Abstract
Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ADP Ribose Transferases / antagonists & inhibitors*
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ADP Ribose Transferases / genetics
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ADP Ribose Transferases / immunology
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ADP Ribose Transferases / metabolism
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Anti-Bacterial Agents / immunology
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Anti-Bacterial Agents / pharmacology*
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Anti-Bacterial Agents / therapeutic use
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Apoptosis / drug effects
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Bacterial Toxins / antagonists & inhibitors*
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Bacterial Toxins / genetics
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Bacterial Toxins / immunology
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Bacterial Toxins / metabolism
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Catalytic Domain / genetics
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Complementarity Determining Regions / immunology
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Complementarity Determining Regions / pharmacology
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Exotoxins / antagonists & inhibitors*
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Exotoxins / genetics
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Exotoxins / immunology
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Exotoxins / metabolism
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HeLa Cells
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Humans
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Molecular Docking Simulation
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Peptide Library
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Pseudomonas Infections / drug therapy*
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Pseudomonas Infections / microbiology
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Pseudomonas aeruginosa / drug effects*
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Pseudomonas aeruginosa / immunology
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Pseudomonas aeruginosa / pathogenicity
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Pseudomonas aeruginosa Exotoxin A
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Recombinant Proteins / genetics
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Recombinant Proteins / immunology
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Recombinant Proteins / metabolism
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Single-Chain Antibodies / immunology
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Single-Chain Antibodies / pharmacology*
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Single-Chain Antibodies / therapeutic use
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Virulence Factors / antagonists & inhibitors*
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Virulence Factors / genetics
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Virulence Factors / immunology
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Virulence Factors / metabolism
Substances
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Anti-Bacterial Agents
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Bacterial Toxins
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Complementarity Determining Regions
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Exotoxins
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Peptide Library
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Recombinant Proteins
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Single-Chain Antibodies
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Virulence Factors
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ADP Ribose Transferases