Dhammika N. Atapattu

Inflammatory Cytokines Enhance the Interaction of Mannheimia haemolytica Leukotoxin with Bovine Peripheral Blood Neutrophils In Vitro

ABSTRACT 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 β2-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-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) 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-1β, TNF-α, or IFN-γ. 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.

Mannheimia haemolytica Leukotoxin Induces Apoptosis of Bovine Lymphoblastoid Cells (BL-3) via a Caspase-9-Dependent Mitochondrial Pathway

ABSTRACT Mannheimia haemolytica is a key pathogen in the bovine respiratory disease complex. It produces a leukotoxin (LKT) that is an important virulence factor, causing cell death in bovine leukocytes. The LKT binds to the β2 integrin CD11a/CD18, which usually activates signaling pathways that facilitate cell survival. In this study, we investigated mechanisms by which LKT induces death in bovine lymphoblastoid cells (BL-3). Incubation of BL-3 cells with a low concentration of LKT results in the activation of caspase-3 and caspase-9 but not caspase-8. Similarly, the proapoptotic proteins Bax and BAD were significantly elevated, while the antiapoptotic proteins Bcl-2, BclXL and Akt-1 were downregulated. Following exposure to LKT, we also observed a reduction in mitochondrial cytochrome c and corresponding elevation of cytosolic cytochrome c, suggesting translocation from the mitochondrial compartment to the cytosol. Consistent with this observation, tetramethylrhodamine ethyl ester perchlorate staining revealed that mitochondrial membrane potential was significantly reduced. These data suggest that LKT induces apoptosis of BL-3 cells via a caspase-9-dependent mitochondrial pathway. Furthermore, scanning electron micrographs of mitochondria from LKT-treated BL-3 cells revealed lesions in the outer mitochondrial membrane, which are larger than previous reports of the permeability transition pore through which cytochrome c is usually released.

Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and Clathrin-Dependent Manner

ABSTRACT Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex. Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the β2-integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demonstrated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3) death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly, expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation, reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internalization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the cytoskeletal network.

Prior exposure to Mannheimia haemolytica leukotoxin or LPS enhances β2-integrin expression by bovine neutrophils and augments LKT cytotoxicity

Mannheimia (Pasteurella) haemolytica serotype1 produces a variety of virulence factors that play an important role during the pathogenesis of bovine pneumonic pasteurellosis. Among these, a leukotoxin (LKT) and lipopolysaccharide (LPS) are thought to be the primary virulence factors that contribute to the characteristic pathology of pasteurellosis. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the beta(2)-integrin CD11a/CD18 (LFA-1), which subsequently induces activation and death of these cells. Exposure of bovine peripheral blood neutrophils (PMNs) to LKT or LPS induces expression of inflammatory cytokines, which in turn can increase LFA-1 expression and conformational activation. In this study we demonstrated, by flow cytometry and Western blot, that bovine PMNs increased their LFA-1 expression following in vitro exposure to M. haemolytica LKT and LPS. Increased LFA-1 expression by PMNs exposed to LKT and LPS was associated with increased LKT binding and cell death. The results of this study suggest that M. haemolytica LKT and LPS might cooperatively increase LFA-1 expression, and by so doing amplify the lung inflammation that characterizes bovine pasteurellosis.

Dynamin-2-dependent targeting of mannheimia haemolytica leukotoxin to mitochondrial cyclophilin D in bovine lymphoblastoid cells.

ABSTRACT 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 (ψm). 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 ψm. 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.

Effects of Lipopolysaccharide and Mannheimia haemolytica Leukotoxin on Bovine Lung Microvascular Endothelial Cells and Alveolar Epithelial Cells

ABSTRACT Bovine respiratory disease resulting from infection with Mannheimia haemolytica commonly results in extensive vascular leakage into the alveoli. M. haemolytica produces two substances, lipopolysaccharide (LPS) and leukotoxin (LKT), that are known to be important in inducing some of the pathological changes. In the present study, we examined bovine pulmonary epithelial (BPE) cell and bovine lung microvascular endothelial cell monolayer permeability, as measured by trans-well endothelial and epithelial cell electrical resistance (TEER), after incubation with LPS, LKT, or LPS-activated neutrophils. Endothelial cell monolayers exposed to LPS exhibited significant decreases in TEER that corresponded with increased levels of proinflammatory cytokines, apoptosis, and morphological changes. In contrast, BPE cells exposed to LPS increased the levels of production of inflammatory cytokines but displayed no changes in TEER, apoptosis, or visible morphological changes. Both cell types appeared to express relatively equal levels of the LPS ligand Toll-like receptor 4. However, TEER in BPE cell monolayers was decreased when the cells were incubated with LPS-activated neutrophils. Although the incubation of BPE cells with LKT decreased TEER, this was not reduced by the incubation of LKT with a neutralizing antibody and was reversed when LKT was preincubated with the LPS-neutralizing compound polymyxin B. Because BPE cells did not express the LKT receptor CD11a/CD18, we infer that contaminating LPS was responsible for the decreased TEER. In conclusion, LPS triggered changes in endothelial cells that would be consistent with vascular leakage, but neither LPS nor LKT caused similar changes in epithelial cells, unless neutrophils were also present.

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

Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase‐9‐dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N‐terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N‐terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagicEscherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N‐terminal region in addressing certain RTX toxins to mitochondria.

Gene expression profiling of bovine bronchial epithelial cells exposed in vitro to bovine herpesvirus 1 and Mannheimia haemolytica.

Abstract Bovine respiratory disease (BRD) often occurs when active respiratory virus infections (BHV-1, etc.) impair resistance to Mannheimia haemolytica infection in the lower respiratory tract. The interactions that occur when the respiratory epithelium encounters these viral and bacterial pathogens are poorly understood. We used Agilent bovine gene microarray chips containing 44,000 transcripts to elucidate bovine bronchial epithelial cell (BBEC) responses following in vitro exposure to BHV-1 alone, M. haemolytica alone, or both BHV-1 and M. haemolytica. Microarray analysis revealed differential regulation (>2-fold) of 978 transcripts by BHV-1 alone, 2040 transcripts by M. haemolytica alone, and 2189 genes by BHV-1 and M. haemolytica in combination. M. haemolytica treatment produced significantly greater inductions (>10-fold) of several inflammation associated genes, such as CXCL2, IL-6, IL-1α, e-selectin, and IL-8, than to BHV-1 alone. Functional analysis of the microarray data revealed a significant upregulation of genes involved in important biological processes such as inflammation (TNF-α, IL-8, Tlr-2, IL-1, CXCL2, CSF2), vascular functions (VEGF, EDN2) and leukocyte migration (ICAM1, IL-16) during a co-infection with BHV-1 and M. haemolytica compared to either pathogen alone. This study provides evidence to support that lung epithelial cells are a source of mediators that may promote inflammatory changes observed during bovine respiratory disease.

Brief heat treatment increases cytotoxicity of Mannheimia haemolytica leukotoxin in an LFA-1 independent manner.

Mannheimia haemolytica is an important respiratory pathogen in cattle. Its predominant virulence factor is a leukotoxin (LKT) that is a member of the RTX family of exotoxins produced by a variety of Gram negative bacteria. LKT binds to the CD18 chain of beta(2) integrins on bovine leukocytes, resulting in cell death. In this study, we show that brief heat treatment of native LKT (95 degrees C for 3 min) results in increased cytotoxicity for BL-3 (bovine lymphoblastoid) cells. Similar heat treatment restored the activity of LKT that had been rendered inactive by incubation at 22 degrees C for 3 days. A hallmark of LKT is that its toxicity is restricted to leukocytes from cattle or other ruminant species. Surprisingly, heat treatment rendered LKT cytotoxic for human, porcine and canine leukocytes. Membrane binding studies suggested that heat-treated LKT binds to membrane proteins other than LFA-1, and is distributed diffusely along the BL-3 cell membrane. Circular Dichroism spectroscopy studies indicate that heat treatment induced a small change in the secondary structure of the LKT that was not reversed when the LKT was cooled to room temperature. Thus, we speculate that these structural changes might contribute to the altered biological properties of heat-treated LKT.

Brief heat treatment causes a structural change and enhances cytotoxicity of the Escherichia coli α-hemolysin.

α-Hemolysin (HLY) is an important virulence factor for uropathogenic Escherichia coli. HLY is a member of the RTX family of exotoxins secreted by a number of Gram-negative bacteria. Recently, it was reported that a related RTX toxin, the Mannheimia haemolytica leukotoxin, exhibits increased cytotoxicity following brief heat treatment. In this article, we show that brief heat treatment (1 min at 100°C) increases cytotoxicity of HLY for human bladder cells, kidney epithelial cells (A498) and neutrophils. Heat treatment also increased hemolysis of human red blood cells (RBCs). Furthermore, heat treatment of previously inactived HLY restored its cytotoxicity. Heat-activated and native HLY both required glycophorin A to lyse RBCs. Native and heat-activated HLY appeared to bind equally well to the surface of A498 cells; although, Western blot analyses demonstrated binding to different proteins on the surface. Confocal microscopy revealed that heat-activated HLY bound more extensively to internal structures of permeabilized A498 cells than did native HLY. Several lines of spectroscopic evidence demonstrate irreversible changes in the structure of heat activated compared to native HLY. We show changes in secondary structure, increased exposure of tryptophan residues to the aqueous environment, an increase in molecular dimension and an increase in hydrophobic surface area. These properties are among the most common characteristics described for the molten globule state, first identified as an intermediate in protein folding. We hypothesize that brief heat treatment of HLY causes a conformational change leading to significant differences in protein–protein interactions that result in increased cytotoxicity for target cells.