Carnocyclin A

Carnocyclin A (CclA) is a potent antimicrobial peptide from Carnobacterium maltaromaticum UAL307 that displays a broad spectrum of activity against numerous Gram-positive organisms. An amide bond links the N and C termini of this bacteriocin, imparting stability and structural integrity to this 60-amino acid peptide. CclA interacts with lipid bilayers in a voltage-dependent manner and forms anion selective pores. Several other circular bacteriocins have been reported, yet only one (enterocin AS-48) has been structurally characterized. We have now determined the solution structure of CclA by NMR and further examined its anion binding and membrane channel properties. The results reveal that CclA preferentially binds halide anions and has a structure that is surprisingly similar to that of AS-48 despite low sequence identity, different oligomeric state, and disparate function. CclA folds into a compact globular bundle, comprised of four helices surrounding a hydrophobic core. NMR studies show two fluoride ion binding modes for CclA. Our findings suggest that although other circular bacteriocins are likely to have diverse mechanisms of action, many may have a common structural motif. This shared three-dimensional arrangement resembles the fold of mammalian saposins, peptides that either directly lyse membranes or serve as activators of lipid-degrading enzymes.

Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and alpha-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.

Bacterial resistance to conventional antibiotics is a major challenge in controlling infectious diseases and has necessitated the development of novel approaches in antimicrobial therapy. One such approach is the use of antimicrobial peptides, such as the bacterially produced bacteriocins. Carnocyclin A (CclA) is a 60-amino acid circular bacteriocin produced by Carnobacterium maltaromaticum UAL307 that exhibits potent activity against many Gram-positive bacteria. Lipid bilayer and single channel recording techniques were applied to study the molecular mechanisms by which CclA interacts with the lipid membrane and exerts its antimicrobial effects. Here we show that CclA can form ion channels with a conductance of 35 pS in 150 mM NaCl solution. This channel displays a linear current-voltage relationship, is anion-selective, and its activation is strongly voltage-dependent. The formation of ion channels by CclA is driven by the presence of a negative membrane potential and may result in dissipation of membrane potential. Carnocyclin A's unique functional activities as well as its circular structure make it a potential candidate for developing novel antimicrobial drugs.