Central Role of the Copper-Binding Motif in the Complex Mechanism of Action of Ixosin: Enhancing Oxidative Damage and Promoting Synergy with Ixosin B

Abstract

Ticks transmit multiple pathogens to different hosts without compromising their health. Their ability to evade microbial infections is largely a result of their effective innate immune response including various antimicrobial peptides. Therefore, a deep understanding of how ticks (and other arthropod vectors) control microbial loads could lead to the design of broad-spectrum antimicrobial agents. In this paper we study the role of the aminoterminal copper and nickel (ATCUN)-binding sequence in the peptide ixosin, isolated from the salivary glands of the hard tick Ixodes sinensis. Our results indicate that the ATCUN motif is not essential to the potency of ixosin, but is indispensable to its oxidative mechanism of action. Specifically, the ATCUN motif promotes dioxygen-and copper-dependent lipid (per)oxidation of bacterial membranes in a temporal fashion coinciding with the onset of bacterial death. Microscopy and studies on model membranes indicate that the oxidized phospholipids are utilized as potential targets of ixosin B (another tick salivary gland peptide) involving its delocalization to the bacterial membrane, thus resulting in a synergistic effect. Our proposed mechanism of action highlights the centrality of the ATCUN motif to ixosin's mechanism of action and demonstrates a novel way in which (tick) antimicrobial peptides (AMPs) utilize metal ions in its activity. This study suggests that ticks employ a variety of effectors to generate an amplified immune response, possibly justifying its vector competence.

Membrane Oxidation in Cell Delivery and Cell Killing Applications

“…The native ixosin B localization was restored when an ixosin derivative harboring a point mutation that abolishes copper-binding and ROS formation was used. 110 Together, these results indicate that the oxidation mediated by ixosin leads to retention of ixosin B at the membrane. It is then possible that membrane oxidation promotes the lytic behavior of the peptide, and ultimately enhances its bactericidal activity.…”

“…107 The relevance of such phenomenon was highlighted in a report that studied two antimicrobial peptides, ixosin and ixosin B, isolated from the tick Ixodes sinensis . 108–110 The study demonstrated the change in the localization of ixosin B as a result of membrane oxidation by ixosin. Ixosin contains a sequence known as the amino-terminal copper and nickel binding (ATCUN) motif that binds copper ions with high affinity (GLHKVMREVLGYERNSYKKFFLR, Figure 6A).…”

How Does Membrane Oxidation Affect Cell Delivery and Cell Killing?

“…The peptides ixosin (GLHKVMREVLGYERNSYKKFFLR-NH 2 ) and ixosin B (QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY-NH 2 ), isolated from the salivary glands of the tick Ixodes sinensis , act synergistically by exploiting bacterial membrane oxidation [15]. The first three amino acids in the sequence of ixosin, Gly-Lys-His, have high affinity for copper ions.…”

“…Metal chelators that outcompete ixosin for copper ions inhibit the oxidation of the lipid membrane. Once the lipids are oxidized, ixosin B exhibits an increased affinity for the membrane resulting in a synergistic effect (assessed via fractional inhibitory concentration, or FIC, indices) between the two ixosins [15]. It should be noted that FIC indices corresponding to synergistic interactions represent a minimum of 4-fold increase in potency.…”

Metal‐ion Binding to Host Defense Peptide Piscidin 3 Observed in Phospholipid Bilayers by Magic Angle Spinning Solid‐state NMR

“…The ATCUN motif is found in many peptides and proteins . The metal‐bound ATCUN complexes have been shown to cleave DNA and RNA and inactivate enzymes ,. The stability of the complex is governed by the basicity of the N‐terminal amino group because of the competition between 1 H and Cu 2+ coordination.…”