Journal: Infection and Immunity
Article Title: Brucella targets the host ubiquitin-specific protease, Usp8 , through the effector protein, TcpB, for facilitating infection of macrophages
doi: 10.1128/iai.00289-23
Figure Lengend Snippet: USP8 affects the invasion of Brucella into macrophages through CXCR4. (A) Levels of CXCR4 on the plasma membrane of HEK293T cells overexpressing HA-Usp8. HEK293T cells were transfected with HA-Usp8 or EV. Twenty-four hours post-transfection, the cells were stained with the anti-CXCR4 antibody, followed by Alexa Fluor 647-conjugated secondary antibody (red). HA-USP8 was stained with FITC-conjugated anti-HA antibody (green), and nuclei were stained with DAPI (blue). The cells were imaged using a laser confocal microscope at 63×. Scale bar, 5 µm. The image represents 12 different fields captured from cells transfected with either empty vector or HA-USP8. The right panel indicates the quantification of fluorescence intensity of CXCR4 using ImageJ software. (Panels B and C) Total levels of CXCR4 in iBMDMs treated with antagonists of USP8 or 14-3-3ζ. iBMDMs treated with DUB_IN-2 (B) or BV02 (C), or DMSO (vehicle) for 24 hours, followed by harvesting the cells and immunoblotting. To detect CXCR4, the membranes were probed with the anti-CXCR4 antibody, followed by HRP-conjugated anti-rabbit IgG. Actin was used as the loading control. The right panels indicate the densitometry of the CXCR4 band with respect to the actin band. (Panels D–F) Levels of plasma membrane-localized CXCR4 in iBMDMs treated with DUB-IN-2 (D) or BV02 (E) or infected with B. neotomae (F). The membrane fractions showing CXCR4 and TNFR1 isolated from the compound-treated or Brucella-infected cells, followed by immunoblotting. The right panel indicates the densitometry of the CXCR4 band with respect to the TNFR1 band. (G) iBMDMs showing membrane-localized CXCR4 upon treatment with DUB_IN-2 or DMSO. iBMDMs were treated with DUB_IN-2 or DMSO for 24 hours, followed by staining membrane-localized CXCR4 (red) as described earlier. Nuclei were stained with DAPI (blue). The cells were imaged using a laser confocal microscope at 63× magnification. Scale bar, 20 µm. The image represents 12 different fields captured from cells treated with either DMSO or DUB-IN-2. Right panel indicates the quantification of fluorescence intensity of CXCR4 using ImageJ software. (Panels H and I) Brucella invasion assay showing the CFU count of B. neotomae (H) or B. melitensis (I) in the presence of the CXCR4 inhibitor. (Panels J and K) The expression of Cxcr4 in iBMDMs transfected with Cxcr4-specific or control shRNA construct. The cells were transfected with shRNA expression constructs for 48 hours, followed by analyzing the levels of Cxcr4 by immunoblotting (J) and qRT-PCR (K). The right panel indicates the densitometry of the CXCR4 band with respect to the actin band. (Panels L and M) Brucella invasion assay in Cxcr4-silenced or control iBMDMs. The cells were transfected with Cxcr4-specific or non-targeting control shRNA. Forty-eight hours post-transfection, the cells were infected with B. neotomae or B. melitensis for 30 min, followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae (L) or B. melitensis (M) by CFU enumeration. (N) Brucella invasion assay using iBMDMs treated with antagonists of USP8 and CXCR4, as indicated in the figure. iBMDMs were treated with DUB_IN-2 or AMD3100 or both DUB_IN-2 and AMD3100 for 24 hours, followed by infection with B. neotomae and enumeration of CFU. A representative result from at least two biological replicates is shown. The data are presented as the mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001, ns > 0.05). WCL, whole-cell lysate; EV, empty vector; ns, non-significant.
Article Snippet: Forty-eight hours post-transfection, the cells were infected with B. neotomae (ATCC 23459–5K33) at an MOI of 1,000:1 or B. melitensis 16M (obtained from Indian Veterinary Research Institute) at an MOI of 200:1.
Techniques: Membrane, Transfection, Staining, Microscopy, Plasmid Preparation, Fluorescence, Software, Western Blot, Control, Infection, Isolation, Invasion Assay, Expressing, shRNA, Construct, Quantitative RT-PCR, Bacteria