Background: Mannheimia haemolytica is the major bacterial infectious agent of bovine respiratory disease complex and causes severe morbidity and mortality during lung infections. M. haemolytica secretes a protein leukotoxin (Lkt) that binds to the CD18 receptor on leukocytes, initiates lysis, induces inflammation, and causes acute fibrinous bronchopneumonia. Lkt binds the 22-amino acid CD18 signal peptide domain, which remains uncleaved in ruminant species. Our aim was to identify missense variation in the bovine CD18 signal peptide and measure the effects on Lkt binding. Methods: Missense variants in the integrin beta 2 gene ( ITGB2) encoding CD18 were identified by whole genome sequencing of 96 cattle from 19 breeds, and targeted Sanger sequencing of 1238 cattle from 46 breeds. The ability of different CD18 signal peptide variants to bind Lkt was evaluated by preincubating the toxin with synthetic peptides and applying the mixture to susceptible bovine cell cultures in cytotoxicity-blocking assays. Results: We identified 14 missense variants encoded on 15 predicted haplotypes, including a rare signal peptide variant with a cysteine at position 5 (C 5) instead of arginine (R 5). Preincubating Lkt with synthetic signal peptides with C 5 blocked cytotoxicity significantly better than those with R 5. The most potent synthetic peptide (C 5PQLLLLAGLLA) had 30-fold more binding activity compared to that with R 5. Conclusions: The results suggest that missense variants in the CD18 signal peptide affect Lkt binding, and animals carrying the C 5 allele may be more susceptible to the effects of Lkt. The results also identify a potent class of non-antibiotic Lkt inhibitors that could potentially protect cattle from cytotoxic effects during acute lung infections.
|State||Published - 2018|
Bibliographical noteFunding Information:
Funding for this research was provided by the USDA, ARS appropriated projects 5438-32000-029-00D (CGCM) and 5438-31320-012-00D (TPLS).
The authors thank Brad Sharp, Stacy Bierman, and Jacky Carnahan for technical support and Stephanie Schmidt for secretarial support. The use of product and company names is necessary to accurately report the methods and results; however, the United States Department of Agriculture (USDA) neither guarantees nor warrants the standard of the products. The use of names by the USDA implies no approval of the product to the exclusion of others that may also be suitable. The USDA is an equal opportunity provider and employer. Funding for this research was provided by the USDA, ARS appropriated projects 5438-32000-029-00D (CGCM) and 5438-31320-012-00D (TPLS).
Grant information: Funding for this research was provided by the USDA, ARS appropriated projects 5438-32000-029-00D (CGCM) and 5438-31320-012-00D (TPLS).
© 2018 Workman AM et al.
- Bacterial leukotoxin
- Bovine respiratory disease
- Integrin beta 2
- Mannheimia haemolytica
- Missense mutation
- Shipping fever
- Signal peptide variants
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (all)
- Immunology and Microbiology (all)
- Pharmacology, Toxicology and Pharmaceutics (all)