Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Philip R. Cunningham

Abstract

The rise of antibiotic resistance necessitates new approaches for the isolation of new antimicrobials with novel inhibitory mechanisms, bypassing the development of rapid resistance by modification of pre-existing resistance mechanisms. In response, we have developed a series of systems for the rapid isolation and identification of peptides that inhibit the growth of Escherichia coli and other bacteria, termed in vivo display (IVD).

IVD harnesses the cellular processes of E. coli for the expression of a library of random peptides at the terminus of a display protein. A library of 12-amino acid random peptide sequences was added to either the C- or N-terminus of Emerald Green Fluorescent Protein (EmGFP) for the isolation of peptides active in the cytoplasm or the N-terminus of Alkaline Phosphatase (PhoA) for the isolation of peptides active in the periplasm. The peptide-display protein fusions were encoded behind an inducible promoter on plasmid vectors. A high-throughput method was implemented to select for peptides that inhibit bacterial growth followed by replica plating to confirm their efficacy.

To date, we have isolated 322 peptides that inhibit the growth of E. coli, exhibiting a number of growth phenotypes including bacteriolytic, bacteriostatic, bactericidal and growth rate reducing or weakly inhibitory. The cytoplasmic selection, in which the peptide library is fused to the C-terminus (cIVD) or the N-terminus (nIVD) of EmGFP produced 39 and 5 peptides, respectively. Of the 39 peptides isolated using the cIVD system, 1 is bacteriolytic, 18 are bacteriostatic/bactericidal and 20 are weakly inhibitory. Similarly, 2 of the peptides isolated using the nIVD system are bacteriostatic/bactericidal and 3 are weakly inhibitory. The periplasmic in vivo display (pIVD) system yielded a total of 192 growth inhibiting peptides, including 53 bacteriostatic/bactericidal, 44 weakly inhibitory and 95 bacteriolytic peptides. Further characterization of the isolated bacteriolytic peptides revealed that 75 of them share common sequence elements suggesting they share a common target. Based on the amino acid distribution of all the bacteriolytic peptides isolated using pIVD, a constrained peptide library enriched for the observed sequence elements was generated and screened isolating 9 bacteriostatic/bactericidal, 4 weakly inhibitory and 68 bacteriolytic peptides. Further, using a rational approach, we successfully designed a series of peptides based on the phenotypic groupings of the bacteriolytic peptides yielding 1 weakly inhibitory and 4 bacteriolytic peptides.

Select peptides were analyzed for activity against a variety of micro-organisms including Gram-negative and Gram-positive bacteria and yeast when added exogenously to live cultures. Three of the peptides, PL098, 84 Pro- and EO1, exhibit different spectra of activity suggesting distinct targets and/or antimicrobial mechanisms which were examined by comparing cell morphologies, growth in hypertonic growth media and PhoA activity assays. The ultimate isolation of antimicrobial peptides from IVD and its derivatives demonstrates proof of principle that IVD is an efficient method of the further isolation of antimicrobial peptides and their characterization for the development of therapeutic anti-infectives.

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