Fig 1: Schematic model for the roles in rMPT against MTB infection. (A) Domain screening of interacting site between MPT63 or MPT64 with TBK1, p47phox and HK2. (B) Construction of rMPT combined TBK1, p47phox, and HK2 interacting domains in MPT63 and MPT64. (C) Regulatory pathway of rMPT in macrophages.
Fig 2: TBK1 and HK2 directly interact with MPT64. (A) Identification of TBK1 and HK2 by mass spectrometry analysis in THP-1 cell lysates treated with rMPT64 or rVector. (B) THP-1 cells were stimulated with rMPT64 (5 μg mL−1) for the indicated times, followed by IP with αHis-agarose bead and IB with αTBK1, αP-TBK1 (S172), αHK2, αHis, αActin. (C,D) Titration of fluorescently labelled MPT64 with TBK1 and HK2 (left), with Kd (193 and 134 nM), determined by curve fit analysis (right). (E,G) Binding mapping. Schematic diagrams of the structures of MPT64 (upper). At 48 h after transfection with GST or GST-MPT63 and truncated mutant constructs together with Flag-TBK1 or V5-p47phox. 293T cells were used for GST pull down, followed by IB with αFlag or αV5. WCLs were used for IB with αFlag or αV5, αGST, and αActin. (F,H) 293T cells expressing Myc-MPT63 and Flag-TBK1 or V5-p47phox and treated with several Tat-MPT64-N or MPT64-C peptides (10 µM) for 6 h, followed by IP with αMyc and IB with αFlag. WCLs were used for IB with αMyc, αFlag, and αActin. The data are representative of four independent experiments with similar results (A–H).
Fig 3: HK2 peptide’s role in signal peptide for targeting the MTB-infected macrophages. (A) Schematic design of HK2 peptide (upper). Empty or HK2 KO THP-1 and BMDM cells were treated with Cy5.5 labelled-HK2 peptide for 1 h in various concentrations. THP-1 and BMDMs were used for IB or counting the number of HK-HK2 peptide+ cells by FACS. (B) Empty or HK2 KO THP-1 cells were infected with MTB for 4 h and treated Cy5.5 labelled-HK2 peptide in various concentrations for 1, 18 or 72 h. After 1 h, the THP-1 cells were used for IB and the number of HK2-Hk2 peptide+ cells were counted by FACS (top). The HK2 peptide treated-supernatants of THP-1 for 18 h were used for ELISA to measure the level of TNF-α and IL-6 (middle). The colony forming units (CFU) of intracellular MTB in THP-1 cells were measured after 3 d (bottom). (C) Mice was infected by MTB through intranasal infection (1 × 103/per mice) and intranasally treated Cy5.5-labelled HK2 peptide (1 mg kg−1) after 3 wks. Lung harvests were used for analysis of the number of HK2 peptide+ cells by FACS. The data are representative of four independent experiments with similar results (A–C).
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