Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis

Authors: Dassanayake, Rohana; Falkenberg, Shollie; Register, Karen; SAMORODNITSKY, DANIEL - US Department Of Agriculture (USDA); Nicholson, Eric; Reinhardt, Timothy

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2018
Publication Date: 5/17/2018
Citation: Dassanayake, R.P., Falkenberg, S.M., Register, K.B., Samorodnitsky, D., Nicholson, E.M., Reinhardt, T.A. 2018. Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis. PLoS One. 13(5):e0197677. https://doi.org/10.1371/journal.pone.0197677.
DOI: https://doi.org/10.1371/journal.pone.0197677

Interpretive Summary: Mycoplasma bovis, one of the bacterial pathogens associated with bovine respiratory disease complex, is also known to cause mastitis, arthritis and otitis media in cattle. This bacterium has recently been recognized as a highly virulent pathogen in North American bison. Bovine respiratory disease complex (or pneumonia), which is commonly known as shipping fever, is the leading cause of economic losses to the United States beef and dairy cattle industry. 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 tested several small proteins (NK-lysins) that are found in certain white blood cells in cattle for killing activities against cattle and bison Mycoplasma bovis isolates. Two small proteins (out of four) showed higher M. bovis killing activity. These small proteins were previously shown to kill other bacterial pathogens involved in shipping fever such as Mannheimia haemolytica, Pasteurella multocida and Histophilus somni. Further studies are needed to assess whether small proteins tested in this study can be used for prophylaxis or treat cattle against shipping fever causing bacterial pathogens.

Technical Abstract: Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on up take of propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality.