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    Home > Biochemistry News > Microbiology News > Gene knockout of Bacillus tuberculosis helps to study the signaling path path of cell coke death

    Gene knockout of Bacillus tuberculosis helps to study the signaling path path of cell coke death

    • Last Update: 2020-12-22
    • Source: Internet
    • Author: User
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    On October 21, 2020, Zhang Xiaolian and Yin Lei of Wuhan University, as co-authors of the study published online at Science Advances, "Mycobacterial EST12 actives a RACK1-NLRP3-gasdermin D pytoropsis-IL-1 beta immuneway path", identified a study that identified a study from
    M. tb
    EST12 (Rv1579c) secreted by H37Rv.
    :
    cell coke death is an inflammatory form of programmed cell death associated with the elimination of pathogen infections. This article describes a TB protein that induces macrophage coke death,
    EST12
    . EST12 combines the active C kinase 1 (RACK1) in macrophages, and the EST12-RACK1 complex collects the de-ubibinase UCHL5 to promote K48 connection de-ubimination of NLRP3, which then leads to the NLRP3-caspase-1/11-GSDMD-IL-1 beta immune process. The analysis of EST12 crystal structure shows that
    amino acid Y80 is
    binding point of RACK1.
    EST12 knock-off strains showed high susceptivity in both in vivo and in vitro MTB infections.
    this study is the first to demonstrate that RACK1 is an endogenetic host-sensing protein for pathogens, and that EST12-RACK1-induced cell coke death plays a key role in M.tb-induced immunity.
    research content:
    1, RD area coded protein screening: 40 kinds of RD region (RD1-3, 11-14) recombinant protein screening (macrophage coke phosphate-related protein), through LDH release experimental cytotoxicity analysis, found that EST12 has significant coke effect on macrophages.
    2, strain construction:
    build
    M. tb
    H37Rv EST12 (Rv1579c knock-out strain)
    , BCG-EST12 (Rv1579c over-expression strain),
    M. smeg
    -EST12 (Rv1579c over-expression strain).
    3, mouse gene knock-out model: NLRP3-/,GSDMD-/?, caspase-1/11-mouse, RACK1-M-.
    4, detection content: LDH (lactic acid dehydrogenase) release experimental detection cytotoxicity, ELISA detection
    cellular factor
    secretion, WB detection, immunosuppression (IP), pull down,
    mass spectrometry
    , EST12 crystal structure, a focus
    microscope
    , mouse nob infection model, etc.
    results:
    1, EST12 (Rv1579c) induced GSDMD-mediated macrophage coke death
    expression purification of 40 RD region coded proteins, through LDH release experiments, found that EST12 (Rv1579c) has significant macrophage toxicity. The EST12 protein causes the death of celiac macrophages (Figures 1A and B), human monocyte THP-1 (Figure 1C) and bone marrow-source macrophages (BMDMs) in mice (Figure 1D). The results showed that EST12 was highly toxic to macrophages.
    1 identification of the macrophage coke-induced protein EST12A-D: EST12 and Rv1577 were used to treat celiac macrophages (A, B), PMA differentiated THP1 cells (C), bone marrow macrophages BMDMS (D) in mice, respectively, using LDH release method analysis. E:WB detection 2 m EST12 or RV1577 treatment macrophage GSDMD active; F:ELISA detection IL-1 beta secretion; G:WB detection H37Rv and H37Rv-EST12 after infection with BMDMs cells caspase-1/GSDMD Activity; H:LDH release experiment detects cytotoxicity; I:ELISA detects IL-1 beta secretion; J:WB detects caspase-1/GSDMD activity after infecting BMDMs cells with BMDMs cells; K:LDH release test detects cytotoxicity; ELISA detects IL-1 beta secretion.
    to determine whether GSDMD and GSDME were involved in EST12-induced cell death, EST12 stimulated macrophages in the abdominal cavity of mice at different times to detect GSDMD and GSDME activity. It was found that the full length of GSDMD, GSDMD N and C ends were detected, and the fission activity of caspase-1 P20 fragments was enhanced by stimulating coeliac macrophages in mice for 0.5 hours (Figure 1E) ), macrophage IL-1 beta secretion increased (1F), EST12 further treated human THP-1 monocyte-source macrophages, and found that caspase-1 and GSDMD were cleavaged by EST12.
    detection of H37Rv and H37Rv-EST12 infection macrophage caspase-1 and GSDMD activity, there was no significant change in the H37Rv∆EST12 and H37Rv groups within 1 hour of infection. After stimulating 6h, H37Rv induces more cracked caspase-1 p20 and GSDMD-N end fragments in BMDMs (Figure 1G). Similarly, H37Rv ∆ LDH release and IL-1 beta secretion were lower than in group H37Rv (Figures 1H, I). The results showed that H37Rv ∆ the inflammatory coke death in the EST12 group was less than that in the H37Rv group. In BMDMs, BCG-EST12 continuously induces more caspase-1 p20 and GSDMD-N end fragments (Figure 1J), cytotoxicity, and higher IL-1 secretions than BCG (Figure 1K). The results showed that recombined BCG-EST12 significantly increased the expression and cytotoxicity of IL-1 induced by BCG.
    results: EST12 mediates macrophage coke death and inflammatory cytokine IL-1 secretion through GSDMD.
    2, EST12 and RACK1 interaction induced macrophage inflammatory coke
    To detect proteins in cells that interact with EST12, using pull down experiments and mass spectrometrography, it was found that the RACK1 protein may be a binding protein to EST12, and the immunoprint further validated this result (Figure 2A). The EST12-green fluorescence and RACK1-red fluorescent protein expression protons were transfected into macrophage RAW264.7, and a confocus fluorescence microscope showed that EST12 and RACK1 were positioned in the cytostype to produce yellow (Figure 2B). q
    PCR
    and WB analysis shows that EST12 stimulates the expression of macrophage RACK1, peaks at 6h and then falls (Figure 2C), presumably EST12 promotes the expression of RACK1 at an early stage.
    2 EST12 interacts with RACK1 to induce macrophage inflammatory coke
    A: RAW264.7 Cells for EST12-His pull-down experiment, WB analysis; B: confocus microscope analysis transfect pEGFP-C1-EST12 and pAsRED2-C1-RACK, respectively RAW264.7 cells for 1; C:WB and qPCR analysis for RAW264.7 cells stimulated by 2μM EST12; D, E: LDH, ELISA analysis 2 xM EST 12 Treatment of WT and RACK1-/Celiac macrophages; F:WB detection of GSDMD and caspase-1 in cell lysate; G:2?M EST12 treatment and PI staining W T, RACK1-/-macrophages; E-J: LDH, ELISA analysis H37Rv and H37Rv-EST12, BCG and BCG-EST12, M. smeg and M. smeg-EST12 infects WT, RACK1,/-BMDMs.studied the interaction between EST12 and RACK1 in macrophages, and stimulated WT and RACK1-celiac macrophages, respectively, compared to WT macrophages, RACK1-/celiac macrophages EST12 induced cytotoxicity (Figure 2D) and IL-1 secretion (Figure 2E) were significantly reduced, and GSDMD N fragments and caspase-1 p10 cleavage were strongly inhibited (Figure 2F). Real-time cofocus microscope analysis shows that EST12 causes celiac macrophages to present a typical cell coke-dead form: a ruptured mass membrane, multiple bubble protrusions, and an "omelette" in the nuclei of the cells (Figure 2G). There is no difference between apoptosis and IL-1 beta secretion of H37Rv-EST12 infected with WT BMDMs and H37Rv infected with RACK1-/BMDMs (Figure 2H). There is no significant difference between cytotoxicity and IL-1 beta secretion between BCG-EST12 and BCG-infected RACK1-/BMDMs (Figure 2I). The same results were found in the M. smeg and M. smeg-EST12 experimental groups (Figure 2J)
    results are condensed: EST12 causes macrophage inflammatory coke death through RACK1.
    3, EST12 protein C and Y80 are key bits in combination with RACK1
    to predict the structure of EST12-RACK1 (Figure 3B), the interaction between EST12 and RACK1 may depend on the third helix structure of EST12 (E55, F76, Y80). Recombinant proteins (Figure 3C) perform a pull down experiment on RAW264.7 cell lyses, and then immunoprint analysis shows that EST12-D1 (missing at the C end) has little interaction with RACK1 (Figure 3D), and the binding of Y80A and F76A to RACK1 is significantly lower than that of EST12 (Figure 3E). Compared with EST12, the cytotoxic effect of EST12-D1 was also significantly reduced (Figure 3F, G), and the cytotoxic effect induced by EST12-E55A, F76A, and Y80A decreased, of which EST12-Y80A was the most obvious (Figure 3H, I). Similarly, M. smeg-EST1-D1 and M. smeg-EST12-Y80A infections result in fewer cell coke deaths than M. smeg-EST12 (Figures 3J, K).
    results: Y80 at the EST12 C end is the key binding point for RACK1 causing coke death.
    figure 3 Y80 is the key binding bit of RACK1
    A:EST12 Crystal Structure; B:EST12-RACK1 Predictive Structure; C: Recombination Protein Full Length, EST12 C End Missing (EST12-D1), N End Missing (EST12-D2), EST12 3 mutant illustrations; D-E: recombinant protein pull down and WB analysis; F-I:LDH analysis recombinant protein treatment of celiac macrophages at different times and doses; J-K:LDH analysis M. smeg and M. smeg-est12, EST12-D1, EST12-D2, EST12-E55A, EST12-F76A, EST12-Y80A infect BMDMs.
    4, EST12-RACK1 through NLRP3 and GSDMD caused macrophage coke death and IL-1 secretion
    inflammatory small body activation is a key defense mechanism against bacterial infection, bacterial infection can induce congenital immune responses such as caspase-1 activation and inflammatory cell death. The mRNA chip analyzed EST12 to treat RAW264.7 cells and showed an increase in expression of NLRP3, IL-1 beta, IL-6, α TNF-α. After 8 PYRIN domain proteins (NLRP1b, NLRP2, NLRP3, NLRP5, NLRP6, NLRP9, NLRP12, PYRIN) were treated with EST12, the mRNA expression of NLRP3 changed significantly in macrophages (Figure 4A). After EST12 stimulation for 6 hours, immunoprint analysis NLRP3 protein expression increased to the highest level (Figure 4B). The confocus microscope analysis shows that the NLRP3 expression is highest 6 hours after EST12 stimulation (Figure 4C). After EST12 is processed, the expression of NLRP3 in RACK1 is significantly impaired (Figure 4D).
    result: EST12 causes the expression of NLRP3 to depend on RACK1.
    4 EST12 induces the activation of the NLRP3 inflammatory small body through RACK1
     A:qPCR analysis
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