Copper-Binding Antimicrobial and Host-Defense Peptides: Microbial Elimination through Lipid and DNA Oxidation

The immune system utilizes a plethora of antimicrobial effectors to ensure efficient microbial eradication. This dissertation focuses on two of these effectors: antimicrobial peptides (AMPs) and copper ions. The work described here examines both fundamental and translational aspects of AMPs that contain the amino terminal copper and nickel (ATCUN) binding unit. The first section details our efforts to define the contribution of the ATCUN motif to the biological activity of peptides that are expressed in nature containing the metal binding sequence. In chapter 2, we show how the metal is utilized by an ATCUN-containing AMP to bring about copper-mediated membrane lipid oxidation. This activity, in turn is utilized by a homologous peptide to achieve synergistic interactions that involve oxidized lipid targeting. In chapter 3, we show the first example of an AMP that can oxidatively cleave microbial DNA in a copper-dependent fashion. This nuclease activity is intimately linked to its expression profile and to that of a homologous, yet mechanistically distinct peptide. These two AMPs show complementary effects associated with oxidation of varying targets in bacteria. The oxidative stress generated by ATCUN-AMPs lead us to speculate that a similar mode of attack can be imparted to other therapeutically relevant peptides. Therefore, our goal in the second section was to generate novel ATCUN-based antibiotics. In chapter 4, we appended the ATCUN motif to membrane-targeting AMPs and in chapter 5, added it to an oligonucleotide-binding peptide. We observed that for both instances, a gain-of-function phenotype resulted from the exogenous addition of the metal-binding sequence, and that microbial killing was directly related to the oxidative activity of the conjugates. Finally, in chapter 6, we developed an ATCUN-based peptidomimetic that can target intracellular Mycobacterium tuberculosis. We reasoned that antibiotics of this nature can exploit the differential trafficking of transition metals during infection, be activated in situ by copper in the cellular milieu and target pathogenic bacteria that evolved to evade the metal’s toxic effects. Overall, this work highlights the biological importance of ATCUN-containing AMPs and expands the catalog by which these peptides utilize transition metals for their activity.