Structure–activity relationships of fowlicidin‐1, a cathelicidin antimicrobial peptide in chicken

Abstract

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti‐infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad‐spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858–2867). Here we report that fowlicidin‐1 mainly adopts an α‐helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin‐1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)‐binding activities. The short C‐terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16–23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5–7) in the N‐terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin‐1 analog, with deletion of the second N‐terminal segment (residues 5–7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti‐infective agent.

Structure and Function of Papiliocin with Antimicrobial and Anti-inflammatory Activities Isolated from the Swallowtail Butterfly, Papilio xuthus

“…Cathelicidin-derived AMPs with outstanding anti-inflammatory activity, such as LL-37, CAP18, SMAP-29, and fowlicidin-1, are also known to have high LPSbinding affinity and neutralize LPS, measured in quantitative chromogenic Limulus amoebocyte lysate assays (79). Recent transferred NOE, STD-NMR, and Carr-Purcell-Meiboom-Gill relaxation-dispersion experiments have determined the threedimensional structure of pardaxin4 (Pa4), identified the amino acid residues responsible for interacting with LPS, and established the binding kinetics of the Pa4-LPS complex (78).…”

Rattusin, an Intestinal α-Defensin-Related Peptide in Rats with a Unique Cysteine Spacing Pattern and Salt-Insensitive Antibacterial Activities

“…Briefly, Caco-2 cells were seeded into the wells of a 96-well plate at 5 ϫ 10 4 /ml of Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and grown overnight in a humidified Rattusin GAAGACACTTGTCCTCCTTTCTG ATGTGGACCTTGATAGCCGATG AY623756 261 MMP7 AGTGGACAAACTGAGGGAAATG ACTAAGAACCGAGGCAAGTCTG NM_012864 210 Anionic trypsin GTTGGAGGATACACCTGCCCG CTTGATGATCTTGGCAGCATTG NM_012635 213 Mesotrypsin GGAGGATACACCTGCCAAGAGA CTTGATGATCTTGGCAGCATTG NM_001108626 210 Cationic trypsin ATTGATGTCGTTGAGGGTGGT GAAGCAGTGAAGGGTAGTTCGT NM_173127 244 GAPDH GTGAAGGTCGGAGTGAACG GAGATGATGACCCTCTTGGC NM_017008 356 5% CO 2 incubator. Cells were washed once with DMEM, and then fresh DMEM containing 100 M rattusin, Crp4, or chicken fowlicidin-1 (32,33) in the presence or absence of 10% FBS was added. After 18 h of incubation, 10 l of alamarBlue dye was added and the cells were incubated for another 6 h at 37°C.…”

“…The toxicity of rattusin to human Caucasian colon adenocarcinoma (Caco-2) cells (ATCC, Manassas, VA) was measured with alamarBlue dye (BioSource, Inc., Camarillo, CA) as described previously (33,34). Briefly, Caco-2 cells were seeded into the wells of a 96-well plate at 5 ϫ 10 4 /ml of Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and grown overnight in a humidified Rattusin GAAGACACTTGTCCTCCTTTCTG ATGTGGACCTTGATAGCCGATG AY623756 261 MMP7 AGTGGACAAACTGAGGGAAATG ACTAAGAACCGAGGCAAGTCTG NM_012864 210 Anionic trypsin GTTGGAGGATACACCTGCCCG CTTGATGATCTTGGCAGCATTG NM_012635 213 Mesotrypsin GGAGGATACACCTGCCAAGAGA CTTGATGATCTTGGCAGCATTG NM_001108626 210 Cationic trypsin ATTGATGTCGTTGAGGGTGGT GAAGCAGTGAAGGGTAGTTCGT NM_173127 244 GAPDH GTGAAGGTCGGAGTGAACG GAGATGATGACCCTCTTGGC NM_017008 356 5% CO 2 incubator.…”

Natural Antimicrobial Peptides from Eukaryotic Organisms

“…Some of them present a proline-induced kink in the middle of the alpha helix (Shin et al, 2001). Others have a glycine in the same relative position that has been suggested to give the flexibility needed for the membrane lysis activity (Xiao et al, 2006). This structural plasticity has been defined as a molecular determinant for the antimicrobial vs eucaryont membrane specificity (Shin et al, 1999;Shin et al, 2000;Shin et al, 2001), together with overall net positive charge and hydrophibic moment.…”