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Infection and Immunity, October 2007, p. 4719-4727, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00534-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and Clathrin-Dependent Manner{triangledown}

Dhammika N. Atapattu and Charles J. Czuprynski*

Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin

Received 13 April 2007/ Returned for modification 15 June 2007/ Accepted 24 July 2007

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 ß2-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.

* Corresponding author. Mailing address: Department of Pathobiological Sciences, University of Wisconsin, 2015, Linden Drive, West, Madison, WI 53706. Phone and fax: (608) 262-8102. E-mail: czuprync{at}svm.vetmed.wisc.edu

{triangledown} Published ahead of print on 6 August 2007.

Editor: D. L. Burns

Infection and Immunity, October 2007, p. 4719-4727, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00534-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Atapattu, D. N., Albrecht, R. M., McClenahan, D. J., Czuprynski, C. J. (2008). Dynamin-2-Dependent Targeting of Mannheimia haemolytica Leukotoxin to Mitochondrial Cyclophilin D in Bovine Lymphoblastoid Cells. Infect. Immun. 76: 5357-5365 [Abstract] [Full Text]  


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