The N-acetyl-d-glucosamine specific adhesin from Mannheimia haemolytica activates bovine neutrophils oxidative burst

ABSTRACTHuman 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 pathogenMannheimia haemolyticaand its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed toM. haemolyticaor its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by anlktCmutant ofM. 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 theM. haemolyticacells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response toM. haemolyticacells. 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 toEscherichia colihemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to theM. haemolyticaleukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins.

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Engineering and characterization of human β-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils

Veterinary Research ◽

10.1186/s13567-021-00956-4 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Harpreet Dhingra ◽

Kamaljit Kaur ◽

Baljit Singh

Keyword(s):

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Bactericidal Effect ◽

Inoculum Size ◽

Mannheimia Haemolytica ◽

Boyden Chamber ◽

Cattle Industry ◽

Bovine Neutrophils ◽

Microcin J25

AbstractMannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human β-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn’t have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-l-methionyl-l-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

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Mannheimia haemolytica and Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils

Infection and Immunity ◽

10.1128/iai.00840-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4454-4466 ◽

Cited By ~ 48

Author(s):

Nicole A. Aulik ◽

Katrina M. Hellenbrand ◽

Heather Klos ◽

Charles J. Czuprynski

Keyword(s):

Respiratory Disease ◽

Bovine Respiratory Disease ◽

Neutrophil Infiltration ◽

Mannheimia Haemolytica ◽

Neutrophil Extracellular Trap ◽

Human Neutrophils ◽

Lymphocyte Function ◽

Dependent Manner ◽

Bacterial Cells ◽

Bovine Neutrophils

ABSTRACT 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.

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