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Infect. Immun. doi:10.1128/IAI.00534-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Mannheimia haemolytica leukotoxin binds to lipid rafts in bovine lymphoblastoid cells (BL-3) and is internalized in a dynamin-2 and clathrin-dependent manner

Department of Pathobiological Sciences, University of Wisconsin-Madison

^* To whom correspondence should be addressed. Email: czuprync{at}svm.vetmed.wisc.edu.

	Abstract

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 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 cells (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 siRNA reduced both LKT internalization and cytotoxicity. Similarly, expression of a dominant negative Eps 15 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.