Title : A novel dry-cured ham broth-derived peptide JHBp2 effectively inhibits Salmonella typhimurium in vitro: Integrated metabolomic, proteomic and molecular simulation analyses
Abstract:
JHBp2 is a peptide purified from Jinhua ham broth with antibacterial activity against Salmonella typhimurium. Untargeted-metabolomics and label-free-quantitative proteomics were used to analyze metabolic and protein expression changes in S. typhimurium after JHBp2 treatment. Based on the integrated nucleic acid and protein leakage result, and metabolomic and proteomic analysis, the growth-inhibitory mechanisms of JHBp2 against S. typhimurium could be summarized as follows:
- JHBp2 destroyed the structure of the cell membrane and interfered with bacterial cell membrane-associated protein synthesis, thus disrupted cell membrane function. The scanning electron microscope indicates that the structure of bacterial cell walls and membranes was disrupted after JHBp2 treatment. Further, subcellular localization analysis revealed that more than 65% of the differential proteins in the AMPT group were localized to the cell membrane. KEGG analysis of the metabolome suggested that the levels of 14 metabolites involved in the ABC transporter pathway were significantly altered. Of these, 10 were significantly downregulated. YehZ (ABC Transporter Substrate-Binding Protein) expression was also significantly downregulated in RT-qPCR experiments. The JHBp2-mediated downregulation of ABC transporter-related metabolites indicated that the important functions of S. typhimurium membrane structural proteins were inhibited, preventing the development of drug resistance. Additionally, GO annotation analysis revealed a significant downregulation of proteins related to periplasmic space and outer membrane periplasmic space, which containing a large number of hydrolytic and synthetic enzymes associated with peptidoglycan synthesis, as well as binding proteins that aid in nutrient transport.
- JHBp2 significantly downregulated energy metabolism-related enzyme and metabolites expression, which affected normal energy metabolism and cellular respiration in bacteria. KEGG analysis of the proteome revealed that 6 of the 12 significantly downregulated pathways were involved in energy metabolism, including pyruvate metabolism, carbon metabolism, glycolysis/gluconeogenesis, dicarboxylic acid metabolism, starch and sucrose metabolism, and TCA cycle with pyruvate metabolism being the most significantly downregulated pathway. Of the five significant metabolic pathways, 2,3-diphosphoglycerate-dependent PGM (dPGM) linked four pathways. Energy metabolism-related enzymes, such as fumarate hydratase II, malate dehydrogenase, fructose diphosphate aldolase, and acetyl coenzyme A synthase, were significantly downregulated in the AMPT group. Among the metabolomics, 24 differential metabolites were involved in the biosynthesis of cofactors pathway, which accounted for the highest number of differential metabolites among all pathways. Meanwhile, 18 differential proteins were involved in the biosynthesis of cofactors pathway in proteomics.
- JHBp2 significantly downregulated the synthesis/metabolism of amino acids, nucleotides-related enzymes, and metabolites, thereby interfering with normal genetic material and protein synthesis. Differential metabolites of amino acids and nucleotide, such as glutamate and 5,6-Dihydrouracil, are important raw materials for protein and nucleic acid synthesis in S. typhimurium. Proteomic and RT-qPCR analysis revealed that structure-related enzymes associated with amino acid and nucleotide anabolism (such as carbamate cinase and 2',3'-Cyclic-Nucleotide-2'-Phosphodiesterase/ 3'-Nucleotidase) were significantly downregulated upon the JHBp2 treatment. Molecular docking results also demonstrated that JHBp2 can bind stably to both enzymes.
Consequently, JHBp2 destroyed the structure of the cell wall and membrane, inhibited the bacterial absorption of nutrients, affected normal energy metabolism, and interfered with regular genetic material and protein synthesis, thereby inhibiting bacterial growth.
