A bovine CD18 signal peptide variant with increased binding activity to Mannheimia hemolytica leukotoxin

Authors: Workman, Aspen; Chitko-Mckown, Carol; Smith, Timothy - Tim; Bennett, Gary; KALBFLEISCH, THEODORE - UNIVERSITY OF LOUISVILLE; BASNAYAKE, VERONICA - GENESEEK INC., A NEOGEN COMPANY; Heaton, Michael - Mike

Submitted to: F1000Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/2018
Publication Date: 12/28/2018
Citation: Workman, A.M., Chitko-McKown, C.G., Smith, T.P.L., Bennett, G.L., Kalbfleisch, T.S., Basnayake, V., Heaton, M.P. 2018. A bovine CD18 signal peptide variant with increased binding activity to Mannheimia hemolytica leukotoxin. F1000Research. 7:1985. https://doi.org/10.12688/f1000research.17187.1.
DOI: https://doi.org/10.12688/f1000research.17187.1

Interpretive Summary: Bovine respiratory disease complex (BRDC) is the most significant illness in U.S. cattle and a major source of economic loss, exceeding a billion dollars annually. BRDC is caused by a variety of infectious agents, including viruses and bacteria, that combine with environmental and management factors to cause disease. Outbreaks are enhanced by the increased stresses of weaning, transportation, comingling, changes in feed, and adverse weather. A common scenario for a BRDC outbreak is that bacteria invade the lungs after animals’ immune systems have been weakened by viral infection and environmental stress factors. During their invasion, rapidly multiplying bacteria produce a toxin that causes a severe and sometimes fatal pneumonia. The bacterial toxin binds to a receptor on white blood cells and kills them, causing rapid inflammation, bleeding, and fluid accumulation in the air sacs of the lungs. The bovine cellular receptor for this bacterial toxin was previously identified; however, we sought to identify genetic variation in the bovine gene that encodes the toxin receptor. Using a genome sequencing approach, 15 different versions of the bovine receptor were found, including versions that may have increased toxin binding activity. In laboratory tests with cattle blood cells, there was a 14-fold difference in toxin binding between different receptor variants. This suggests that cattle with variant receptors may be at increased risk for toxin-related respiratory disease. The discoveries reported here also provide new possibilities for disease intervention including: identifying high-risk animals through genetic testing, selectively breeding for less susceptible animals, and developing non-antibiotic treatments that may neutralize the bacterial toxin.

Technical Abstract: 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 (C5) instead of arginine (R5). Preincubating Lkt with synthetic signal peptides with C5 blocked cytotoxicity significantly better than those with R5. The most potent synthetic peptide (C5 PQLLLLAGLLA) had 30-fold more binding activity compared to that with R5. Conclusions: The results suggest that missense variants in the CD18 signal peptide affect Lkt binding, and animals carrying the C5 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.