title: Recombinant expression of bovine LFA-1 and characterization of its role as a receptor for Mannheimia haemolytica leukotoxin.
abstract: Mannheimia (Pasteurella) haemolytica leukotoxin (LktA) is the primary virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM), a disease which causes major economic loss to the US cattle industry annually. Recent work from our laboratory using an antibody-based approach has shown that LktA binds to bovine LFA-1 in target cells. While this study suggests that LFA-1 might be a specific receptor, it remains to be conclusively shown that LFA-1 is sufficient to induce susceptibility to LktA. It was of interest to determine if functionally active bovine LFA-1 could be reconstituted on a LFA-1 negative cell line and reconstitute susceptibility to LktA. Here, we report the successful recombinant expression of bovine LFA-1 on the cell surface of the human erythroleukemic K562 cell line. The BoLFA-1 transductant expresses bovine CD18 and CD11a as a heterodimer. We found that LktA binds to both the CD18 and CD11a subunits of BoLFA-1 cells. Exposure of BoLFA-1 cells to LktA, induced tyrosine phosphorylation of the CD18 tail, elevation of intracellular calcium, and cytolysis. This is the first report on recombinant expression of functionally active bovine LFA-1 by transduction into an LktA-non-susceptible human cell line.
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA.
authors.forename: Thamotharampillai
authors.initials: T
authors.lastname: Dileepan
authors.forename: Praveen
authors.initials: P
authors.lastname: Thumbikat
authors.forename: Bruce
authors.initials: B
authors.lastname: Walcheck
authors.forename: Mathur S
authors.initials: MS
authors.lastname: Kannan
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Maheswaran
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism; D040881:CD11a Antigen / Q000235:genetics / Q000378:metabolism; D018821:CD18 Antigens / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000032:analysis; D002417:Cattle; D016923:Cell Death; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry; D005098:Exotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D016169:Lymphocyte Function-Associated Antigen-1 / Q000235:genetics / Q000502:physiology*; D016978:Mannheimia haemolytica; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D037521:Virulence Factors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D040881:CD11a Antigen; D018821:CD18 Antigens; D005098:Exotoxins; D006460:Hemolysin Proteins; D016169:Lymphocyte Function-Associated Antigen-1; D011994:Recombinant Proteins; D037521:Virulence Factors; C102677:lktA protein, bacteria; D002118:Calcium