Histophilus somni causes extracellular trap formation by bovine neutrophils and macrophages.

Histophilus somni (formerly Haemophilus somnus) is a Gram-negative pleomorphic coccobacillus that causes respiratory, reproductive, cardiac and neuronal diseases in cattle. H. somni is a member of the bovine respiratory disease complex that causes severe bronchopneumonia in cattle. Previously, it has been reported that bovine neutrophils and macrophages have limited ability to phagocytose and kill H. somni. Recently, it was discovered that bovine neutrophils and macrophages produce extracellular traps in response to Mannheimia haemolytica, another member of the bovine respiratory disease complex. In this study, we demonstrate that H. somni also causes extracellular trap production by bovine neutrophils in a dose- and time-dependent manner, which did not coincide with the release of lactate dehydrogenase, a marker for necrosis. Neutrophil extracellular traps were produced in response to outer membrane vesicles, but not lipooligosacchride alone. Using scanning electron microscopy and confocal microscopy, we observed H. somni cells trapped within a web-like structure. Further analyses demonstrated that bovine neutrophils trapped and killed H. somni in a DNA-dependent manner. Treatment of DNA extracellular traps with DNase I freed H. somni cells and diminished bacterial death. Treatment of bovine monocyte-derived macrophages with H. somni cells also caused macrophage extracellular trap formation. These findings suggest that extracellular traps may play a role in the host response to H. somni infection in cattle.

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.

Mannheimia haemolytica and its leukotoxin cause macrophage extracellular trap formation by bovine macrophages.

Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins.

Mannheimia haemolytica leukotoxin binds cyclophilin D on bovine neutrophil mitochondria.

Mannheimia haemolytica is an important member of the bovine respiratory disease (BRD) complex that causes fibrinous and necrotizing pleuropneumonia in cattle. BRD is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. The most important virulence factor of M. haemolytica is its leukotoxin. Previous research in our laboratory has shown that the leukotoxin is able to enter into and traffic to the mitochondria of a bovine lymphoblastoid cell line (BL-3). In this study, we evaluated the ability of LKT to be internalized and travel to mitochondria in bovine neutrophils. We demonstrate that LKT binds bovine neutrophil mitochondria and co-immunoprecipitates with TOM22 and TOM40, which are members of the translocase of the outer mitochondrial (TOM) membrane family. Upon entry into mitochondria, LKT co-immunoprecipitates with cyclophilin D, a member of the mitochondria permeability transition pore. Unlike BL-3 cells, bovine neutrophil mitochondria are not protected against LKT by the membrane-stabilizing agent cyclosporin A, nor were bovine neutrophil mitochondria protected by the permeability transition pore antagonist bongkrekic acid. In addition, we found that bovine neutrophil cyclophilin D is significantly smaller than that found in BL-3 cells. Bovine neutrophils were protected against LKT by protein transfection of an anti-cyclophilin D antibody directed at the C-terminal amino acids, but not an antibody against the first 50 N-terminal amino acids. In contrast, BL-3 cells were protected by antibodies against either the C-terminus or N-terminus of cyclophilin. These data confirm that LKT binds to bovine neutrophil mitochondria, but indicate there are distinctions between neutrophil and BL-3 mitochondria that might reflect differences in cyclophilin D.

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 enterohaemorrhagic Escherichia 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.

Mannheimia haemolytica and its leukotoxin cause neutrophil extracellular trap formation by bovine neutrophils.

Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of 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.