Antibacterial mechanism of the Asp - Asp - Asp - Tyr peptide [P. aeruginosa]

Previously, we found that ASP-ASP-ASP-TYR (DDDY) from Dendrobium aphyllum has a minimum inhibitory concentration of 36.15 mg/mL against Pseudomonas aeruginosa. Here, we explored the antibacterial mechanism of DDDY and its potential preservation applications. Metabolomic and transcriptomic analyses revealed that DDDY mainly affects genes involved in P. aeruginosa membrane transport and amino acid metabolism pathways. Molecular dynamics simulation revealed that DDDY had a stronger effect on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine phospholipid membranes than on 1-palmitoyl-2-oleoyl-lecithin or 1-palmitoyl-2-oleoyl phosphatidylglycerol membranes, with high DDDY concentrations displaying stronger efficacy on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. Mechanistically, the N-terminal of DDDY first bound to the phospholipid head group, while its C-terminal amino acid residue bound the hydrophobic tail, thereby creating a gap in the membrane when the phospholipids were clustered by hydrogen bonding. Finally, DDDY inhibited the growth of food microorganisms inoculated onto chestnut kernels, suggesting that DDDY is a promising antibacterial agent against multidrug-resistant gram-negative bacteria. Inhibitory effect of ASP-ASP-ASP-TYR (DDDY) from Dendrobium on Pseudomonas aeruginosa Overall design: P. aeruginosa was incubated in TSB at 37 C for 8 h. During the logarithmic phase, 200 µL of bacterial solution was added to 200 mL of liquid medium, 200 µL of sterile water (control group; P_CK), or 50 mg/mL of DDDY solution (treatment group; P_DDDY). The bacteria were then cultured on a shaking table at 150 R/min and 37 C for 24 h, centrifuged at 6000 R/min and 4 C for 10 min, washed with PBS three times, and then collected and stored at -80 C.