Targeting nutritional immunity: microplusin fragments as potent copper chelators for antimicrobial activity

The rapid emergence of antimicrobial resistance (AMR) poses a critical global public health crisis. As pathogens evolve powerful resistance mechanisms, the effectiveness of once-reliable drugs is quickly diminishing, making many infectious diseases increasingly difficult to treat. Consequently, an important aim for researchers is the urgent identification and design of novel compounds capable of fighting these resistant strains. Antimicrobial peptides (AMPs) represent highly promising candidates for drug development due to their broad-spectrum activity, different mechanisms of action, and, crucially, their low propensity to induce 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 α-helices with disordered N- and C-termini. It contains six cysteine residues forming three disulfide bonds and histidine-rich regions in both the N- and C-terminal domains [1]. This peptide shows a broad spectrum of activity, being effective against Gram-positive and Gram-negative bacteria, fungi and yeasts at micromolar and submicromolar concentrations [2]. Different studies indicate that microplusin does not disrupt bacterial membranes, but it acts as a potent chelator of copper ions. The ability of an AMP to chelate metal ions can confer it an antimicrobial ability: by sequestrating these essential micronutrients from the surrounding environment, the 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]. In order to shed light on the action mechanism of microplusin as antimicrobial peptide, we decided to study two short fragments of this protein, corresponding to the N-terminal (HHQEL) and C-terminal (DPEAHHEHDH) domains. Both terminal sequences are naturally unfolded and rich in histidine residues and lie on the same side of the three-dimensional structure. Previous NMR studies suggested a preferred binding site at the N-terminus, but an unequivocal and complete description of the bioinorganic chemistry of microplusin is still lacking. By means of different experimental techniques (mass spectrometry, potentiometry, UV-Vis spectrophotometry and circular dichroism) we have characterized the complexes formed by the Cu(II) ion with the selected fragments and we have been able to compare the Cu(II) binding behaviour of the two putative N- and C- terminal sequences of microplusin.