123. Engineering Polycationic Nanopeptide-fused Endolysins to Target Mannheimia Haemolytica Associated with Bovine Respiratory Disease.

Abstract Background Bovine respiratory disease (BRD), associated with multidrug resistant bacterial pathogens, is one of the major health concerns with significant impact on feedlot cattle industry in North America. Among the bacterial pathogens implicated in BRD, M. haemolytica is of prime importance. The primary approaches for controlling M. haemolytica are vaccination and the use of antibiotics. However, vaccines often provide incomplete protection due to antigenic variation among strains and the complex, multifactorial nature of BRD. Furthermore, reliance on antibiotics is increasingly challenged by the rapid emergence of antimicrobial resistance (AMR), growing regulatory and consumer pressure to minimize antibiotic use. These limitations highlight an urgent need for alternative treatment strategies that can effectively target M. haemolytica while mitigating development of AMR. Endolysins are enzymes naturally produced by bacteriophages at the end of their lytic cycle to hydrolyze peptidoglycan layers in bacterial cell wall and release from host cell. Endolysins have emerged as promising alternatives to antibiotics, but their activity against Gram-negative bacteria is hindered by the presence of outer membrane (OM) barrier. However, Gram-negative endolysins are found to have improved antibacterial activity when engineered with OM permeabilizers such as polycationic nanopeptides (PCNPs). Polycationic nanopeptides are short, highly positively charged peptides that can disrupt negatively charged cell wall of Gram-negative bacteria where the OM blocks the entry of endolysins.Objectives: The present study was designed to engineer prophage derived endolysins and make their PCNP-fused variants to employ them as alternative antimicrobial agent against M. haemolytica. Methodology In this study, we screened five distinct prophage-derived endolysins from M. haemolytica and successfully expressed two (185 and 587AP2) along with their engineered PCNP fusions (PCNP-185 and PCNP-587AP2). The endolysins were cloned in pBAD plasmid and expressed in Escherichia coli BL21(DE3) expression system. Purification, quantification, and confirmation through SDS-PAGE was performed before using the expressed endolysins in muralytic/peptidoglycan degradation and antibacterial activity assays. Results All four recombinant proteins demonstrated muralytic activity, with original endolysins exhibiting higher enzymatic efficiency highlighting the role of PCNP as OM permeabilizer and not as the enhancer of peptidoglycan degradation ability of endolysins. However, PCNP-fused variants showed superior antibacterial effects as compared to those without. Especially, PCNP-185 achieved the most consistent multi-log reductions of up to 4.5 log10 CFU/mL in bacterial counts across M. haemolytica strains, particularly when combined with EDTA which further increased permeability of the OM by acting as chelating agent. Conclusion These findings highlight the synergistic potential of PCNP-fused engineered endolysins and OM chelators in overcoming the Gram-negative barrier and serve as alternative antimicrobial agent. To our knowledge, this is the first report of engineering and evaluating prophage-derived endolysins against M. haemolytica, underscoring their potential as innovative therapeutics for BRD associated bacterial pathogens. For image description, please refer to the figure legend and surrounding text.