Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage β2 integrins

Abstract

-Mannheimia (Pasteurella) haemolytica, the etiologic agent of bovine pneumonic mannheimiosis, produces an exotoxic leukotoxin. The leukotoxin (LktA) is a member of the RTX (repeats in toxin) family of bacterial cytolysins and is distinguished from other toxins by its unique target cell specificity to ruminant leukocytes occurring through binding to a specific receptor. We have demonstrated previously that the β 2 integrin LFA-1 is a receptor for LktA in bovine leukocytes and is involved in leukotoxin-induced biological effects. However the subunits within LFA-1 involved in binding to LktA, and post-binding signaling leading to cellular activation have not been well characterized. The purpose of our study was to pinpoint these precise subunits on bovine alveolar macrophages and to characterize their interaction with LktA. The results in this study indicate that although LktA can efficiently bind to the CD18 subunit of both LFA-1 and Mac-1, post-binding signaling events including elevation of intracellular calcium and CD18 tail phosphorylation are only observed through LFA-1. Furthermore, LktA also binds to the CD11a subunit of LFA-1. LktA binding to CD11a could be inhibited by a small molecule inhibitor of the I(inserted)-domain, the major ligand binding interface on CD11a. I-domain inhibition significantly blunts LktA-induced intracellular calcium elevation and tyrosine phosphorylation of the CD18 tail. Based on our results we suggest that LFA-1 serves as the functional leukotoxin receptor on bovine alveolar macrophages.Mannheimia haemolytica leukotoxin / receptor / lymphocyte function associated antigen-1 / signaling

Monomeric Expression of Bovine β2-Integrin Subunits Reveals Their Role inMannheimia haemolyticaLeukotoxin-Induced Biological Effects

“…This was not an unexpected observation since our earlier studies have shown that Lkt-LFA-1 binding is inhibited by anti-CD11a or anti-CD18 MAbs (17). We have also demonstrated Lkt-CD11a and Lkt-CD18 binding by affinity chromatography-based binding assays with bovine alveolar macrophages and LFA-1 transductants (10,38). Therefore, it is very likely that at least one or perhaps more Lkt-binding sites are present in the bovine CD11a and CD18 subunits.…”

“…Previous studies by us and others confirmed ␤ 2 -integrins as the receptors for Lkt on the leukocytes of cattle, bighorn sheep, and domestic sheep (1,10,17,23,24,38,40). In further studies in our laboratory, the recombinant expression of CD18 from cattle or domestic sheep or bighorn sheep rendered a Lkt-nonsusceptible murine cell line susceptible to Lkt-induced cytolysis (7,8,26), prompting us to propose that CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.…”

“…In related studies, Lkt also bound to the CD11a subunit of LFA-1 (17). In addition, antibodies specific for CD18 or CD11a were equally effective in inhibiting signaling events, leading to the elevation of intracellular [Ca 2ϩ ], tyrosine phosphorylation of the cytosolic domain of CD18, and cytolysis of bovine leukocytes (10,18,38). These discordant findings underscore the need for additional studies to identify the precise functional receptor to which Lkt binds and initiates signaling events leading to activation and cytolysis of bovine leukocytes.…”

RTX cytotoxins recognize β 2 integrin receptors through N-linked oligosaccharides

“…LktA was shown initially to bind most ␤ 2 integrins (CD11a/CD18, CD11b/CD18, and CD11c/CD18), very likely through their CD18 subunit (14,15). Later reports, however, have shown that only CD11a/CD18 is the receptor for LktA and is involved in leukotoxin-induced biological effects (20,21).…”

Dynamin-2-Dependent Targeting ofMannheimia haemolyticaLeukotoxin to Mitochondrial Cyclophilin D in Bovine Lymphoblastoid Cells

“…It was originally thought that M. haemolytica LKT damages cells by inserting into the cell membrane, resulting in pore formation and necrosis (53). At lower LKT concentrations, however, susceptible cells die via caspase-dependent apoptotic pathways (8,10,45,46). LKT was first reported to bind to the ␤ 2 integrin CD18/CD11a, also known as leukocyte functional antigen 1 (LFA-1) (1,24,28).…”