Fina Blade Dissertation 8_23_19.pdf (8.28 MB)
Download file

STRATEGIC MODIFICATIONS TO OPTIMIZE A CELL PENETRATING ANTIMICROBIAL PEPTIDE

Download (8.28 MB)
thesis
posted on 31.01.2022, 15:24 by Reena BladeReena Blade

Pathogenic bacteria are evolving to drug resistant strains at alarming rates. The threat posed by drug resistant bacterial infections emphasize the need to establish new antimicrobial agents. Of immediate concern regarding the dangers of antibiotic resistance is the existence of intracellular bacteria, which find refuge from bactericidal devices by hiding within mammalian cells. Unfortunately, many therapeutics, such as vancomycin, do not possess membrane penetrating abilities to achieve efficacious eradication of bacteria at the subcellular level, allowing infections to persist. In an effort to target pathogens that thrive within mammalian cells, features of cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) were combined to develop a dual action antimicrobial CPP, cationic amphiphilic polyproline helices (CAPHs). CAPHs have proven to be an effective antimicrobial agent to combat an array of both Gram negative and Gram positive bacteria.

Herein, to improve CAPHs activity, we have demonstrated how the incorporation of strategic modifications has resulted in increased cell uptake, alternative subcellular locations for CAPHs, and advanced antimicrobial potency. By simultaneously extending the helical length of CAPHs while incorporating different hydrophobic groups in place of the original isobutyl moiety that compose CAPHs we have created a FL-P17-5R series of peptides with five carbon aliphatic motifs: Fl-P17-5B, Fl-P17-5C and Fl-P17-5L. Through these modifications the peptides proved to be 2 to 5-fold more efficient in accumulating in macrophage cells than parent peptide Fl-P14LRR and where able to clear intracellular pathogenic bacteria, such as Listeria, from infected macrophages by 26 to 54%.

In addition to making the Fl-P17-5R series of CAPHs to potentiate CAPHs activity, modifications to the cationic moiety of CAPHs were explored. By incorporating a new cationic monomer into the CAPHs sequence, a guanylated amino proline (GAP) residue, we produced Fl-P14GAP, a CAPHs peptide with an organized cationic charge display. This modification resulted in a 5-fold increase in cell uptake and a 2 to 16-fold decrease in minimum inhibitory concentration (MIC) values against strains of enteric and ESKAPE pathogens in comparison to Fl-P14LRR. Fl-P14GAP also executed superior clearance of intracellular pathogenic bacteria that resulted in the complete eradication of a drug resistant strain of A. baumannii from infected macrophage cells. Overall, our efforts with the Fl-P17-5R series of CAPHs and Fl-P14GAP have strengthened the therapeutic potential of CAPHs in the hopes of addressing the need for novel antibiotics with the propensity to eradicate intracellular pathogens.

Funding

National Science Foundation

National Cancer Institute

Find out more...

History

Degree Type

Doctor of Philosophy

Department

Chemistry

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Jean Chmielewski

Advisor/Supervisor/Committee co-chair

David Thompson

Additional Committee Member 2

Betsy Parkinson

Additional Committee Member 3

Herman Sintim