A dodecapeptide (YQVTQSKVMSHR) exhibits antibacterial effect and induces cell aggregation in Escherichia coli

Abstract

Antimicrobial peptides play an important role in the innate immune response and host defense mechanism. In the present study, we employed phage display technique to screen for inhibitors which may block the phosphoenolpyruvatedependent phosphotransferase system (PTS) pathway and hence retard cell growth. The recombinant histidine-containing phosphocarrier HPr protein was prepared as the target to screen for the tight binders from the phage-displayed random peptide library Ph.D.-12. The biopanning processes were performed and the binding capabilities of the selected phage were further estimated by enzyme-linked immunosorbent assay (ELISA). The single-stranded DNAs of the 20 selected phages were isolated, sequenced, and five corresponding peptides were synthesized. Only one of the five peptides, AP1 (YQVTQSK VMSHR) was found to inhibit the growth of Escherichia coli cells efficiently (IC₅₀~50 μM). Molecular modeling reveals that AP1 may block the EI-HPr interaction and phosphotransfer. Interestingly, AP1 was also found to induce cell aggregation in a concentration-dependent manner. Since glycogen accumulation has been attributed to biofilm formation, the effects of AP1 on the intracellular glycogen levels were measured. The results strongly indicate that the cell aggregation may be caused by the binding of peptide AP1 with HPr to block the interaction of dephosphorylated HPr with glycogen phosphorylase (GP). Because glycogen phosphorylase activity can be activated by HPr-GP interaction, the binding of AP1 to HPr would cause a decreasing rate of glycogen breakdown in M9 medium and accumulation of glycogen, which may lead to eventual cell aggregation. To the best of our knowledge, this is the first study to demonstrate that an inhibitor bound to a dephosphorylated HPr can decouple its regulatory function and induce cell aggregation.

Exploration of Peptide Inhibitors of Human Squalene Synthase through Molecular Modeling and Phage Display Technique

“…Then, phage clones were picked randomly for the binding assay. To evaluate the binding capabilities of phage particles to the recombinant hSQS, enzyme-linked immune-sorbent assay (ELISA) experiments were performed [16,20]. It started with coating the wells with recombinant hSQS protein (100 μl, 100 μg/ml), incubated overnight at 4°C, blocked (with 200-μl blocking buffer), and washed with 200-μl TBS buffer containing 0.5 % Tween-20 (TBST) six times.…”

“…Over-expression of the cloned gene was induced by the addition of 2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD 600 =0.8-1.0, and grown for 4 h [16]. The recombinant His-hSQS protein was purified via Ni-NTA column and AKTAprime (GE Healthcare, USA).…”

“…DNA sequencing and sequence analysis were accomplished by Genomics BioScience and Tech Company (Taiwan). Peptides were synthesized by an automated PSIII synthesizer (Nature Gene Corp, USA) as described previously [16,18].…”

“…Biopanning and Phage-hSQS Binding Assay by ELISA Biopanning (affinity selection) the phage library was performed following the manufacturer's protocol [16,20]. After three rounds of selection, the eluents were amplified and plated.…”

“…These factors prompted us to focus on the development of peptide drugs as hypocholesterolemic agents. In the present study, we employed phage display technique to screen for tight peptide binders against recombinant hSQS [13][14][15][16]. The potentials of the selected peptides were further evaluated through molecular docking and absorption, distribution, metabolism, excretion, toxicity (ADMET) predictions.…”