Computational protocol: Phagosomal membrane targeting of Irgm1 via PtdIns(3,4)P2 and PtdIns(3,4,5)P3 promotes mycobacterial immunity

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Protocol publication

[…] For live imaging, 2 × 105 macrophages were nucleoporated, dispensed onto MatTek glass-bottom dishes and activated for 16-24 h with IFN-γ (100U.ml-1). rBGP-GFP or Cy5-BCG were pulse-chased for 40-60 min before recording at 37°C under oil (100x objective) on a Zeiss LSM510 confocal microscope in MetaDetector mode. Compressed Z-series were deconvolved using AutoQuant 3D (InVision) or ImageJ (NIH) software to yield 4D data over time. When applied, pseudocoloring used original pinhole, Airy disc and intensity gain settings. Static images were captured on 3% PFA-fixed samples. Cardiolipin was detected via a nonyl acridine orange stain . EM cryosections were processed as described and stained with A19 or anti-EGFP followed by either rabbit anti-goat or anti-mouse (Jackson Immunochemicals) and then Protein A-10nm gold. BSA-gold (15nm) tracer was pulse-chased into IFN-γ activated macrophage lysosomes overnight and unsonicated BCG (MOI 10:1) added for 60 min at 16°C. Mycobacterial uptake was initiated by shifting to 37°C to allow phagosomal trafficking for 6 h . [...] An Irgm1 homology model was generated in Modeller (9v1, 1) using the crystal structure of Iigp1 (Irga6) bound to Mg2+ and GNP (phosphoaminophosphonic acid guanylate ester; PDB code 1TQ2; 2) with a manually curated sequence alignment based on Probcons and 3DPSSM. The GROMOS96 force-field in SwissPDB Viewer (v3.7; http://www.expasy.org/spdbv/) was used for energy minimization. Structures were rendered using PyMol (www.pymol.org) . […]

Pipeline specifications

Software tools ProbCons, PyMOL
Application Amino acid sequence alignment
Organisms Mycobacterium tuberculosis
Diseases Infection, Tuberculosis
Chemicals Phosphatidylinositols, Potassium