Biological effects of two genetically defined leukotoxin mutants of Mannheimia haemolytica

Mannheimia(Pasteurella)haemolytica serotype 1 is the primary causative agent responsible for bovine pneumonic mannheimiosis, also known as shipping fever in cattle. The bacterium produces a variety of virulence factors, foremost of which is the exotoxic leukotoxin. The leukotoxin is a calcium-dependent cytolysin that is a member of the RTX (repeats in toxin) family and exhibits a narrow cell-type and species specificity and has biological effects only on ruminant leukocytes and platelets. The genetic organization of the leukotoxin is comprised of four genes: lktC, lktA, lktB and lktD. The lktA structural gene encodes the protoxin (pro-LktA) and lktC encodes a transacylase that post-translationally modifies the inactive pro-LktA to a biologically active wild-type leukotoxin (LktA). The LktA has been implicated as the key factor that contributes to the pathogenesis of lung injury associated with the disease and considerable efforts have been employed in abrogating toxin function while retaining immunogenicity, with an eye towards design of attenuated vaccines. We hypothesized that the pro-LktA retains the ability to cause biological effects on target cells as has been reported in the case of the closely related RTX toxin α-hemolysin (HlyA). We also examined the biological effects of an amino-terminal truncation mutant leukotoxin ΔLktA on target cells. Thus the objectives of our study were to investigate whether two different mutant leukotoxins, one a nonacylated pro-LktA, and the other lacking 344 amino acids at the N-terminal end of the LktA protein; ΔLktA, are capable of (i) binding to the β2-integrin leukotoxin receptor, (ii) inducing the elevation of second messenger intracellular calcium ([Ca²⁺]_i), and (iii) inducing inflammatory gene expression, reactive oxygen metabolites (ROMs) and cytolysis in target cells. Our results demonstrate that neither acylation nor the amino terminal 344 amino acids are required for LktA binding but are essential for LktA-induced [Ca²⁺]_i elevation, generation of ROM, generation of the inflammatory cytokine IL-8 and cytolysis in target cells.