title: Brief heat treatment increases cytotoxicity of Mannheimia haemolytica leukotoxin in an LFA-1 independent manner.
abstract: Mannheimia haemolytica is an important respiratory pathogen in cattle. Its predominant virulence factor is a leukotoxin (LKT) that is a member of the RTX family of exotoxins produced by a variety of Gram negative bacteria. LKT binds to the CD18 chain of beta(2) integrins on bovine leukocytes, resulting in cell death. In this study, we show that brief heat treatment of native LKT (95 degrees C for 3 min) results in increased cytotoxicity for BL-3 (bovine lymphoblastoid) cells. Similar heat treatment restored the activity of LKT that had been rendered inactive by incubation at 22 degrees C for 3 days. A hallmark of LKT is that its toxicity is restricted to leukocytes from cattle or other ruminant species. Surprisingly, heat treatment rendered LKT cytotoxic for human, porcine and canine leukocytes. Membrane binding studies suggested that heat-treated LKT binds to membrane proteins other than LFA-1, and is distributed diffusely along the BL-3 cell membrane. Circular Dichroism spectroscopy studies indicate that heat treatment induced a small change in the secondary structure of the LKT that was not reversed when the LKT was cooled to room temperature. Thus, we speculate that these structural changes might contribute to the altered biological properties of heat-treated LKT.
authors.affiliation: Department of Pathobiological Sciences, University of Wisconsin, 2015, Linden Drive, West, Madison, WI 53706, USA.
authors.forename: Dhammika N
authors.initials: DN
authors.lastname: Atapattu
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Aulik
authors.forename: Darrell R
authors.initials: DR
authors.lastname: McCaslin
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Czuprynski
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D002942:Circular Dichroism; D004285:Dogs; D005098:Exotoxins / Q000528:radiation effects* / Q000633:toxicity*; D006358:Hot Temperature; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D016978:Mannheimia haemolytica / Q000472:pathogenicity*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013552:Swine; D037521:Virulence Factors / Q000528:radiation effects* / Q000633:toxicity*
chemical_list: D005098:Exotoxins; D016169:Lymphocyte Function-Associated Antigen-1; D037521:Virulence Factors; C018579:leukotoxin