Gallinacin 8

Antimicrobial peptides (AMPs), essential components of innate immunity, are found in a range of phylogenetically diverse species and are thought to act by disrupting the membrane integrity of microbes. In this paper, we used evolutionary signatures to identify sites that are most relevant during the functional evolution of these molecules and introduced amino acid substitutions to improve activity. We first demonstrate that the anti-microbial activity of chicken avian beta-defensin-8, previously known as gallinacin-12, can be significantly increased against Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Salmonella typhimurium phoP- mutant and Streptococcus pyogenes through targeted amino acid substitutions, which confer increased peptide charge. However, by increasing the AMP charge through amino acid substitutions at sites predicted to be subject to positive selection, antimicrobial activity against Escherichia coli was further increased. In contrast, no further increase in activity was observed against the remaining pathogens. This result suggests that charge-increasing modifications confer increased broad-spectrum activity to an AMP, whilst positive selection at particular sites is involved in directing the antimicrobial response against specific pathogens. Thus, there is potential for the rational design of novel therapeutics based on specifically targeted and modified AMPs.

The intestinal immune system in Gallus species must rapidly adapt to the omnivorous onset of an adult diet and to colonization by commensal bacteria. Yet, acquired immune functions in Gallus digestive tract fully develop only towards the end of the first week post-hatch. This raises the question of immune protection in the digestive tract during the first week of life. We postulated that in addition to protection conferred by maternal antibodies, the gut is protected by a functionally sufficient innate immune system at hatch. We studied granulocyte distribution in the gut as well as expression of functional genes representing different cells and activities of the innate immune system in chicken hatchlings. These included pro-inflammatory cytokines and chemokines (IL-1beta, IL-8, K203), antibacterial beta-defensins, Gallinacin 1 and 2, and presenilin 1. We demonstrate innate preparedness in the developing chick gut in two circumstances: The first is independent of intestinal exposure to feed and bacteria and is manifested by heterophil maturation in situ. This gut-specific extramedullary granulopoietic process is reported for the first time in the chick, and is supported by beta-defensin and presenilin 1 gene expression. The second is responsive to environmental stimuli, and is demonstrated by gradual development of pro-inflammatory functions: Exposure of the gut to feed and bacteria triggered a low but significant increase in IL-1beta, IL-8 and K203. This resulted in the possible recruitment of bone marrow-derived heterophils as demonstrated by elevation of beta-defensin gene expression. The pro-inflammatory activity in the developing gut also explains the later recruitment of lymphocytes.

Gallinacins (Gal) are antimicrobial peptides that play significant roles in innate immunity in chickens. Two Gal genes--Gal-8 and Gal-9--were cloned and sequenced from chicken liver and tongue, respectively, by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, the mRNA expression of these genes has been demonstrated across a panel of chicken tissues. It was demonstrated that Gal-9 mRNA was highly expressed in the tongue and small intestine and moderately expressed in the chicken proventriculus, lung, liver, heart, spleen, and thymus. However, Gal-8 mRNA was highly expressed in the chick small intestine and liver, and moderately expressed in the chick tongue, and lung. The recombinant fusion proteins containing Gal-9 or Gal-9 and Gal-8, namely rGal-9 and rGal-9-Gal-8, were produced and purified, respectively. Both rGal-9 and rGal-9-Gal-8 were expressed as insoluble bodies and exhibited the expected antimicrobial activity against Escherichia coli and pathogenic Streptococci suis CAB strain, as determined by the measurement of the inhibition zone and a liquid growth inhibition assay.