Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Doron C. Greenbaum

Abstract

Antimicrobial peptides (AMPs) are small (2-8kDa) peptides that have remained an important part of innate immunity over evolutionary time. AMPs vary greatly in sequence and structure, but display broad-spectrum activity against bacteria, yeast, and fungi via direct perturbation of the pathogen membrane. AMPs are typically amphipathic, and contain 2-4 positively charged amino acid residues. It has been previously shown that many chemokines also display AMP activity, both via their canonical function recruiting immune cells to the site of an infection and by direct interaction with the pathogen itself. Many antimicrobial chemokines have the same tertiary structure: an N-terminal loop responsible for receptor recognition; a three-stranded antiparallel β-sheet domain that provides a stable scaffold; and a C-terminal α-helix that folds over the β-sheet and helps to stabilize the overall structure. The α-helix of these chemokines tends to be amphipathic and retains AMP activity on its own. The work presented here focuses on two antimicrobial chemokines - platelet factor 4 (CXCL4/PF4) and macrophage inflammatory protein-3α (CCL20/MIP-3α) - and shows that they have activity against the malaria parasite. Chapter 2 details the exploration into the mechanism of action of PF4 against P. falciparum, and the translation of this mechanism to small molecules that mimic antimicrobial peptides. Additionally, this antimalarial activity is also present in a mouse model of malaria. Chapter 3 focuses on finding additional chemokines with antimalarial activity, and explores the structure-function relationship between the conserved domains and how they modulate i) the antimalarial activity, and ii) provide a protective scaffold to prevent harm to host cells. We provide evidence that PF4 and small molecule mimics of AMPs kill parasites by exclusively lysing the digestive vacuole. Furthermore, the C-terminal α-helix of PF4 and CCL20 are the active components of each chemokine, and are necessary for parasite killing. The N-terminal loop is necessary to stabilize the tertiary structure of each chemokine, providing a stable scaffold that aides in the selectivity to protect host cells. Taken together, these data suggest that antimicrobial chemokines and small molecule mimics may provide an interesting scaffold and mechanism for potential therapeutics for the fight against malaria.

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