Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae

Abstract

Several members of the Rubiaceae and Violaceae plant families produce a series of cyclotides or macrocyclic peptides of 28 -37 aa with an embedded cystine knot. The cyclic peptide backbone together with the knotted and strongly braced structure confers exceptional chemical and biological stability that has attracted attention for potential pharmaceutical applications. Cyclotides display a diverse range of biological activities, such as uterotonic action, anti-HIV activity, and neurotensin antagonism. In plants, their primary role is probably protection from insect attack. Ingestion of the cyclotide kalata B1 severely retards the growth of larvae from the Lepidopteran species Helicoverpa armigera. We examined the gut of these larvae after consumption of kalata B1 by light, scanning, and transmission electron microscopy. We established that kalata B1 induces disruption of the microvilli, blebbing, swelling, and ultimately rupture of the cells of the gut epithelium. The histology of this response is similar to the response of H. armigera larvae to the Bacillus thuringiensis delta-endotoxin, which is widely used to control these insect pests of crops such as cotton. circular peptides ͉ insecticidal ͉ kalata B1 ͉ Lepidoptera ͉ microscopy C yclotides are a series of cyclic minipeptides of 28-37 aa that are expressed at high levels in the leaves, stems, and roots of several plant species (1, 2). They have an unusual topology based on a cyclic peptide backbone together with a cystine knot in which two disulfide bonds are threaded by a third disulfide bond (2, 3). This structure forces hydrophobic amino acids onto the surface of the molecule, creating a hydrophobic face on the otherwise hydrophilic surface. The cyclotides are thus soluble in both organic and aqueous solvents and are very stable at extremes of pH and temperature (4). The absence of free N and C termini, together with the tightly packed cystine knot, also renders the cyclotides resistant to the activity of proteases (4). The cyclotides are the largest family of circular proteins, although others have been described in bacteria, plants, and animals (5).Cyclotides were first isolated from the African plant Oldenlandia affinis. Two peptides, kalata B1 and B2, were recognized as the active components in a traditional medicine used to accelerate childbirth (6). Additional members of the cyclotide family were subsequently identified in screening studies directed toward discovery of bioactive molecules such as neurotensin antagonists (7), inhibitors of HIV replication (8), and hemolytic agents (9). More than 100 cyclotides have since been isolated from various members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families (10, 11). A single plant may have at least 12 cyclotide genes and produce dozens of different cyclotides (12)(13)(14). Although an earlier study reported antimicrobial activity (15), it appears that their predominant activity in plants is insecticidal (13, 16). Jennings et al. (13) demonstrated that Helicoverpa punctigera failed to devel...

Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions

“…The physiological role of cyclotides has been suggested to be as insecticidal agents (69) with a mode of action involving membrane disruption (70), and this is consistent with multiple cyclotides having communal lipid recognition. The observation that one plant produces many cyclotides is puzzling in light of these findings if insecticidal activity is the sole function of cyclotides.…”

Decoding the Membrane Activity of the Cyclotide Kalata B1

“…Such a mechanism of action is consistent with their hemolytic properties (6) and ability to disrupt gut epithelial cells in lepidopteron larvae (3) and is further supported by a range of biophysical studies (12)(13)(14)(15). The prototypic cyclotide kalata B1 (kB1) 7 is the most well studied cyclotide, and it has been shown to bind to (12) and disrupt phospholipid bilayers by a pore-forming mechanism (15).…”

“…2 A National Health and Medical Research Council Biomedical Career Development Award Fellow. 3 Recipient of a Fundaçao para a Ciê ncia e Tecnologia scholarship (SFRH/BD/39039/2007). 4 A National Health and Medical Research Council Senior Research Fellow.…”

“…insecticidal agents (3), but many other activities have also been reported, including uterotonic (4), anti-HIV (5), hemolytic (6), antibacterial (7), anti-cancer (8), and pesticidal action (9). These activities reflect the ability of cyclotides to target cells of different compositions.…”

Structure and Activity of the Leaf-Specific Cyclotide vhl-2

“…[15] In addition, kalata B1 and B2 have been shown to inhibit the growth and development of Helicoverpa punctigera and H. armigera larvae, suggesting that cyclotides function as insecticidal agents as part of plant defence systems. [16][17][18][19] A presumed role in plant defence is also supported by recent reports of their molluscidal activity against the Golden Apple snail, a major pest of rice in South East Asia, [20] and also by their anthelmintic activities. [21,22] More broadly, their unique structural features, stability and diverse bioactivities suggest that cyclotides might be promising templates for drug design and agrochemical applications.…”