title: Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution.
abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized α-helical and β-sheet (CSαβ) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76μM. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.
authors.affiliation: Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Gao
authors.forename: Shunyi
authors.initials: S
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology*; D001410:Bacillus megaterium / Q000187:drug effects / Q000648:ultrastructure; D002462:Cell Membrane / Q000187:drug effects* / Q000648:ultrastructure; D023082:Defensins / Q000737:chemistry* / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D017433:Protein Structure, Secondary
chemical_list: D000900:Anti-Bacterial Agents; D023082:Defensins; D010455:Peptides; C574456:micasin