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

Abstract

Mannheimia 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.

Bovine neutrophil chemotaxis to Listeria monocytogenes in neurolisteriosis depends on microglia-released rather than bacterial factors

“…While data on the effect of Lm -derived formyl peptides on bovine PMN chemotaxis is lacking, available data on the chemotactic effect of E. coli -derived fMLP toward bovine PMN is contradicting. Some authors reported a chemotactic effect of fMLP [ 53 – 55 ], while others, in line with our results, failed to identify a bovine PMN migratory response toward bacterial peptides (formylated and non-formylated), bacterial culture supernatants, and viable or sonicated Gram-positive and Gram-negative bacteria [ 50 – 52 ]. The latter studies and our findings are supported by the reported lack of FPRs on bovine PMNs [ 52 ].…”

“…To select an effective positive control for PMN chemotaxis, we tested bovine PMN chemotaxis towards bovine IL-8 and fMLP, using, for the latter, concentrations reported to efficiently elicit chemotaxis of human and murine PMN (i.e., 0.1 and 1 µM) [ 45 , 47 , 48 ]. While bovine IL-8 has been shown to strongly elicit bovine PMN chemotaxis [ 49 ], studies on the chemotactic effect of fMLP are contradictive with some indicating that bovine PMN do not migrate towards formyl peptides [ 50 – 52 ], while others claim a chemotactic effect of fMLP on bovine PMN [ 53 – 55 ]. As illustrated in Fig.…”

Evaluation of human β‑defensins in the cerebrospinal fluid of suspected meningitis

“…HBD3 is an important AMP involved in protection against bacterial and viral infections, and is also known to have immunomodulatory functions ( 20 ). Regarding its antibacterial effects, studies have shown significant bactericidal activity of HBD3 against different G+ve and G-ve bacteria and this may be related to the cationic charges of HBD3 molecules ( 30 , 31 ). Additionally, high expression of HBD3 significantly enhanced wound closure in diabetic animal models infected with Staphylococcus aureus ( 32 ).…”

Stimulating immunoglobulin response by intramuscular delivery of exopolysaccharides-adjuvanted mannheimiosis vaccine in goats

“…Vitek 2 ® System compact (BioMériuex Available at www.veterinaryworld.org/Vol.15/December-2022/25.pdf SA, USA) was used for identification. Culture plates were grown at 37°C in a rotatory shaking incubator for 16 h [17,18]. About 30 colonies of pure bacteria were transferred into 250 mL of brain heart infusion broth (Oxoid).…”