Abstract
Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.
Highlights
In addition to cationic antimicrobial peptide (AMP), there are some anionic AMPs with documented activity against a range of microorganisms (6 –11)
The membrane has been described as the main target for many AMPs, there are several reports showing that AMPs may translocate through the bacterial membrane and act on intracellular targets, leading to inhibition of specific
Native and recombinant microplusin were active against M. luteus, with MIC100 of 0.38 M for the recombinant and 0.78 M for the native peptide
Summary
The recombinant and native microplusin were obtained as previously described [27, 29]. M. luteus was preincubated with 0.18 M microplusin for 24 h, and 2-fold serial dilutions of CuCl21⁄76H2O (0.18 – 0.03 M) were added After another 24 h of incubation, the bacterial growth was recorded. After 24 h of incubation, bacteria were washed three times in sterile phosphate-buffered saline (136 mM NaCl, 2.6 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.4) by centrifugation (2500 ϫ g, 5 min) and suspended in LB broth (with or without microplusin). After another 24-h incubation at 30 °C, aliquots were taken for CFU evaluation.
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