Mannheimia haemolytica leukotoxin binds to lipid rafts in bovine lymphoblastoid cells and is internalized in a dynamin-2- and clathrin-dependent manner.
Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex. Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the beta(2)-integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demonstrated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3) death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly, expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation, reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internalization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the cytoskeletal network.
Department of Pathobiological Sciences, University of Wisconsin, 2015, Linden Drive, West, Madison, WI 53706, USA.
Dhammika N
DN
Atapattu
Charles J
CJ
Czuprynski
D000199:Actins / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism / Q000633:toxicity; D002417:Cattle; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005098:Exotoxins / Q000378:metabolism* / Q000633:toxicity; D020868:Gene Silencing; D016978:Mannheimia haemolytica / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics
D000199:Actins; D001426:Bacterial Proteins; D002966:Clathrin; D005098:Exotoxins; D034741:RNA, Small Interfering; C018579:leukotoxin; D034285:Dynamin II