Nowadays, the phenomenon of antimicrobial resistance is a serious public health challenge. The emergence of strong resistance mechanisms among bacteria is rendering many diseases difficult to treat, consequently diminishing the efficacy of previously effective drugs. One of the main aims of researchers is to identify and design novel drugs effective against pathogens. Among potential candidates, antimicrobial peptides (AMPs) represent a very interesting substrate for drug design due to their broad spectrum of activity, different mechanisms of action and low propensity to induce antimicrobial resistance. Natural AMPs are abundant across all kingdoms of life; microplusin is a representative example. Microplusin is an antimicrobial peptide isolated from the thick Rhipicephalus microplus. Structurally, microplusin consists of five -helices with disordered N- and C-termini. It contains six cysteine residues forming three disulfide bonds and a histidine-rich region in both the N- and C-terminal domains [1]. This peptide shows a broad spectrum of activity, being efficacy against Gram-positive and Gram-negative bacteria, fungi and yeast at micromolar and submicromolar concentrations [2]. Different studies indicate that while microplusin does not disrupt bacterial membranes, it acts as a potent chelator of copper ions. The ability of an AMP to chelate metal ions can confer an antimicrobial effect by sequestrating these essential micronutrients from the surrounding environment, thereby depriving pathogens of nutrients for their survival and growth. This mechanism is known as “nutritional immunity”. Furthermore, microplusin has been shown to negatively affect cellular respiration in Micrococcus luteus, most likely through the removal of copper ions from heme-copper terminal oxidases [3]. We decided to study short fragments of this protein, corresponding to the N-terminal (HHQEL) and C-terminal (DPEAHHEHDH) domains. Both terminal sequences are unfolded and rich in histidine residues and lie on the same side of the structure. Previous NMR studies suggest a preferred binding site in the N-terminus, however an unequivocably determination of the binding residues has not been obtained and the bioinorganic chemistry of microplusin still remains unravelled. By means of different experimental techniques (mass spectrometry, potentiometry, UV-Vis spectrophotometry and circular dichroism) we have characterized the formed copper complexes with the selected fragments and compared the Cu(II) binding behavior of the two putative N- and C- terminal sequences of microplusin. Financial support of the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.1 – NextGenerationEU (grant PRIN PNRR 2022-P2022EMY52; CUP F53D23008960001; project title: “EnzyMime – Biocompatible and sustainable enzyme mimics based on metal complexes with peptide derivatives: synthesis, characterization and potential biological applications”) is gratefully acknowledge.
Microplusin: a strong copper-chelating and effective antimicrobial, natural peptide
Silvia LEVERARO;Denise BELLOTTI;Maurizio REMELLI
2025
Abstract
Nowadays, the phenomenon of antimicrobial resistance is a serious public health challenge. The emergence of strong resistance mechanisms among bacteria is rendering many diseases difficult to treat, consequently diminishing the efficacy of previously effective drugs. One of the main aims of researchers is to identify and design novel drugs effective against pathogens. Among potential candidates, antimicrobial peptides (AMPs) represent a very interesting substrate for drug design due to their broad spectrum of activity, different mechanisms of action and low propensity to induce antimicrobial resistance. Natural AMPs are abundant across all kingdoms of life; microplusin is a representative example. Microplusin is an antimicrobial peptide isolated from the thick Rhipicephalus microplus. Structurally, microplusin consists of five -helices with disordered N- and C-termini. It contains six cysteine residues forming three disulfide bonds and a histidine-rich region in both the N- and C-terminal domains [1]. This peptide shows a broad spectrum of activity, being efficacy against Gram-positive and Gram-negative bacteria, fungi and yeast at micromolar and submicromolar concentrations [2]. Different studies indicate that while microplusin does not disrupt bacterial membranes, it acts as a potent chelator of copper ions. The ability of an AMP to chelate metal ions can confer an antimicrobial effect by sequestrating these essential micronutrients from the surrounding environment, thereby depriving pathogens of nutrients for their survival and growth. This mechanism is known as “nutritional immunity”. Furthermore, microplusin has been shown to negatively affect cellular respiration in Micrococcus luteus, most likely through the removal of copper ions from heme-copper terminal oxidases [3]. We decided to study short fragments of this protein, corresponding to the N-terminal (HHQEL) and C-terminal (DPEAHHEHDH) domains. Both terminal sequences are unfolded and rich in histidine residues and lie on the same side of the structure. Previous NMR studies suggest a preferred binding site in the N-terminus, however an unequivocably determination of the binding residues has not been obtained and the bioinorganic chemistry of microplusin still remains unravelled. By means of different experimental techniques (mass spectrometry, potentiometry, UV-Vis spectrophotometry and circular dichroism) we have characterized the formed copper complexes with the selected fragments and compared the Cu(II) binding behavior of the two putative N- and C- terminal sequences of microplusin. Financial support of the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.1 – NextGenerationEU (grant PRIN PNRR 2022-P2022EMY52; CUP F53D23008960001; project title: “EnzyMime – Biocompatible and sustainable enzyme mimics based on metal complexes with peptide derivatives: synthesis, characterization and potential biological applications”) is gratefully acknowledge.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
