Date Approved

3-22-2017

Embargo Period

3-22-2017

Document Type

Thesis

Degree Name

MS Pharmaceutical Sciences

Department

Chemistry & Biochemistry

College

College of Science & Mathematics

Advisor

Caputo, Gregory

Committee Member 1

Vaden, Timothy

Committee Member 2

Keck, Thomas

Keywords

Antimicrobial peptides, membrane permeabilization, pore-forming peptides, Silver Ions

Subject(s)

Peptide antibiotics

Disciplines

Pharmacy and Pharmaceutical Sciences

Abstract

Antimicrobial peptides are produced by multicellular organisms as a defense against competing pathogenic microbes. The mechanism of action for positively charged antimicrobial peptides is widely believed to occur when the positively charged peptide interacts with the negatively charged outer lipid membrane followed by the insertion of the peptide into the membrane. This insertion into the outer or cytoplasmic membranes leads to disruption of membrane integrity. Alternatively, silver based antimicrobials, which have been well known for centuries, are thought to act by inhibiting the proton motive force, the respiratory electron transport chain, and by affecting membrane permeability resulting in cell death. Considering the different proposed modes of action, we investigated the combination of these two antimicrobial species in an attempt to enhance efficacy by targeting different cellular processes. We performed biophysical and microbiological characterization using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, vesicle permeabilization assays, bacterial permeabilization assays, and minimal inhibitory concentration (MIC) assays. While all five peptides tested in this study exhibited binding to model lipid membranes, the truncated peptides showed no measurable antimicrobial activity. The most active peptide proved to be the parent peptide AP3 with the highest degree of leakage and bacterial membrane permeabilization. Moreover it was found that the ability to permeabilize model and bacterial membranes correlated most closely with the ability to predict antimicrobial activity. The mechanism of enhancement is under investigation along with expansion to other strains and various other antimicrobial peptides. Combinatorial delivery of antimicrobials with different sizes and modes of action appears to be a promising approach while minimizing potent toxicity and resistance concerns.

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