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

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 (C5PQLLLLAGLLA) 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.

Ruminant-specific retrotransposons shape regulatory evolution of bovine immunity

“…We investigated whether Bov-A2 elements are a source of modern variation by using MELT (Gardner et al 2017) to identify polymorphic mobile element insertions in a diverse panel of 38 cattle whole-genome sequences (Workman et al 2018;Tsuda et al 2013;Stothard et al 2015;Shin et al 2014;Bickhart et al 2016;Heaton et al 2016;Chen et al 2018). Across all individuals, our analysis revealed 1017 Bov-A2 elements with read-supported evidence for insertional polymorphism, including 740 non-reference insertions and 277 deletions relative to the reference Hereford bosTau9/ARS-UCD1.2 assembly (Fig.…”

Ruminant-specific retrotransposons shape regulatory evolution of bovine immunity

“…We investigated whether Bov-A2 elements are a source of modern variation by using MELT ( Gardner et al 2017 ) to identify polymorphic mobile element insertions in a diverse panel of 38 cattle whole-genome sequences ( Tsuda et al 2013 ; Shin et al 2014 ; Stothard et al 2015 ; Bickhart et al 2016 ; Heaton et al 2016 ; Chen et al 2018 ; Workman et al 2018 ). Across all individuals, our analysis revealed 1017 Bov-A2 elements with read-supported evidence for insertional polymorphism, including 740 nonreference insertions and 277 deletions relative to the reference Hereford bosTau9/ARS-UCD1.2 assembly ( Fig.…”

RTX Toxins of Animal Pathogens and Their Role as Antigens in Vaccines and Diagnostics

“…A recent study analyzing missense variants in the integrin β 2 gene (ITGB2) encoding CD18 of more than 1200 cattle from 50 different breeds revealed that missense variants in the CD18 signal peptide affect LktA binding. Consequently, animals carrying certain alleles can be more susceptible to the effects of LktA, and hence more prone to mannheimiosis [57]. These findings could be important to cattle breeding since M. haemolytica is spread widely in cattle herds.…”