An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides

Abstract

Four macrocyclic cystine-knot peptides of 29-31 residues, kalata, circulin A and B (CirA and CirB), and cyclopsychotride, have been isolated from coffee plants but have undetermined physiological functions. These macrocycles and 10 of their analogs prepared by chemical synthesis were tested against nine strains of microbes. Kalata and CirA were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of Ϸ0.2 M. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, CirB and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, CirB showed potent activity against E. coli with a minimum inhibitory concentration of 0.41 M. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans. These macrocycles are cytotoxic and lysed human red blood cell with a lethal dose 50% of 400 M. Modifying the Arg residue in kalata with a keto aldehyde significantly reduced its activity against S. aureus whereas blocking the arg in CirA produced no significant effect. The two-disulfide variants and their scrambled disulfide isomers exhibited antimicrobial profiles and potency similar to their native peptides. However, in high-salt assays (100 mM NaCl), few of these macrocyclic peptides, natives or analogs, retained antimicrobial activity. These results show that the macrocyclic peptides possess specific and potent antimicrobial activity that is saltdependent and that their initial interactions with the microbial surfaces may be electrostatic, an effect commonly found in defensin antimicrobial peptides. Furthermore, their endto-end cyclic structure with a cystine-knot motif represents a molecular structure of antimicrobials and may provide a useful template for the design of novel peptide antibiotics.Although cyclic peptides are produced naturally in microbes and plants, they are usually restricted to small and medium size peptides of Ͻ15 amino acids. Thus, it is interesting to note the recent discovery of four large end-to-end cyclic peptides of 29-31 amino acids from plants of the Rubiacease family. These peptides contain six cysteines that can be aligned and share Ϸ45% of sequence homology (Fig.

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

“…Boc-, 38 microwave-enhanced 24 and Fmoc- 14,30,34,35 based solid-phase peptide synthesis (SPPS) have all been used for cyclotide backbone construction, whilst recent work has shown that bacterial expression can also be an effective approach for 20 smaller scale production of the cyclotide backbone. 25,28 Backbone cyclisation may then be achieved by exposing a suitably side chain protected backbone peptide bearing an amino N-terminus and a carboxy C-terminus to standard peptide coupling conditions, but this approach suffers from 25 multiple drawbacks including poor peptide solubility and the need for high dilution.…”

Dissecting Defense-Related and Developmental Transcriptional Responses of Maize during Ustilago maydis Infection and Subsequent Tumor Formation

“…Cyclotides are structurally related to plant defensins, which occur ubiquitously throughout the plant kingdom (Craik et al, 1999, and refs. therein;Trabi and Craik;, and antimicrobial activity against several fungal species has been shown (Tam et al, 1999;Jennings et al, 2001). Thus, it will be interesting to determine whether this also accounts for the putative maize cyclotide.…”

Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae

“…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 develop past the second instar stage when raised on artificial diets containing the cyclotide kalata B1 (13).…”