title: Immunostimulating and Gram-negative-specific antibacterial cyclotides from the butterfly pea (Clitoria ternatea).
abstract: Cyclotides are plant-derived, cyclic miniproteins with three interlocking disulfide bonds that have attracted great interests because of their excellent stability and potential as peptide therapeutics. In this study, we characterize the cyclotides of the medicinal plant Clitoria ternatea (butterfly pea) and investigate their biological activities. Using a combined proteomic and transcriptomic method, we identified 41 novel cyclotide sequences, which we named cliotides, making C. ternatea one of the richest cyclotide-producing plants to date. Selected members of the cationic cliotides display potent antibacterial activity specifically against Gram-negative bacteria with minimal inhibitory concentrations as low as 0.5 μm. Remarkably, they also possess prominent immunostimulating activity. At a concentration of 1 μm, cationic cliotides are capable of augmenting the secretion of various cytokines and chemokines in human monocytes at both resting and lipopolysaccharide-stimulated states. Chemokines such as macrophage inflammatory proteins 1α and 1β, interferon γ-induced protein 10, interleukin 8 and tumor necrosis factor α were among the most upregulated with up to 129-fold increase in secretion level. These findings suggest cyclotides can serve as potential candidates for novel immunomodulating therapeutics.
  
  
  
  The protein sequences reported in this paper (cT13-cT21) are available in the UniProt Knowledgebase under the accession numbers C0HJS0, C0HJS1, C0HJS2, C0HJS3, C0HJS4, C0HJS5, C0HJS6, C0HJS7 and C0HJS8, respectively. The transcriptome data in this paper are available at the Sequence Read Archive database (NCBI) under accession number SRR1613316. The protein precursors reported in this paper (ctc13, ctc15, ctc17-ctc19, ctc21-ctc53) are available at GenBank under the accession numbers KT732712, KT732713, KT732714, KT732715, KT732716, KT732717, KT732718, KT732719, KT732720, KT732721, KT732722, KT732723, KT732724, KT732725, KT732726, KT732727, KT732728, KT732729, KT732730, KT732731, KT732732, KT732733, KT732734, KT732735, KT732736, KT732737, KT732738, KT732739, KT732740, KT732741, KT732742, KT732743, KT732744, KT732745, KT732746, KT732747, KT732748 and KT732749, respectively.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: Kim Ngan T
authors.initials: KN
authors.lastname: Nguyen
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: Giang Kien Truc
authors.initials: GK
authors.lastname: Nguyen
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: Phuong Quoc Thuc
authors.initials: PQ
authors.lastname: Nguyen
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: Koon Hwee
authors.initials: KH
authors.lastname: Ang
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.
authors.forename: James P
authors.initials: JP
authors.lastname: Tam
mesh_terms: D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D031292:Clitoria / Q000737:chemistry / Q000235:genetics*; D047169:Cyclotides / Q000235:genetics* / Q000627:therapeutic use; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006801:Humans; D007114:Immunization; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000627:therapeutic use; D011498:Protein Precursors / Q000235:genetics; D040901:Proteomics
chemical_list: D000900:Anti-Bacterial Agents; D047169:Cyclotides; D010936:Plant Extracts; D010940:Plant Proteins; D011498:Protein Precursors
keywords: antimicrobial peptide; cyclotide; cytokine; immunomodulatory; transcriptomic