Date of Award

Spring 5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Jason Yamaki

Second Advisor

Rakesh Tiwari

Third Advisor

Ahmed Aftab

Fourth Advisor

Keykavous Parang

Abstract

Antimicrobial peptides (AMPs) are being explored as a potential solution to combat antibiotic resistance, as they have shown promise in reducing susceptibility to antibiotics. This study explores if [R4W4] peptide is bacteriostatic or bactericidal using modified two-fold serial dilution and evaluates synergism of gentamicin and [R4W4] against Escherichia coli (E. coli) and Methicillin-resistant Staphylococcus aureus (MRSA) by checkered board assay. The MRSA resistant to [R4W4] and possibility with other antibiotics was evaluated with serial passage. Moreover, we investigated the mechanism of action of [R4W4] against MRSA by applying biophysical assays to evaluate zeta potential, cytoplasmic membrane depolarization, and Lipoteichoic acid (LTA) binding affinity. [R4W4] exhibits bactericidal activity against bacterial isolates (MBC/MIC £ 4), with a synergistic effect with gentamicin against E. coli (FICI=0.3), but not against MRSA (FICI=0.75). [R4W4] at 16 µg/ml concentration stabilizes the zeta potential of MRSA -33 ± 0.88 mV to -8.37 mV. Also, [R4W4] at 2 x MIC and 16 x MIC revealing a membrane perturbation process associated with concentration-dependent effects. Lastly, in the presence of BODIPY-Cadaverine fluorescence dyes, [R4W4] reveals binding affinity to LTA comparable with melittin, the positive control. In addition, a change in the antibacterial activity of [R4W4] against MRSA in the absence and presence of LTA did not change the MIC 8µg/ml. Therefore, the [R4W4] mechanism of action is bactericidal with the potential to interact with bacterial cell membranes, causing concentration-dependent cytoplasm membrane perturbation.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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