Dissertation
Mannheimia haemolytica leukotoxin: host cell receptor interactions
Washington State University
Doctor of Philosophy (PhD), Washington State University
05/2010
DOI:
https://doi.org/10.7273/000005612
Abstract
Mannheimia haemolytica is the primary bacterial pathogen of bovine pneumonic pasteurellosis, an economically important disease of cattle worldwide. Leukotoxin (Lkt) produced by M. haemolytica is the major virulence factor of this organism. The cytolytic activity of Lkt is specific for ruminant leukocytes. Lkt utilizes CD18, the [beta] subunit of [beta]2-integrins, as its receptor on ruminant leukocytes. Previously, our laboratory mapped the Lkt-binding domain to lie between amino acids (aa) 1-291 of CD18. Therefore, the next logical step was to identify the precise Lkt binding site within this domain and to determine whether co-administration of CD18 peptide analogs would inhibit / mitigate M. haemolytica-caused lung injury. In this study, by using synthetic peptides spanning aa1-291 of bovine CD18 in Lkt-induced cytolysis assays, the precise binding site of Lkt was mapped to aa 5-17 of ruminant CD18. Surprisingly, all the aa of this peptide belong to the predicted signal peptide of CD18. This observation led to the finding that the signal peptide of ruminant CD18 is not cleaved, and that the intact signal peptide renders ruminants susceptible to M. haemolytica Lkt. Site-directed mutagenesis of a single aa in the signal peptide resulted in the cleavage of signal peptide and abrogation of Lkt-induced cytolysis of target cells. This finding indicates that engineering cattle and other ruminants to contain this mutation would provide a novel technology to render them less susceptible to pneumonic pasteurellosis and concomitant economic losses. The peptide spanning aa 5-17 (P17) was used in a calf challenge study which was designed as a "proof of concept" experiment. Even though the difference in percent volume of lungs exhibiting gross pneumonic lesions between P17-inoculated calves and control peptide-inoculated calves was not statistically significant, M. haemolytica isolated from the lungs of P17-inoculated calves was 100- to 1000-fold less than those from the control peptide-inoculated calves, suggesting that P17 reduced leukotoxic activity in the lungs which enhanced bacterial clearance by phagocytes. It is likely that prolonging the presence and activity of CD18 peptide analog in the lungs, for example by means of a nanoparticle delivery system such as dextran nanospheres, would enhance its protective activity.
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Details
- Title
- Mannheimia haemolytica leukotoxin
- Creators
- Sudarvili Shanthalingam
- Contributors
- Subramaniam Srikumaran (Chair)Thomas Eugene Besser (Committee Member) - Washington State University, Department of Veterinary Microbiology and PathologyWendy C Brown (Committee Member)Douglas Ruben Call (Committee Member) - Washington State University, Paul G. Allen School for Global Animal HealthTerry F McElwain (Committee Member) - Washington State University, Paul G. Allen School for Global Animal Health
- Awarding Institution
- Washington State University
- Academic Unit
- College of Veterinary Medicine
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 114
- Identifiers
- 99901054738801842
- Language
- English
- Resource Type
- Dissertation