Bovine NK-lysin-derived peptides are bactericidal against Mycobacterium avium subspecies paratuberculosis

Authors: Dassanayake, Rohana; WHERRY, TAYLOR - Iowa State University; Falkenberg, Shollie; Reinhardt, Timothy; Casas, Eduardo; Stabel, Judith

Submitted to: Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2021
Publication Date: 1/21/2021
Citation: Dassanayake, R.P., Wherry, T.L., Falkenberg, S.M., Reinhardt, T.A., Casas, E., Stabel, J.R. 2021. Bovine NK-lysin-derived peptides are bactericidal against Mycobacterium avium subspecies paratuberculosis. Veterinary Research. 52. Article 11. https://doi.org/10.1186/s13567-021-00893-2.
DOI: https://doi.org/10.1186/s13567-021-00893-2

Interpretive Summary: Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or chronic enteritis in cattle and other ruminants. Due to the emergence of antibiotic-resistant bacteria, there is a need for the identification and development of reagents for an alternative to antibiotics. In this regard, we investigated antimicrobial activity of bovine NK-lysin-derived peptides (small antimicrobial proteins which are not antibiotics but identified as alternative to antibiotics) against MAP. Two of the four small antimicrobial proteins (NK-lysins) were able to kill MAP very efficiently and the killing of MAP was mediated by membrane pore formation. Furthermore, a small antimicrobial protein (NK-lysins) fused with a cell-penetrating peptide was able to kill intracellular MAP within the infected macrophages. Further studies are needed to assess whether small antimicrobial proteins (NK-lysins) tested in this study can be used as a therapeutic agent against MAP.

Technical Abstract: Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease affecting cattle and other ruminants. Bovine NK-lysins (bNK-lysins; bNK1, bNK2A, bNK2B, and bNK2C) are antimicrobial proteins produced by T-lymphocytes and natural killer (NK) cells. We and others have previously demonstrated that cationic 30-mer synthetic peptides of bNK-lysins show strong antimicrobial activity against bacterial pathogens, however, bNK-lysin activity has not been studied against MAP. In the present study, live MAP was incubated with bNK-lysin peptides at 10, 50 and 100 µM concentrations for 72 h and impacts on bacterial viability were determined by a novel propidium monoazide-based qPCR and culture (CFU) assays. Viability qPCR assay revealed that all four bNK-lysin peptides were bactericidal, with 48-99% MAP killing at 100 µM final concentration, however, bNK1 and bNK2A were more potent than bNK2B and bNK2C. Anti-MAP activities of peptides were also confirmed by CFU and flow cytometry. Transmission electron microscopy of MAP incubated with bNK2A showed MAP with damaged cell membranes, cytosolic content leakage, and cell debris. Further, confocal microscopy was performed on monocyte-derived macrophages 24 h post-infection with MAP after staining with bacterial viability dyes indicated that bNK2A peptide fused with a cell-penetrating peptide improved intracellular MAP killing activity as compared to bNK2A alone. These findings suggest that bNK-lysin peptides elicit anti-MAP activities, albeit at higher concentrations than noted for extracellular microorganisms, and that killing of MAP is mediated by a membranolytic pore-formation mechanism.