Activation of bovine neutrophils by partially purified Pasteurella haemolytica leukotoxin

Abstract

In this study we developed a new method for the partial purification of Pasteurella haemolytica leukotoxin by size-exclusion high-performance liquid chromatography. The partially purified leukotoxin had a molecular weight of 104,000, as estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reacted on an immunoblot with an antileukotoxin monoclonal antibody. As expected, high concentrations of the leukotoxin were inhibitory or lethal to bovine neutrophils. Incubation of bovine neutrophils with diluted leukotoxin, however, resulted in significant neutrophil activation that was comparable in magnitude to that obtained with standard activating agents such as opsonized zymosan or zymosan-activated serum. Dilute leukotoxin (1:128 to 1:8,192 dilutions) stimulated an oxidative burst (luminol-dependent chemiluminescence) by bovine neutrophils that was comparable in magnitude to that obtained with opsonized zymosan. Preincubation with leukotoxin did not significantly prime the neutrophils for an enhanced oxidative burst when they were then exposed to opsonized zymosan as a second stimulus. Dilute leukotoxin (1:100 to 1:1,000 dilutions) also stimulated cytoskeletal alterations in bovine neutrophils, as measured by a significant shape change response. Preferential release of secondary granule constituents (lactoferrin) occurred when neutrophils were incubated with 1:100 to 1:500 dilutions of leukotoxin. Significant release of primary granules, as measured by I-glucosaminidase activity, was not observed except at low dilutions (1:20) of leukotoxin that resulted in significant release of cytosolic constituents (i.e., lactate dehydrogenase activity). The neutrophil-activating activity of the leukotoxin was heat labile, unaffected by polymyxin B, and abrogated by a leukotoxinneutralizing monoclonal antibody. These data indicate that P. haemolytica leukotoxin, like the closely related Escherichia coli hemolysin, is a potent neutrophil-activating agent. Leukotoxin-stimulated release of neutrophil oxygen intermediates and granule constituents may contribute to the intense inflammation that characterizes bovine pulmonary pasteurellosis. * Corresponding author. trophil oxidative activity, degranulation, and shape change. Our results indicate that the P. haemolytica leukotoxin, like the E. coli hemolysin, is a potent neutrophil-modulating agent.

Polymorphonuclear leukocyte degranulation induced by leukotoxin from Actinobacillus actinomycetemcomitans

“…Occupancy of integrin receptors by ligands such as matrix proteins can activate signaling systems in neutrophils (15). RTX toxins from E. coli and Pasteurella hemolytica can induce certain biological activities in PMNL, such as production of cytokines, leukotrienes and superoxide, cytoskeleton re-arrangement and receptor shedding, beside being cytotoxic for the target cells (7,16,17). These eects are probably the result of an intracellular signal transduction concomitant to interactions between receptors and RTX toxins that act as their ligands.…”

Occurrence of [copper, zinc]-cofactored Superoxide dismutase in Pasteurella haemolytica and its serotype distribution

“…The leukotoxin of P. haemolytica Al, implicated in the pathogenesis of bovine pasteurellosis. is a potent neutrophil activator [2,3], and similarly neutrophil activation is a feature of ovine pneumonic pasteurellosis caused by a range of P. haemolytica serotypes. This activation of neutrophils is attended by a burst of production of superoxide and more toxic free-radical products of its further reaction with hydrogen peroxide, but factors in pasteurella strains conferring resistance to their bactericidal action remain to be characterized.…”

“…Leukotoxin production has been shown to lead to activation of lung neutrophils [2], resulting in generation of an oxidative burst, production of oxygen-free radicals [3], and consequent host tissue damage. While the damage caused to host cells may facilitate bacterial survival, the products of the oxidative burst are potentially highly detrimental to EA.…”

N-terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells

“…At high concentrations, M. haemolytica LktA creates a pore in the cell membrane leading to cellular swelling and lysis (Shewen and Wilkie, 1982;Clinkenbeard et al, 1989;Cudd et al, 2001). At sublytic concentrations, target cells are activated or stimulated to undergo apoptosis (Czuprynski et al, 1991;Maheswaran et al, 1992). Previous studies in our laboratory have shown that LktA induces apoptosis in bovine lymphoblastoid (BL-3) cells via a caspase-9-dependent pathway (Atapattu and Czuprynski, 2005).…”

“…One of the major virulence factors of M. haemolytica that contributes to the pathogenesis of bovine respiratory disease is a leukotoxin (LktA). LktA both activates and kills bovine leukocytes (Shewen and Wilkie, 1982;Clinkenbeard et al, 1989;Czuprynski et al, 1991;Maheswaran et al, 1992;Cudd et al, 2001).…”