantly inhibited when RAW264.7 cells were infected with MS-mcelF.We also generated mcelF up-regulated expression of stable macrophage cell line(RAW264.7-meelF).The TLR4 downstream signaling pathway was significantly inhibited in RAW264.7-mcelF cells activated by TLR4 agonist LPS,whereas TLR2 downstream signaling pathway activated by TLR2 agonist Pam3csk4 in RAW264.7-mce 1F cells had no significant change.Similar to the infection of MS-mcelF,TLR4 downstream signaling pathway activated by LPS could be inhibited by RAW264.7-mcelF.The MS-mcelF could inhibit the expression of NF-κB signaling pathway in BMDMs from WT and TLR2 knock-out mice.By using purified mcelF protein to stimulate the RAW264.7 cells,we found that it could inhibit the phosphorylation of p65 and the expression of iNOS similar with the MS-mcelF.In summary,our results indicate that the Mtb mcelF protein can reduce the expression of pro-inflammatory faetors and iNOS through inhibiting the TLR4 signaling pathway,and promote the survival of Mtb in the host.