ETD135

Both vertebrates and invertebrates employ alpha-helical antimicrobial peptides (AMPs) as an essential component of their innate immune system. However, evolutionary relation of these immune molecules remains unresolved. Venoms, as key weapons of venomous arthropods for prey and defense, receive increasing recognition as an emerging source of such peptides. From a cDNA library prepared from the venom gland of the scorpion Mesobuthus eupeus, clones encoding precursors of two new AMPs, named meucin-13 (IFGAIAGLLKNIF-NH(2)) and meucin-18 (FFGHLFKLATKIIPSLFQ), have been isolated. The precursor of meucins consists of a signal peptide, a mature peptide, and an acidic propeptide, in which dibasic residues as the typical processing signal are located between the mature and propeptide. Meucin-13 is an ortholog of several previously described AMPs from scorpion venom and has also detectable sequence similarity to temporins, a large family of AMPs from frog skin, whereas meucin-18 displays some similarity to AMPs from diverse origin including arthropod venoms, fish mast cells, and frog skins. These two meucin peptides form alpha-helical structure in the presence of 50% trifluoroethanol (TFE), a membrane-mimicking environment, as identified by circular dichroism (CD) spectroscopy. This finding is further verified by their NMR structures that show a typical alpha-helical amphipathic design, a structural prerequisite for cytolytic activity. Meucins exhibit extensive cytolytic effects on both prokaryotic and eukaryotic cells (gram(+) and gram(-) bacteria, fungi, yeasts, rabbit erythrocytes, and rat dorsal root ganglion cells) at micromolar concentrations. It is remarkable that muecin-18 was 2- to >14-fold more potent than meucin-13 against nearly all the cells tested. Structural differences in hydrophilic/hydrophobic balance and cationic amino acid location between two meucins could account for their differential potency. Despite these differences, commonalities at precursor organization, three-dimensional structure, and biological function suggests that meucins are two evolutionarily related AMPs and likely originated from a common ancestor by gene duplication. Our work presented here also provides new insights into an evolutionary link among AMPs from invertebrates and vertebrates and clues for evolutionary convergence between AMPs and virus fusion domains.

Insect peptides are key elements of the innate immunity against bacteria and fungi. These molecules offer remarkable properties: high efficacy, a low probability of resistance, limited toxicity, and immunogenicity. In this context, we are investigating several classes of peptides, and we have been successful in identifying biologically important classes of peptides and small molecules that will provide a stream of drug candidates for treating severe, life-threatening, hospital-acquired infections and other pathologies of high medical need. Recently, we have isolated a new class of antifungal peptides from the coleopteran Acrocinus longimanus. Three homologous peptides, Alo-1, Alo-2, and Alo-3, with sequence identity above 80% and active against the Candida glabrata yeast strain were identified. Alo-3 displayed the highest activity against Candida glabrata and was thus chosen for structure determination using NMR spectroscopy and molecular modeling. Alo-3 contains six cysteine residues forming three disulfide bridges. The pairing of the cysteines was assessed using ambiguous disulfide restraints within the ARIA software, allowing us to establish that Alo-3 belongs to the inhibitor cystine-knot family. It exhibits all the structural features characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first strand to the second strand and a series of turns. To our knowledge, Alo-3 is the first peptide from insects with antimicrobial activity adopting the knottin fold. Alo-3 shows a level of activity significantly higher against C. glabrata than Alo-1 or Alo-2. It has no negatively charged residues and displays on its surface a cationic pole that may account for its antifungal activity. This finding is validated by the comparison of the structure of Alo-3 with the structure of other structurally related peptides from other sources also showing antifungal activity.

Candida albicans is one of the most prevalent fungal pathogens, causing both mucosal candidiasis and invasive candidemia. Antimicrobial peptides (AMPs), part of the human innate immune system, have been shown to exhibit antifungal activity but have not been effective as pharmaceuticals because of low activity and selectivity in physiologically relevant environments. Nevertheless, studies on α-peptide AMPs have revealed key features that can be designed into more stable structures, such as the 14-helix of β-peptide-based oligomers. Here, we report on the ways in which two of those features, hydrophobicity and helicity, govern the activity and selectivity of 14-helical β-peptides against C. albicans and human red blood cells. Our results reveal both antifungal activity and hemolysis to correlate to hydrophobicity, with intermediate levels of hydrophobicity leading to high antifungal activity and high selectivity toward C. albicans. Helical structure-forming propensity further influenced this window of selective antifungal activity, with more stable helical structures eliciting specificity for C. albicans over a broader range of hydrophobicity. Our findings also reveal cooperativity between hydrophobicity and helicity in regulating antifungal activity and specificity. The results of this study provide critical insight into the ways in which hydrophobicity and helicity govern the activity and specificity of AMPs and identify criteria that may be useful for the design of potent and selective antifungal agents.