Veterinary Research Forum

Abstract The rise of antimicrobial resistance poses a severe global health challenge, with multidrug-resistant bacteria causing millions of deaths annually and diminishing the effectiveness of traditional antibiotics. Bacteriophage-derived endolysins, such as PVP-SE1gp146 from Salmonella phage PVP-SE1, emerge as promising alternatives due to their high specificity, low toxicity, and minimal resistance development. However, their activity against Gram-negative bacteria is limited by poor outer membrane (OM) penetration. This study investigates the structural and functional impacts of fusing the polycationic nonapeptide, PCNP to PVP-SE1gp146 to create engineered endolysin that enhance OM permeability while preserving enzymatic function. Using advanced computational tools including Phyre2, GalaxyWEB, and AlphaFold2, three-dimensional models of native and engineered endolysins were generated and validated with Verify3D. Sequence analysis via Basic Local Alignment Search Tool identified homologs, and structural superposition with template modelin-align (TM-align) confirmed high conservation (TM-score 0.921, Root Mean Square Deviation 1.84 Å) despite the PCNP insertion. Active sites were predicted using PrankWeb, and molecular docking with AutoDock assessed interactions with peptidoglycan components (e.g., N-acetylmuramic acid - N-acetylglucosamine dimer, tetramer). Results revealed superior binding affinities in the native endolysin (e.g., – 10.35 kcal mol^-1 for N-acetylmuramic acid -L-alanine) compared to the engineered variant (– 9.65 kcal mol^-1 for N-acetylmuramic acid - N-acetylglucosamine -L-alanine trimer), with positive energies for larger ligands indicating steric hindrance. This trade-off reduced affinity for improved OM penetration supports potential enhancement of OM penetration while preserving bactericidal function like Pseudomonas aeruginosa. The findings highlight the potential of rational engineering for superior antimicrobials, emphasizing the need for molecular dynamics simulations and experimental validation to optimize Artilysin^® design in combating antimicrobial resistance.

Abstract Aflatoxins are toxic chemicals produced by Aspergillus fungi. Reports exist on the relationship of aflatoxin exposure via contaminated food and feed to hepatotoxicity and liver cancer. Aflatoxin B1 (AFB1) and Aflatoxin G1 (AFG1) are two dangerous types of aflatoxins for human health. Bovine α-lactalbumin (ALA) is the second major whey protein in milk which bear diverse biological functions. In this study, the interaction of AFB1 and AFG1 with the ALA protein was studied using fluorescence spectroscopy, molecular docking and molecular dynamic (MD) simulation. The spectroscopy experiments showed that the interaction with AFB1 and AFG1significantly quenched the intrinsic fluorescence emission of ALA via a static quenching mechanism. The free energy of binding and binding constant (Ka) obtained from the intrinsic fluorescence results were –5.32 kcal per mol and 0.80 × 10⁴ L mol^-1 for AFB1 and -5.64 kcal per mol and 1.35 × 10⁴ l mol^-1 for AFG1, respectively. Molecular docking studies were conducted before and after the MD simulation to estimate the binding sites, Ka s and binding mode. Results from the molecular docking showed that AFB1 and AFG1 bound to ALA via hydrophobic interaction and hydrogen bond. After MD simulation, the precision of the Ka obtained from the docking results was improved and it was more similar to the experimental results of fluorescence spectroscopy. Other simulation results were aligned well with the molecular docking and fluorescence spectroscopy results. Accordingly, AFB1and AFG1 could form complex with ALA, however, AFG1 showed higher affinity for binding to ALA and more compact complex structure.