Study On Protein Interactions Between Macrophages And Mycobacterium Tuberculosis H37Rv

Mycobacterium tuberculosis(Mtb)is the intracellular pathogen of Tuberculosis,whose main human host cells are macrophages.Macrophages can kill intracellular Mtb by the fusion of phagosome and lysosome,killing of cytokines and radical,apoptosis in the progress of innate immunity or antigen presentation to initiate adaptive immune.Meanwhile,Mtb has generated kinds of mechanisms of escaping and latency to avoid being killed in the long-term evolutionary process.It is critical to interpret the relation between Mtb and macrophages for the study of pathogenesis and the treatment of Tuberculosis.In this work,THP-1 cell line(monocytic cell line of human acute leukemia,differentiating into macrophage after PMA induction)was used.And the study is based on Mtb proteome chip as well as quantitative proteomics technique to explore the interactions between Mtb H37Rv and THP-1 macrophages.THP-1 monocytes differentiate into THP-1 macrophages successfully after inducted with 25 ng/mL phorbol-12-myristate-13-acetate(PMA)for 48 h followed by flow cytometry detection,ELISA etc.After stimulating THP-1 macrophages by various MOI for 12 h,apoptosis related factors of infected macrophages at the level of RNA was detected by RT-qPCR,it is found that MOI 10:1-50:1 infection all can obtain macrophages infected successfully.After stimulating THP-1 macrophages with H37Rv or Latex beads,hybridizing the THP-1 total protein with Mtb proteome chips and processioning data,we got 150 differential proteins of Mtb interacting with macrophage proteins from the two groups.By comprehensive analysis of preliminary bioinformatics,function research and in vivo interaction between macrophages and Mtb,proteins with incomplete research and potential interesting predicted functions,Rv1815 and Rv2538c,were chosen for following study to further explore their actual function in Mtb infection.After being overexpressed in THP-1 monocyte,Rv1815 and Rv2538c were found to be related with the expression level of apoptosis related genes.The results still need more in vitro and in vivo verifications.The Mtb proteome chip we used has advantages of high-throughput,miniaturization,parallelism and rapid analysis,which can be used to globally analyze protein interactions between Mtb and macrophages.The interaction networks between macrophages and Mtb constructed in this work will help find some key proteins related with Mtb infection,latency and pathogenesis,which can be used as drug targets or diagnostic markers for diagnosis,development of drug and vaccines against TB.