Mapping of the binding site for Mannheimia haemolytica leukotoxin within bovine CD18.
ABSTRACT: To map the site involved in Mannheimia haemolytica leukotoxin (LktA) binding and biological activity within bovine CD18, bovine x human CD18 chimeric constructs were generated and coexpressed with bovine CD11a in K562 cells. Studies with the chimeric leukocyte function-associated antigen 1 transductants demonstrate that the site required for LktA binding and biological effects resides within amino acid residues 500 and 600 of the extracellular region of bovine CD18.
Project description:Previously, we identified bovine CD18 as the receptor for leukotoxin secreted by Mannheimia (Pasteurella) haemolytica. In this study, we constructed bovine-murine CD18 chimeras to locate the leukotoxin binding domain on CD18. Leukotoxin specifically lysed transfectants expressing bovine CD18 fragment encompassing amino acids 1 to 291, indicating that leukotoxin binding domain lies within amino acids 1 to 291 of bovine CD18.
Project description:Mannheimia haemolytica is a bacterial pathogen that secretes leukotoxin (LktA) which binds to leukocyte membranes via CD18, causing bacterial pneumonia in ruminants. We report the complete closed genome sequences of a leukotoxin mutant and its parent strain that are frequently used in respiratory disease studies.
Project description:The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394-1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M. haemolytica. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M. glucosida and P. trehalosi. In contrast, the lktD gene is highly conserved in M. haemolytica. The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M. haemolytica. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5' and 3' ends of the operon are highly conserved in M. haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.
Project description:Signal peptides of membrane proteins are cleaved by endoplasmic reticulum-resident signal peptidase, and thus, are not present on mature membrane proteins. Here, we report that, contrary to the paradigm, the signal peptide of ruminant CD18, the beta-subunit of beta(2)-integrins, is not cleaved. Intriguingly, the intact signal peptide of CD18 is responsible for the susceptibility of ruminant leukocytes to Mannheimia (Pasteurella) haemolytica leukotoxin (Lkt). Inhibition of Lkt-induced cytolysis of ruminant leukocytes by CD18 peptide analogs revealed that the Lkt-binding site is formed by amino acids 5-17 of CD18, which, surprisingly, comprise most of the signal sequence. Flow cytometric analysis of ruminant leukocytes indicated the presence of the signal peptide on mature CD18 molecules expressed on the cell surface. Analysis of transfectants expressing CD18 containing the FLAG epitope at the putative cleavage site confirmed that the signal peptide of bovine CD18 is not cleaved. Analysis of the signal sequence of CD18 of eight ruminants and five nonruminants revealed that the signal sequence of CD18 of ruminants contains "cleavage-inhibiting" Q, whereas that of nonruminants contains "cleavage-conducive" G at position -5 relative to the cleavage site. Site-directed mutagenesis of Q to G at position -5 of the signal peptide of bovine CD18 resulted in the cleavage of the signal peptide and abrogation of cytolysis of transfectants expressing bovine CD18 carrying the Q(-5)G mutation. We propose that engineering cattle and other ruminants to contain this mutation would provide a novel technology to render them less susceptible to pneumonic pasteurellosis and concomitant economic losses.
Project description:The molecular evolution of the leukotoxin structural gene (lktA) of Mannheimia (Pasteurella) haemolytica was investigated by nucleotide sequence comparison of lktA in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in M. haemolytica. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the lktA gene of the two related species Mannheimia glucosida and Pasteurella trehalosi, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess lktA alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of lktA in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.
Project description:Background: Mannheimia haemolytica is the major bacterial infectious agent of bovine respiratory disease complex and causes severe morbidity and mortality during lung infections. M. haemolytica secretes a protein leukotoxin (Lkt) that binds to the CD18 receptor on leukocytes, initiates lysis, induces inflammation, and causes acute fibrinous bronchopneumonia. Lkt binds the 22-amino acid CD18 signal peptide domain, which remains uncleaved in ruminant species. Our aim was to identify missense variation in the bovine CD18 signal peptide and measure the effects on Lkt binding. Methods: Missense variants in the integrin beta 2 gene ( ITGB2) encoding CD18 were identified by whole genome sequencing of 96 cattle from 19 breeds, and targeted Sanger sequencing of 1238 cattle from 46 breeds. The ability of different CD18 signal peptide variants to bind Lkt was evaluated by preincubating the toxin with synthetic peptides and applying the mixture to susceptible bovine cell cultures in cytotoxicity-blocking assays. Results: We identified 14 missense variants encoded on 15 predicted haplotypes, including a rare signal peptide variant with a cysteine at position 5 (C 5) instead of arginine (R 5). Preincubating Lkt with synthetic signal peptides with C 5 blocked cytotoxicity significantly better than those with R 5. The most potent synthetic peptide (C 5PQLLLLAGLLA) had 30-fold more binding activity compared to that with R 5. Conclusions: The results suggest that missense variants in the CD18 signal peptide affect Lkt binding, and animals carrying the C 5 allele may be more susceptible to the effects of Lkt. The results also identify a potent class of non-antibiotic Lkt inhibitors that could potentially protect cattle from cytotoxic effects during acute lung infections.
Project description:The Pasteurella haemolytica leukotoxin gene cluster (lktCABD) is homologous to the Escherichia coli hemolysin locus (hlyCABD). Since the cloned leukotoxin (LktA) is not secreted from E. coli cells, a heteroplasmid complementation system was developed that permits secretion of the leukotoxin from cells expressing the hemolysin transport proteins HlyB and HlyD. We observed that the secreted leukotoxin protein had weak hemolytic activity when activated by either the HlyC or LktC proteins and that LktC expressed in E. coli could confer weak hemolytic activity upon hemolysin. Thus, it appears that the accessory proteins of the leukotoxin and hemolysin gene clusters are functionally similar, although their expression in E. coli is not equivalent. Northern (RNA) blot analysis of the P. haemolytica leukotoxin gene cluster revealed a major 3.5-kilobase transcript that includes the lktC and lktA genes. The start site for this transcript mapped to a cytosine residue 30 nucleotides upstream from the putative start of lktC; a similar initiation site was observed in E. coli, although adjacent cytosine and adenine residues were also utilized. The 3.5-kilobase transcript terminated near the rho-independent terminator structure between lktA and lktB, but transcription may continue, via antitermination or de novo transcription initiation, into the downstream lktB and lktD genes. We propose that the lack of LktB and LktD function in E. coli is a result, at least in part, of poor lktBD transcription and suggest that a P. haemolytica-specific regulator is required for optimal expression of the leukotoxin genes.
Project description:Mannheimia (Pasteurella) haemolytica A1 produces several virulence factors that play an important role in the pathogenesis of bovine pneumonic pasteurellosis. Foremost among these is a leukotoxin (LKT) that specifically kills ruminant leukocytes. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the beta(2)-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 [LFA-1]), which subsequently induces activation and cytolysis of these cells. Inflammatory cytokines, which are released during viral and bacterial infection, are reported to increase LFA-1 expression and conformational activation. We investigated the effects of the inflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) on the interaction of M. haemolytica LKT with bovine peripheral blood neutrophils (PMNs). In this study we demonstrated, by flow cytometry, that bovine PMNs increased their binding to an anti-bovine LFA-1 monoclonal antibody (BAT75A) following in vitro incubation with IL-1beta, TNF-alpha, or IFN-gamma. Incubation with cytokines also increased CD18 expression, as assessed by real-time PCR and by Western blotting. Increased LFA-1 expression by PMNs exposed to cytokines was associated with increased LKT binding and cytotoxicity. The latter represented, at least in part, enhanced PMN apoptosis, as assessed by propidium iodine staining and caspase-3 activation. The results of this study suggest that inflammatory cytokines may play an important role in enhancing the biological response of bovine PMNs to M. haemolytica LKT.
Project description:All sixteen serotypes of Pasteurella haemolytica were shown to produce a leukotoxin protein which is immunologically related to the well-characterized serotype 1 leukotoxin. All of the leukotoxins were weakly hemolytic and were able to bind to BL-3 target cells. The leukotoxin determinants were characterized by Southern blot hybridization by use of the previously cloned serotype 1 determinant as the probe, and a number of distinct classes were identified. The leukotoxin determinants from serotypes 2, 3, and 11 were cloned. Nucleotide sequence analysis of the lktC and lktA genes of the serotype 3 and 11 determinants revealed nucleotide substitutions throughout the coding sequences. A comparison of the lktC and lktA genes and deduced proteins of serotypes 1, 3, and 11 showed that they are highly homologous.
Project description:Exotoxins which belong to the family containing the RTX toxins (repeats in toxin) contribute to a variety of important human and animal diseases. One example of such a toxin is the potent leukotoxin (LKT) produced by the bovine respiratory pathogen Mannheimia haemolytica. LKT binds to CD18, resulting in the death of bovine leukocytes. In this study, we showed that internalized LKT binds to the outer mitochondrial membrane, which results in the release of cytochrome c and collapse of the mitochondrial membrane potential (psi(m)). Incubation of bovine lymphoblastoid cells (BL-3 cells) with the mitochondrial membrane-stabilizing agent cyclosporine (CSA) reduced LKT-mediated cytotoxicity, cytochrome c release, and collapse of the psi(m). Coimmunoprecipitation and intracellular binding studies suggested that LKT binds to the mitochondrial matrix protein cyclophilin D. We also demonstrated that LKT mobilizes the vesicle scission protein dynamin-2 from mitochondria to the cell membrane. Incubation with CSA depleted mitochondrial dynamin-2 in BL-3 cells, making it unavailable for vesicle scission and LKT internalization. The results of this study show that LKT trafficking and LKT-mediated cell death involve dynamin-2 and cyclophilin D, in a process that can be prevented by the mitochondrial membrane-protecting function of CSA.