Discovery, Isolation, and Bactericidal Activity of a Cyclotide from Spigelia anthelmia L. (Loganiaceae)

Cyclotides are plant-derived macrocyclic peptides stabilized by a cystine-knot motif, found in a limited number of angiosperm plants. This study reports the discovery of the cyclotide, Spat1, from Spigelia anthelmia (Loganiaceae), expanding the phylogenetic range of known cyclotide-producing plants. Spat1, a 30-residue bracelet-type cyclotide, was isolated, purified, and sequenced de novo. It demonstrated strong bactericidal activity against the Gram-positive Bacillus subtilis (LC_99.9 = 20 μM) via rapid membrane disruption but showed no activity against Staphylococcus aureus or Gram-negative Escherichia coli (LC_99.9 > 400 μM). The selective lack of activity against S. aureus is unusual for antimicrobial peptides. The data suggest that Spat1's activity is independent of lipoteichoic acid (LTA) in B. subtilis, suggesting that its mechanism involves interactions with cytoplasmic membrane phospholipids. The lack of phosphatidylethanolamine (PE) in S. aureus membranes and Spat1's weak binding to LTA, combined with its low net positive charge (+1), likely explains its inefficacy against this bacterial species. Structural modeling using AlphaFold AfCycDesign indicated that Spat1 adopts a cyclotide-typical β-sheet architecture and a 3₁₀-helix within its loop regions. Overall, Spat1 broadens understanding of cyclotide diversity and evolution, highlighting their functional specialization and the convergent evolutionary pressures that shape their distribution across plant lineages.