Commonality of Virulence-Promoting Function in Rhodococcus equi Virulence Associated Proteins (Vaps)

Figure 7

V-ATPase recruitment and activity to VapA-positive compartments is unaffected by VapA. (a) Top panels: immunofluorescence of macrophages fed -/+ VapA and immunolabelled for lysosomal-associated membrane protein-1 (LAMP-1) (cyan) or ATP6 subunits (magenta). Bottom panels: immunofluorescence of macrophages infected with 103- and 103+ R. equi and immunolabelled for ATP6 subunits (magenta) and DNA stained with DAPI (cyan). Scale . Arrows indicate regions used for magnification in the insets. (b). Western blotting postnuclear supernatants (PNS) and isolated lysosomes after incubation +/- VapA-His for LAMP1, His₆-tag, and ATP6 subunits. (c). Measurement of ATPase activity in isolated lysosomes (as in (b)) ± luminal VapA using an ATP/NADH coupled-enzyme assay (schematic shown). ATPase activity specific to the V-ATPase is inhibited by concanamycin-A (ConA). Data are mean ().

(a)

(b)

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