Induction of Cationic Chicken Liver-Expressed Antimicrobial Peptide 2 in Response to Salmonella enterica Infection

Abstract

Cationic antimicrobial peptides constitute part of the innate immune system and provide an essential role in the defense against infection. At present there is a paucity of information regarding the antimicrobial profile of the chicken (Gallus gallus). Using in silico studies, an expressed sequence tag (EST) clone was identified which encodes a novel cationic antimicrobial peptide, chicken liver-expressed antimicrobial peptide 2 (cLEAP-2). The predicted amino acid sequence composed a prepropeptide, and the active peptide contained four conserved cysteine amino acids. The gene was localized to chromosome 13, and analysis of the genome revealed three exons separated by two introns. The cLEAP-2 gene was expressed in a number of chicken epithelial tissues including the small intestine, liver, lung, and kidney. Northern analysis identified liver-specific cLEAP-2 splice variants, suggesting some degree of tissue-specific regulation. To investigate whether cLEAP-2 expression was constitutive or induced in response to microbial infection, 4-day-old birds were orally infected with Salmonella. Analyses of cLEAP-2 expression by semiquantitative reverse transcription-PCR indicated that cLEAP-2 mRNA was upregulated significantly in the small intestinal tissues and the liver, indicative of direct and systemic responses. The antimicrobial activity of cLEAP-2 against Salmonella was analyzed in vitro with a time-kill assay and recombinant cLEAP-2. Interestingly Salmonella enterica serovar Typhimurium SL1344 showed increased susceptibility to the active cationic peptide (amino acids 37 to 76) compared to S. enterica serovar Typhimurium C5 and Salmonella enteritidis. Taken together, these data suggest that cationic cLEAP-2 is part of the innate host defense mechanisms of the chicken.

The β-Defensin Gallinacin-6 Is Expressed in the Chicken Digestive Tract and Has Antimicrobial Activity against Food-Borne Pathogens

“…Recombinant chicken LEAP-2 was effective at microgram amounts against Salmonella enterica serovar Typhimurium SL1344, but not against Salmonella enterica serovar Typhimurium C5 and Salmonella enterica serovar Enteritidis (39). Chicken myeloid ␤-defensins gallinacin 1, 1␣, and 2, isolated from chicken heterophils, showed activity against gram-positive, gram-negative bacteria and yeast (10).…”

“…Currently, several chicken antimicrobial peptides, belonging to the cathelicidin, liver-expressed antimicrobial peptide (LEAP), and ␤-defensin families have been discovered (24,39,41,46), but little is known about their roles in the chicken digestive tract.…”

“…High levels of chicken LEAP-2 expression were observed in the small intestine and liver (24) and upregulated in these tissues when challenged with Salmonella enterica serovar Enteritidis (39). Apart from gallinacin 11 (Gal-11), which is highly expressed in the small intestine, liver, gallbladder, and spleen, and Gal-13, which is found in colon, no significant ␤-defensin levels have been detected in the digestive tract (18,24,46).…”

Identification of an antimicrobial peptide from human methionine sulfoxide reductase B3

“…2A). In contrast, cells containing the Δ (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) mutant form showed no significant inhibition of growth by IPTG induction (Fig. 2B).…”

“…The next 12 amino acids (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) were highly predicted to form a coiled α-helix. To test which motif (or both) causes the antibacterial activity against E. coli cells, we made two deletion mutants, Δ(1-10) and Δ (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), by site-directed mutagenesis. Then we performed the same growth experiments as above for Δ(1-10) and Δ(11-22) mutants.…”