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

## TL;DR

A new cyclotide called Spat1 was discovered in Spigelia anthelmia and shown to kill certain bacteria by disrupting their membranes.

## Contribution

Spat1 expands the known range of cyclotide-producing plants and reveals unique antimicrobial properties.

## Key findings

- Spat1 is a 30-residue bracelet-type cyclotide with strong bactericidal activity against Bacillus subtilis.
- Spat1 shows no activity against Staphylococcus aureus or Escherichia coli.
- Structural modeling suggests Spat1 has a typical cyclotide β-sheet and a 310-helix in its loops.

## Abstract

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 (LC99.9 = 20 μM)
via rapid membrane disruption but showed no activity against Staphylococcus aureus or Gram-negative Escherichia coli (LC99.9 > 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.