Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates

Authors: Dassanayake, Rohana; Atkinson, Briony; MULLS, ADAM - Iowa State University; Falkenberg, Shollie; Nicholson, Eric; Casas, Eduardo; NARASIMHAN, BALAJI - Iowa State University; Bearson, Shawn

Submitted to: Nature Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/14/2021
Publication Date: 9/29/2021
Citation: Dassanayake, R.P., Atkinson, B.M., Mulls, A.S., Falkenberg, S.M., Nicholson, E.M., Casas, E., Narasimhan, B., Bearson, S.M. 2021. Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates. Nature Scientific Reports. 11(1). Article 19276. https://doi.org/10.1038/s41598-021-98860-6.
DOI: https://doi.org/10.1038/s41598-021-98860-6

Interpretive Summary: Among the pathogens causing food-borne illnesses in the U.S., Salmonella has been identified as the second most common cause. Centers for Disease Control and Prevention has classified drug resistant Salmonella as a serious threat to human health. Therefore, it is important to identify alternatives to antibiotics to limit Salmonella in food animals, their environment, and their food products. In this regard, we investigated antimicrobial activity of four small antimicrobial proteins (bovine NK-lysin-derived peptides) as alternatives to conventional antibiotics against MDR Salmonella outbreak isolates. Two of the four small antimicrobial proteins (NK2A and NK2B) were able to efficiently kill Salmonella isolates. Furthermore, small antimicrobial protein (NK2A) encapsulated into biodegradable nanoparticles were also able to kill bacteria very efficiently. Further studies are needed to assess whether small antimicrobial proteins encapsulated nanoparticles can be used as a sustainable delivery platform to control MDR Salmonella colonization and shedding in animals.

Technical Abstract: Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasure to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic a-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed no-to-reduced growth inhibition. Scrambled-NK2A, which is devoid of a-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A a-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.