BACKGROUND: Binding and internalization of Pasteurella multocida dermonecrotic toxin (PMDT) by toxin-sensitive canine osteosarcoma and monkey kidney (vero) cells was examined ultrastructurally. EXPERIMENTAL DESIGN: Purified PMDT was conjugated to 20 nm colloidal gold particles in order to observe binding and internalization in the two cell lines at the ultrastructural level. The effects of various compounds on PMDT-vero binding were investigated to help elucidate the nature of putative vero cell receptors. RESULTS: Colloidal gold-labeled PMDT was located at cell surfaces within 1 minute of its addition and rapidly transported to coated and noncoated invaginations of the plasma membrane. After extended incubation, gold particles were observed in endocytic vesicles, but not in any other intracellular structures. The magnitude of gold-PMDT cell association correlated with the cytotoxic sensitivity of the two cell lines. Early, but not late, addition of the lysosomotropic agent methylamine protected vero cells from the cytotoxic effects of PMDT without affecting binding. Biochemical and ultrastructural inhibition studies suggested the requirement for a ganglioside-type vero cell receptor. CONCLUSIONS: This is the first report describing binding and internalization of PMDT in host cells. Biochemical and ultrastructural results suggest that PMDT interacts with a ganglioside-type receptor on vero cells and is transported to the cytosol in endocytic vesicles which do not appear to fuse with lysosomes.
BACKGROUND: Binding and internalization of Pasteurella multocida dermonecrotic toxin (PMDT) by toxin-sensitive canineosteosarcoma and monkey kidney (vero) cells was examined ultrastructurally. EXPERIMENTAL DESIGN: Purified PMDT was conjugated to 20 nm colloidal gold particles in order to observe binding and internalization in the two cell lines at the ultrastructural level. The effects of various compounds on PMDT-vero binding were investigated to help elucidate the nature of putative vero cell receptors. RESULTS: Colloidal gold-labeled PMDT was located at cell surfaces within 1 minute of its addition and rapidly transported to coated and noncoated invaginations of the plasma membrane. After extended incubation, gold particles were observed in endocytic vesicles, but not in any other intracellular structures. The magnitude of gold-PMDT cell association correlated with the cytotoxic sensitivity of the two cell lines. Early, but not late, addition of the lysosomotropic agent methylamine protected vero cells from the cytotoxic effects of PMDT without affecting binding. Biochemical and ultrastructural inhibition studies suggested the requirement for a ganglioside-type vero cell receptor. CONCLUSIONS: This is the first report describing binding and internalization of PMDT in host cells. Biochemical and ultrastructural results suggest that PMDT interacts with a ganglioside-type receptor on vero cells and is transported to the cytosol in endocytic vesicles which do not appear to fuse with lysosomes.
Authors: Michael C Brothers; Mengfei Ho; Ram Maharjan; Nathan C Clemons; Yuka Bannai; Mark A Waites; Melinda J Faulkner; Theresa B Kuhlenschmidt; Mark S Kuhlenschmidt; Steven R Blanke; Chad M Rienstra; Brenda A Wilson Journal: FEBS J Date: 2011-10-20 Impact factor: 5.542