Abstract
Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain. Here we report that the entire chicken genome encodes three cathelicidins, namely fowlicidin-1 to -3, which are densely clustered within a 7.5-kb distance at the proximal end of chromosome 2p. Each fowlicidin gene adopts a fourexon, three-intron structure, typical for a mammalian cathelicidin. Phylogenetic analysis revealed that fowlicidins and a group of distantly related mammalian cathelicidins known as neutrophilic granule proteins are likely to originate from a common ancestral gene prior to the separation of birds from mammals, whereas other classic mammalian cathelicidins may have been duplicated from the primordial gene for neutrophilic granule proteins after mammals and birds are diverged. Similar to ovine cathelicidin SMAP-29, putatively mature fowlicidins displayed potent and salt-independent activities against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range of 0.4-2.0 microm for most strains. Fowlicidin-1 and -2 also showed cytotoxicity, with 50% killing of mammalian erythrocytes or epithelial cells in the range of 6-40 microm. In addition, two fowlicidins demonstrated a strong positive cooperativity in binding lipopolysaccharide (LPS), resulting in nearly complete blockage of LPS-mediated proinflammatory gene expression in RAW264.7 cells. Taken together, fowlicidin-1 and -2 are clearly among the most potent cathelicidins that have been reported. Their broad spectrum and salt-insensitive antibacterial activities, coupled with their potent LPS-neutralizing activity, make fowlicidins excellent candidates for novel antimicrobial and anti-sepsis agents.
Highlights
Phagocytic cells and to a lesser extent in many other cell types such as mucosal epithelial cells and skin keratinocytes [7,8,9]
Because the N-terminal sequence including the start codon of fowlicidin-2 was missing in GenBankTM, a genome walking approach known as vectorette PCR was performed by using chicken genomic DNA as previously described [30, 31]
Three chicken cathelicidins consist of linear cationic sequences at the C termini, which are expected to be freed from the cathelin domain to become biologically active
Summary
Computational Search for Novel Chicken Cathelicidins—To identify potential novel cathelicidins in the chicken, all known cathelicidin peptide sequences discovered in the hagfish and mammals were individually queried against the translated chicken expressed sequence tags (EST), nonredundant sequences, unfinished high throughput genomic sequences, and whole genome shotgun sequences (WGS) in GenBankTM by using the TBLASTN program [25] as we described (26 –28). The missing first intron sequence of the fowlicidin-3 gene (i.e. the gap between AADN01005055 and AADN01005056) was cloned from chicken genomic DNA by PCR using primers Of the Chicken Cathelicidin Gene Cluster—To confirm the orientation of three fowlicidin genes on the chromosome, additional PCRs were performed to clone the intergenic sequences with chicken genomic DNA and combinations of gene-specific primers located in the first and last exons of each fowlicidin gene. Bacteria were subcultured to the midlog phase, washed with 10 mM sodium phosphate buffer, and suspended to 5 ϫ 105 CFU/ml in 1% cation-adjusted Mueller Hinton broth (BBL, Cockeysville, MD) with and without 100 mM of NaCl. Bacteria (90 l) were dispensed into 96-well plates, followed by the addition in duplicate of 10 l of serially diluted peptides in 0.01% acetic acid. Melting curve analysis (55–95 °C) was performed and confirmed no visible nonspecific amplification of any PCR products from genomic DNA or primer dimers
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
