Acylation Enhances, but Is Not Required for, the Cytotoxic Activity of Mannheimia haemolytica Leukotoxin in Bighorn Sheep

Abstract

c Mannheimia haemolytica causes pneumonia in domestic and wild ruminants. Leukotoxin (Lkt) is the most important virulence factor of the bacterium. It is encoded within the four-gene lktCABD operon: lktA encodes the structural protoxin, and lktC encodes a trans-acylase that adds fatty acid chains to internal lysine residues in the protoxin, which is then secreted from the cell by a type 1 secretion system apparatus encoded by lktB and lktD. It has been reported that LktC-mediated acylation is necessary for the biological effects of the toxin. However, an LktC mutant that we developed previously was only partially attenuated in its virulence for cattle. The objective of this study was to elucidate the role of LktC-mediated acylation in Lkt-induced cytotoxicity. We performed this study in bighorn sheep (Ovis canadensis) (BHS), since they are highly susceptible to M. haemolytica infection. The LktC mutant caused fatal pneumonia in 40% of inoculated BHS. On necropsy, a large number of necrotic polymorphonuclear leukocytes (PMNs) were observed in the lungs. Lkt from the mutant was cytotoxic to BHS PMNs in an in vitro cytotoxicity assay. Flow cytometric analysis of mutant Lkt-treated PMNs revealed the induction of necrosis. Scanning electron microscopic analysis revealed the presence of pores and blebs on mutant-Lkt-treated PMNs. Mass spectrometric analysis confirmed that the mutant secreted an unacylated Lkt. Taken together, these results suggest that acylation is not necessary for the cytotoxic activity of M. haemolytica Lkt but that it enhances the potency of the toxin. Mannheimia haemolytica is a respiratory pathogen of domestic and wild ruminants (1-3). It is the most important bacterial pathogen of bovine respiratory disease complex, which costs the U.S. cattle industry alone more than $1 billion (4). M. haemolytica is also an important pathogen of pneumonia in bighorn sheep (Ovis canadensis) (BHS), which is the primary disease responsible for the drastic decline of BHS populations in North America from an estimated 2 million animals in the 1800s to less than 70,000 at the present time (5). Under the experimental conditions, M. haemolytica consistently caused 100% mortality in BHS within 2 to 3 days (6-8). The bacterium possesses several virulence factors, including the capsule, outer membrane proteins, lipopolysaccharide, and leukotoxin (Lkt). Based on the fact that Lkt deletion mutants do not cause mortality (6) or cause reduced mortality and milder lung lesions (9, 10), Lkt has been accepted as the primary virulence factor of M. haemolytica. The 104-kDa Lkt is absolutely specific for ruminant leukocytes (11). Previously, we have shown that the molecular basis for the ruminant specificity of Lkt rests in its binding to the signal peptide of CD18; the signal peptide remains intact in mature CD18 molecules on ruminant leukocytes, unlike that of nonruminants, which is cleaved (12). Although all ruminant leukocyte subsets are susceptible to Lkt-induced cytolysis, polymorphonuclear leukocytes (PMNs) are the most su...

RTX Toxins Ambush Immunity’s First Cellular Responders

“…Several groups have examined the post-translational modification of one or two lysine residues by the "C" gene for each RTX toxin, and determined that this modification is required for full lytic activity (LtxA [75], LktA [76], CyaA [77,78], HlyA [14,15,79]), with two notable exceptions reported. Pro-LktA is cytotoxic to PMNs from Bighorn Sheep, at 128-fold lower levels than mature LktA [80]. A few reports exist of pro-CyaA binding to cells and inducing the same intracellular effects as seen for mature CyaA, i.e., transient cAMP increase, ATP decrease, and annexin V labeling, albeit at 100-1000-fold higher concentrations than the mature form of the toxin [59,73,81].…”