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A peptide derived from phage display library exhibits antibacterial activity against E. coli and Pseudomonas aeruginosa.

Sainath Rao S, Mohan KV, Atreya CD - PLoS ONE (2013)

Bottom Line: Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents.The peptide was highly active against gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log(10) CFU/ml.Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell Biology, Laboratory of Cellular Hematology, Division of Hematology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT
Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents. Antimicrobial peptides (AMPs) are receiving attention as alternatives to antibiotics. In this study, we used phage-display random peptide library to identify peptides binding to the cell surface of E. coli. The peptide with sequence RLLFRKIRRLKR (EC5) bound to the cell surface of E. coli and exhibited certain features common to AMPs and was rich in Arginine and Lysine residues. Antimicrobial activity of the peptide was tested in vitro by growth inhibition assays and the bacterial membrane permeabilization assay. The peptide was highly active against gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log(10) CFU/ml. In homologous plasma and platelets, incubation of EC5 with the bacteria resulted in significant reduction of E. coli and P. aeruginosa, compared to the peptide-free controls. The peptide was non-hemolytic and non-cytotoxic when tested on eukaryotic cells in culture. EC5 was able to permeabilize the outer membrane of E. coli and P. aeruginosa causing rapid depolarization of cytoplasmic membrane resulting in killing of the cells at 5 minutes of exposure. The secondary structure of the peptide showed a α-helical conformation in the presence of aqueous environment. The bacterial lipid interaction with the peptide was also investigated using Molecular Dynamic Simulations. Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides.

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Binding efficiency of EC5 to different bacteria.A. ELISA based assay– 96 well microtiter plates were coated with six bacteria and incubated with the biotinylated peptide and the binding was detected using strepatavidin–HRP and developed using TMB substrate. (**–p<0.001, *–p<0.05). B. Fluorometry based assay– binding of EC5 to different bacteria was detected using streptavidin-conjugated Q dots. Results are presented as Mean±SD. (** – p<0.0001, *– p<0.001). Bac – Bacteria without peptide, Pep – Peptide without bacteria were used as controls.
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pone-0056081-g001: Binding efficiency of EC5 to different bacteria.A. ELISA based assay– 96 well microtiter plates were coated with six bacteria and incubated with the biotinylated peptide and the binding was detected using strepatavidin–HRP and developed using TMB substrate. (**–p<0.001, *–p<0.05). B. Fluorometry based assay– binding of EC5 to different bacteria was detected using streptavidin-conjugated Q dots. Results are presented as Mean±SD. (** – p<0.0001, *– p<0.001). Bac – Bacteria without peptide, Pep – Peptide without bacteria were used as controls.

Mentions: ELISA-based binding analysis of EC5-Binding affinities and specificity of the synthesized peptide was analyzed by whole-cell ELISA. Peptide showed significant binding efficiency to E. coli cells (p<0.001) as seen in the Fig. 1A. Interestingly the peptide also showed binding to P. aeruginosa cells (p<0.05). However, EC5 did not bind to the Gram-positive S. aureus, S. epidermidis, B. cereus and to the Gram-negative K. pneumoniae.


A peptide derived from phage display library exhibits antibacterial activity against E. coli and Pseudomonas aeruginosa.

Sainath Rao S, Mohan KV, Atreya CD - PLoS ONE (2013)

Binding efficiency of EC5 to different bacteria.A. ELISA based assay– 96 well microtiter plates were coated with six bacteria and incubated with the biotinylated peptide and the binding was detected using strepatavidin–HRP and developed using TMB substrate. (**–p<0.001, *–p<0.05). B. Fluorometry based assay– binding of EC5 to different bacteria was detected using streptavidin-conjugated Q dots. Results are presented as Mean±SD. (** – p<0.0001, *– p<0.001). Bac – Bacteria without peptide, Pep – Peptide without bacteria were used as controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569419&req=5

pone-0056081-g001: Binding efficiency of EC5 to different bacteria.A. ELISA based assay– 96 well microtiter plates were coated with six bacteria and incubated with the biotinylated peptide and the binding was detected using strepatavidin–HRP and developed using TMB substrate. (**–p<0.001, *–p<0.05). B. Fluorometry based assay– binding of EC5 to different bacteria was detected using streptavidin-conjugated Q dots. Results are presented as Mean±SD. (** – p<0.0001, *– p<0.001). Bac – Bacteria without peptide, Pep – Peptide without bacteria were used as controls.
Mentions: ELISA-based binding analysis of EC5-Binding affinities and specificity of the synthesized peptide was analyzed by whole-cell ELISA. Peptide showed significant binding efficiency to E. coli cells (p<0.001) as seen in the Fig. 1A. Interestingly the peptide also showed binding to P. aeruginosa cells (p<0.05). However, EC5 did not bind to the Gram-positive S. aureus, S. epidermidis, B. cereus and to the Gram-negative K. pneumoniae.

Bottom Line: Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents.The peptide was highly active against gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log(10) CFU/ml.Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell Biology, Laboratory of Cellular Hematology, Division of Hematology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT
Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents. Antimicrobial peptides (AMPs) are receiving attention as alternatives to antibiotics. In this study, we used phage-display random peptide library to identify peptides binding to the cell surface of E. coli. The peptide with sequence RLLFRKIRRLKR (EC5) bound to the cell surface of E. coli and exhibited certain features common to AMPs and was rich in Arginine and Lysine residues. Antimicrobial activity of the peptide was tested in vitro by growth inhibition assays and the bacterial membrane permeabilization assay. The peptide was highly active against gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log(10) CFU/ml. In homologous plasma and platelets, incubation of EC5 with the bacteria resulted in significant reduction of E. coli and P. aeruginosa, compared to the peptide-free controls. The peptide was non-hemolytic and non-cytotoxic when tested on eukaryotic cells in culture. EC5 was able to permeabilize the outer membrane of E. coli and P. aeruginosa causing rapid depolarization of cytoplasmic membrane resulting in killing of the cells at 5 minutes of exposure. The secondary structure of the peptide showed a α-helical conformation in the presence of aqueous environment. The bacterial lipid interaction with the peptide was also investigated using Molecular Dynamic Simulations. Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides.

Show MeSH
Related in: MedlinePlus