The leukotoxin of Pasteurella haemolytica binds to β2 integrins on bovine leukocytes

Abstract

The putative receptor proteins of Pasteurella haemolytica leukotoxin were isolated from bovine polymorphonuclear neutrophil lysate by affinity chromatography on a leukotoxin-specific monoclonal antibody column to which the leukotoxin was pre-bound. SDS-PAGE of the purified proteins showed four bands at 180 kDa, 170 kDa, 150 kDa and 95 kDa, in addition to the expected 102-kDa leukotoxin band and a series of bands with molecular masses lower than 102 kDa representing the disintegrated leukotoxin. N-terminal amino acid sequencing of the 170-kDa band showed homology with human and murine CD11b. The purified proteins reacted specifically with monoclonal antibodies specific for CD11a, CD11b, CD11c (the K chains of L 2 integrins), and CD18 (the L chain of L 2 integrins). Pre-incubation of polymorphonuclear neutrophils with a monoclonal antibody specific for CD18 reduced the cytotoxicity of the leukotoxin to the cells. These results indicate that the leukotoxin binds to the L 2 integrins on bovine leukocytes, very likely via CD18. ß

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

“…The narrow target cell specificity of LktA effects had led to the hypothesis that it acts through a specific receptor in ruminant leukocytes. In this regard, previous studies using bovine leukocytes have identified β2 integrins as the receptors for LktA [1,12,21,32,52]. The β 2 integrins have a common β subunit, the CD18 that associates with four distinct α subunits, CD11a, CD11b, CD11c, and CD11d to give rise to four different β 2 integrins.…”

“…Studies from another laboratory [1] indicated that the CD18 subunit of all three β 2 integrins served as the receptor for LktA. However, data generated from our laboratory using Western blot analysis of proteins eluted from an affinity chromatographybased binding assay and blocking experiments with anti-β 2 integrin monoclonal antibodies, concluded that LktA binds to CD11a and CD18, but not to CD11b or CD11c subunits [21].…”

How Mannheimia haemolytica defeats host defence through a kiss of death mechanism

“…Indeed, inactivation of M. haemolytica leukotoxin by a gene knockout hardly causes any further pulmonary lesions although the wild-type and mutant strains were equally capable of colonising the upper respiratory tracts of the calves [165]. Moreover, necrosis of neutrophils can be reproduced in vitro with purified LKT [5,34,40,46,88,89,161,162,176]. Biologically, it is worth noting that even if LKT is able to bind leukocytes from various animal species, it is only cytotoxic for ruminant leukocytes, suggesting that the interaction specificity between LKT and ruminant leukocytes could be responsible for the ruminant-specificity of M. haemolytica [92,149,161].…”

“…These data suggest that the binding of LKT on ruminant LFA-1 is liable for the virulence specificity of M. haemolytica against ruminants. The precise identification of the subunit that binds LKT appears controversed [88], even if CD18 seems probable [5,46,109]. Fortunately, the recent cloning, sequencing and characterisation of the Bos taurus CD11a [56] will give the first opportunity to express homologous and heterologous LFA-1 in vitro to definitely answer the question.…”

Bighorn Sheep β2-Integrin LFA-1 Serves as a Receptor for Mannheimia haemolytica Leukotoxin

“…It induces the cytolysis of all subsets of leukocytes, of which polymorphonuclear leukocytes (PMNs) are the most susceptible subset (Silflow and Foreyt, 1994;Liu et al, 2007). In earlier studies, we, and others, have independently shown that the cytotoxic effect of Lkt on ruminant leukocytes is mediated by Lkt-b 2 -integrin interactions (Wang et al, 1998;Ambagala et al, 1999;Li et al, 1999;Jeyaseelan et al, 2000;Deshpande, 2002).…”