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Abstract Cathelicidins are a major family of antimicrobial peptides present in vertebrate animals with potent microbicidal and immunomodulatory activities. Four cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1, have been identified in chickens. As a first step to understand their role in early innate host defense of chickens, we examined the tissue and developmental expression patterns of all four cathelicidins. Real-time PCR revealed an abundant expression of four cathelicidins throughout the gastrointestinal, respiratory, and urogenital tracts as well as in all primary and secondary immune organs of chickens. Fowlicidins 1 to 3 exhibited a similar tissue expression pattern with the highest expression in the bone marrow and lung, while cathelicidin B1 was synthesized most abundantly in the bursa of Fabricius. Additionally, a tissue-specific regulatory pattern was evident for all four cathelicidins during the first 28 days after hatching. The expression of fowlicidins 1 to 3 showed an age-dependent increase both in the cecal tonsil and lung, whereas all four cathelicidins were peaked in the bursa on day 4 after hatching, with a gradual decline by day 28. An abrupt augmentation in the expression of fowlicidins 1 to 3 was also observed in the cecum on day 28, while the highest expression of cathelicidin B1 was seen in both the lung and cecal tonsil on day 14. Collectively, the presence of cathelicidins in a broad range of tissues and their largely enhanced expression during development are suggestive of their potential important role in early host defense and disease resistance of chickens.

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 Chem 281, 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. [ABSTRACT FROM AUTHOR]

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 four-exon, 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 μM 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 μM. 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 antisepsis agents. [ABSTRACT FROM AUTHOR]

Fowlicidins are a group of newly identified chicken cathelicidin host defense peptides. We have shown that the putatively mature fowlicidin-2 of 31 amino acid residues possesses potent antibacterial and lipopolysaccharide (LPS)- neutralizing activities, but with a noticeable toxicity to mammalian cells. As a first step in exploring the structure-activity relationships of fowlicidin-2, in this study we determined its tertiary structure by nuclear magnetic resonance spectroscopy. Unlike the majority of cathelicidins, which are composed of a predominant alpha-helix with a short hinge sequence near the center, fowlicidin-2 consists of 2 well-defined alpha-helical segments (residues 6-12 and 23-27) connected by a long extensive kink (residues 13-20) induced by proline. To further investigate the functional significance of each of these structural components, several N- and C-terminal deletion analogs of fowlicidin-2 were synthesized and analyzed for their antibacterial, cytotoxic and LPS-neutralizing activities. Our results indicated that neither the N- nor C-terminal alpha-helix alone is sufficient to confer any function. Rather, fowlicidin-2(1-18) and fowlicidin-2(15-31), 2 alpha-helical segments with inclusion of the central cationic kink region, retained substantial capacities to kill bacteria and neutralize the LPS-induced proinflammatory response, relative to the parent peptide. More desirably, these 2 peptide analogs showed substantially reduced toxicity to human erythrocytes and epithelial cells, indicative of improved potential as antibacterial and antisepsis agents. To our knowledge, fowlicidin-2 is the first alpha-helical cathelicidin, with the central kink region shown to be critically important in killing bacteria and neutralizing LPS., (Copyright 2008 S. Karger AG, Basel.)

Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities. Fowlicidin-1 and fowlicidin-2 are two newly identified chicken cathelicidins with potent antibacterial activities. Here we report structural and functional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin-3, for exploration of its therapeutic potential. NMR spectroscopy revealed that fowlicidin-3 comprises 27 amino-acid residues and adopts a predominantly alpha-helical structure extending from residue 9 to 25 with a slight kink induced by a glycine at position 17. It is highly potent against a broad range of Gram-negative and Gram-positive bacteria in vitro, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range 1-2 microM. It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them. Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin-3 retains bacteria-killing activities in the presence of 50% serum or physiological concentrations of salt. Furthermore, it is capable of suppressing lipopolysaccharide-induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 microM. Fowlicidin-3 appears to be an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic-resistant pathogens.