A bovine CD18 signal peptide variant with increased binding activity to Mannheimia hemolytica leukotoxin.
ABSTRACT: 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: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: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: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.
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: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:<i>Pasteurella</i> (<i>P</i>.) <i>multocida</i> and <i>Mannheimia</i> (<i>M.</i>) <i>haemolytica</i> are the most two common pathogenic bacterial agents causing pneumonia in calves. Both bacteria are associated with significant economic losses in the cattle industry due to high morbidity and mortality rates, especially in the case of severe infections. The objectives of the present study were to perform serotyping and genotyping, as well as characterization of the virulence-associated genes in 48 bacterial isolates; 33 <i>P. multocida</i> and 15 <i>M. haemolytica</i>. All strains were isolated from pneumonic cattle calves showing respiratory manifestations such as fever, nasal discharges, and rapid breathing in North Upper Egypt governorates (Beni-Suef and El-Fayoum). PCR was applied as a confirmatory test using a specific universal gene, <i>kmt</i>1, and <i>rpt</i>2 for <i>P. multocida</i> and <i>M. haemolytica</i>, respectively. The results show that 29 (87.9%) <i>P. multocida</i> and 15 (100%) <i>M. haemolytica</i> isolates were positive for the corresponding universal gene. The results of serotyping indicate that 86.2% of <i>P. multocida</i> isolates belonged to serotype B:2, while 13.8% were untyped. Meanwhile, 60% and 40% of <i>M. haemolytica</i> isolates belonged to serotype 2 and serotype 1, respectively. Investigation of virulence-associated genes showed that all the tested <i>P. multocida</i> isolates harbored <i>nan</i>B, <i>omp</i>87, and <i>tox</i>A genes. Four <i>M. haemolytica</i> isolates harbored both <i>gcp</i> and <i>lkt</i>C genes and of these, three isolates harbored the <i>ssa</i> gene. Sequencing of <i>tox</i>A gene of <i>P. multocida</i> and <i>lkt</i>C gene of <i>M. haemolytica</i> in the current strains indicated a great homology with strains uploaded in gene banks from different hosts and localities worldwide.
Project description:Mannheimia haemolytica, a commensal organism of the upper respiratory tract in cattle, is the principal bacterial pathogen associated with the bovine respiratory disease complex. Adherence to the respiratory mucosa is a crucial event in its pathogenesis. However, the bacterial components that contribute to this process are not fully characterized. In this study, we demonstrated that M. haemolytica adhered to bovine bronchial epithelial cells (BBEC) in vitro and that adherence was inhibited by anti-M. haemolytica antibody. Western blot analysis of M. haemolytica proteins that bind to BBEC showed a dominant protein band with an apparent molecular mass of approximately 30 kDa. Peptide sequences for the 30-kDa BBEC-binding proteins, as determined by liquid chromatography-tandem mass spectrometry, matched two M. haemolytica surface proteins: heat-modifiable outer membrane protein A (OmpA) and lipoprotein 1 (Lpp1). Western blotting showed that the 30-kDa protein band is recognized by both anti-M. haemolytica OmpA and anti-Lpp1 antibodies. Furthermore, incubation with anti-OmpA and anti-Lpp1 antibodies significantly inhibited M. haemolytica binding to BBEC monolayers. In summary, these results suggest that OmpA and Lpp1 contribute to adherence of M. haemolytica to bovine respiratory epithelial cells.
Project description:Mannheimia haemolytica is the most important bacterial agent associated with the bovine respiratory disease complex (BRDC), which causes worldwide economic losses to the cattle industry. M. haemolytica cells initially colonize the tonsillar crypts in the upper respiratory tract of cattle, from where they can subsequently descend into the lungs to cause disease. Many bacteria exist as biofilms inside their hosts. We hypothesize that M. haemolytica colonization of cattle during its commensal state may include biofilm formation. To begin to assess this possibility, we developed an in vitro system to study biofilm formation directly on bovine respiratory epithelial cells. Using fixed primary bovine bronchial epithelial cells, we observed M. haemolytica biofilm formation after a 48h incubation period at 37°C. Addition of mucin, the main component of mucus present in the upper respiratory tract, decreased M. haemolytica biofilm formation on bovine epithelial cells. We investigated the effects of prior viral infection of the epithelial cells on subsequent biofilm formation by M. haemolytica and found negligible effects. Utilization of this model system will provide new insights into the potential role of biofilm formation by M. haemolytica in the pathogenesis of BRDC.
Project description:Pasteurella haemolytica serotype 1 is the bacterium most commonly associated with bovine shipping fever. The presence of antibodies against P. haemolytica outer membrane proteins (OMPs) correlates statistically with resistance to experimental P. haemolytica challenge in cattle. Until now, specific P. haemolytica OMPs which elicit antibodies that function in host defense mechanisms have not been identified. In this study, we have cloned and sequenced the gene encoding one such protein, PlpE. Analysis of the deduced amino acid sequence revealed that PlpE is a lipoprotein and that it is similar to an Actinobacillus pleuropneumoniae lipoprotein, OmlA. Affinity-purified, anti-PlpE antibodies recognize a protein in all serotypes of P. haemolytica except serotype 11. We found that intact P. haemolytica and recombinant E. coli expressing PlpE are capable of absorbing anti-PlpE antibodies from bovine immune serum, indicating that PlpE is surface exposed in P. haemolytica and assumes a similar surface-exposed conformation in E. coli. In complement-mediated killing assays, we observed a significant reduction in killing of P. haemolytica when bovine immune serum that was depleted of anti-PlpE antibodies was used as the source of antibody. Our data suggest that PlpE is surface exposed and immunogenic in cattle and that antibodies against PlpE contribute to host defense against P. haemolytica.
Project description:The pneumonic lesions and mortality caused by Mannheimia haemolytica in bighorn sheep (BHS; Ovis canadensis) are more severe than those in the related species, domestic sheep (DS; Ovis aries), under both natural and experimental conditions. Leukotoxin (Lkt) and lipopolysaccharide (LPS) are the most important virulence factors of this organism. One hallmark of pathogenesis of pneumonia is the influx of polymorphonuclear leukocytes (PMNs) into the lungs. Lkt-induced cytolysis of PMNs results in the release of cytotoxic compounds capable of damaging lung tissue. Interleukin-8 (IL-8) is a potent PMN chemoattractant. The objective of the present study was to determine if there is differential expression of IL-8 by the macrophages and PMNs of BHS and DS in response to M. haemolytica. Macrophages and PMNs of BHS and DS were stimulated with heat-killed M. haemolytica or LPS. IL-8 expression by the cells was measured by enzyme-linked immunosorbent assays and real-time reverse transcription-PCR (RT-PCR). The PMNs of BHS expressed severalfold higher levels of IL-8 than those of DS upon stimulation. Lesional lung tissue of M. haemolytica-infected BHS contained significantly higher levels of IL-8 than nonlesional tissue. The bronchoalveolar lavage (BAL) fluid of infected BHS also contained higher levels of IL-8 than that of infected DS. Depletion of IL-8 reduced migration of PMNs toward BAL fluid by approximately 50%, indicating that IL-8 is integral to PMN recruitment to the lung during M. haemolytica infection. Excessive production of IL-8, enhanced recruitment of PMNs, and PMN lysis by Lkt are likely responsible for the severity of the lung lesions in M. haemolytica-infected BHS.