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

ABSTRACT: 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

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE192340 | GEO | 2021/12/24

REPOSITORIES: GEO

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Project description:Serum samples were randomized and extracted using a modified version of the previously-published Folch procedure, as applied recently [20]. The maternal samples were analysed as one batch and the cord blood samples as a second batch. In short, 10 µL of 0.9% NaCl and, 120 µL of CHCl3: MeOH (2:1, v/v) containing the internal standards (c = 2.5 µg/mL) was added to 10 µL of each serum sample. The standard solution contained the following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and, triheptadecanoylglycerol (TG(17:0/17:0/17:0)) was purchased from Larodan AB (Solna, Sweden). The samples were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min). 60 µL from the lower layer of each sample was then transferred to a glass vial with an insert and 60 µL of CHCl3: MeOH (2:1, v/v) was added to each sample. The samples were stored at -80 °C until analysis. Calibration curves using 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0e/18:1(9Z))), 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:0p/18:1(9Z))), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:0)), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1)), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)), 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol (DG(18:0/18:2)), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (LPE(18:1)), N-(9Z-octadecenoyl)-sphinganine (Cer(d18:0/18:1(9Z))), 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)) from Avanti Polar Lipids, 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine (LPC(16:0)), 1,2,3 trihexadecanoalglycerol (TG(16:0/16:0/16:0)), 1,2,3-trioctadecanoylglycerol (TG(18:0/18:0/18:)) and 3β-hydroxy-5-cholestene-3-stearate (ChoE(18:0)), 3β-Hydroxy-5-cholestene-3-linoleate (ChoE(18:2)) from Larodan, were prepared to the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng/mL (in CHCl3:MeOH, 2:1, v/v) including 1250 ng/mL of each internal standard. The samples were analyzed by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used in this work was a 1290 Infinity II system from Agilent Technologies (Santa Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 °C), a quaternary solvent manager and a column thermostat (maintained at 50 °C). Injection volume was 1 µL and the separations were performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 QTOF from Agilent Technologies interfaced with a dual jet stream electrospray (Ddual ESI) ion source. All analyses were performed in positive ion mode and MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. Quality control was performed throughout the dataset by including blanks, pure standard samples, extracted standard samples and control serum samples, including in-house serum and a pooled QC with an aliquot of each sample was collected and pooled and used as quality control sample. Relative standard deviations (% RSDs) for identified lipids in the control serum samples (n = 13) and in the pooled serum samples (n = 54) were on average 22.4% and 17.5%, respectively.

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

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