title: Precise gene editing paves the way for derivation of Mannheimia haemolytica leukotoxin-resistant cattle.
abstract: Signal peptides of membrane proteins are cleaved by signal peptidase once the nascent proteins reach the endoplasmic reticulum. Previously, we reported that, contrary to the paradigm, the signal peptide of ruminant CD18, the β subunit of β2integrins, is not cleaved and hence remains intact on mature CD18 molecules expressed on the surface of ruminant leukocytes. Leukotoxin secreted by Mannheimia (Pasteurella) haemolytica binds to the intact signal peptide and causes cytolysis of ruminant leukocytes, resulting in acute inflammation and lung tissue damage. We also demonstrated that site-directed mutagenesis leading to substitution of cleavage-inhibiting glutamine (Q), at amino acid position 5 upstream of the signal peptide cleavage site, with cleavage-inducing glycine (G) results in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of target cells. In this proof-of-principle study, we used precise gene editing to induce Q(‒5)G substitution in both alleles of CD18 in bovine fetal fibroblast cells. The gene-edited fibroblasts were used for somatic nuclear transfer and cloning to produce a bovine fetus homozygous for the Q(‒5)G substitution. The leukocyte population of this engineered ruminant expressed CD18 without the signal peptide. More importantly, these leukocytes were absolutely resistant to leukotoxin-induced cytolysis. This report demonstrates the feasibility of developing lines of cattle genetically resistant to M. haemolytica-caused pneumonia, which inflicts an economic loss of over $1 billion to the US cattle industry alone.
authors: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164.
authors: Sudarvili
authors: S
authors: Shanthalingam
authors: Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164.
authors: Ahmed
authors: A
authors: Tibary
authors: Department of Animal Sciences, University of Illinois, Urbana-Champaign, IL 61801.
authors: Jonathan E
authors: JE
authors: Beever
authors: TransOva-ViaGen, Sioux Falls, SD 57106.
authors: Poothapillai
authors: P
authors: Kasinathan
authors: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164.
authors: Wendy C
authors: WC
authors: Brown
authors: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164; ssrikumaran@vetmed.wsu.edu.
authors: Subramaniam
authors: S
authors: Srikumaran
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D018821:CD18 Antigens / Q000235:genetics* / Q000378:metabolism; D002417:Cattle / Q000235:genetics; D002460:Cell Line; D060467:Disease Resistance; D005098:Exotoxins / Q000633:toxicity*; D005333:Fetus / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D000072669:Gene Editing; D007962:Leukocytes / Q000378:metabolism; D008297:Male; D016978:Mannheimia haemolytica; D048089:Pneumonia of Calves, Enzootic / Q000517:prevention & control*
chemical_list: D018821:CD18 Antigens; D005098:Exotoxins; C018579:leukotoxin
keywords: Mannheimia haemolytica; cattle; cloning; leukotoxin-resistant