Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge

Abstract

We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) · striped bass (M. saxatilis). After the intraperitoneal injection of Micrococcus luteus and Escherichia coli, the peptide was purified from HPLC fractions with antimicrobial activity against Escherichia coli. Sequencing by Edman degradation revealed a 21-residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative cysteines. Molecular mass measurements of the native peptide and the reduced and alkylated peptide confirmed the sequence with four intramolecular disulfide bridges. Peptide sequence homology to human hepcidin and other predicted hepcidins, indicated that the peptide is a new member of the hepcidin family. Nucleotide sequences for cDNA and genomic DNA were determined for white bass. A predicted prepropeptide (85 amino acids) consists of three domains: a signal peptide (24 amino acids), prodomain (40 amino acids) and a mature peptide (21 amino acids). The gene has two introns and three exons. A TATA box and several consensus-binding motifs for transcription factors including C/EBP, nuclear factor-jB, and hepatocyte nuclear factor were found in the region upstream of the transcriptional start site. In white bass liver, hepcidin gene expression was induced 4500-fold following challenge with the fish pathogen, Streptococcus iniae, while expression levels remained low in all other tissues tested. A novel antimicrobial peptide from the gill, bass hepcidin, is predominantly expressed in the liver and highly inducible by bacterial exposure.Keywords: antimicrobial peptide; fish; hepcidin; innate immunity; Streptococcus iniae.Antimicrobial peptides (AMPs) are a broadly distributed group of molecules that are important in host defense against microbial invasion. A growing number of peptides involved in innate immunity have been isolated from plants, invertebrates, and higher vertebrates. Human hepcidin and liver-expressed antimicrobial peptide (LEAP-1) are identical AMPs, which were isolated independently from urine and human blood ultrafiltrate, respectively [1,2]. Peptide sequences of additional hepcidins have been predicted from expressed sequence tag databases from the liver of mouse Fish have evolved to thrive in an aqueous environment with a rich microbial flora, and several AMPs have been isolated from fish [7]. During our search for AMPs from gills of hybrid striped bass, three RP-HPLC fractions with antimicrobial acitivity were found [8]. One contained moronecidin, a 22-residue AMP with an amphipathic a-helical structure. From two other adjacent fractions, we isolated another novel AMP, bass hepcidin, a 21-residue, cysteine-rich peptide, which is a homologue of human hepcidin. We report here the first hepcidin to be isolated from a nonhuman vertebrate, the first cysteine-rich AMP isolated from fish, and the first demonstration of hepcidin gene expression induced by live bacterial challenge. M A T E R I A L S A N D M E T H O D STissue collection and purification of ba...

C/EBPα Regulates Hepatic Transcription of Hepcidin, an Antimicrobial Peptide and Regulator of Iron Metabolism

“…Sequence analysis of the 5Ј-flanking region of the human and mouse HEPC gene identified several binding sites for liver-enriched transcription factors C/EBP and HNF4 known to be involved in the regulation of liver-specific genes (18,32,33). Interestingly, four putative C/EBP binding sites were also described in 5Ј-flanking region of bass hepcidin gene (11). The C/EBP family of transcription factors includes at least six members, although two of them, C/EBP␣ and C/EBP␤, are enriched in the liver.…”

“…In addition, expression of hepcidin was enhanced in mouse hepatocytes both in vivo and in vitro after exposure to bacterial lipopolysaccharides (7) and was dramatically induced following bacterial challenge in bass liver (11). Induction of hepcidin in these two unrelated physiopathological situations could indicate that besides its antimicrobial functions this peptide exhibits other functions.…”

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout

“…An exception is LEAP-1 (hepcidin), which has been reported recently in many species including salmonids (rainbow trout and Atlantic salmon, Salmo salar) Patrzykat and . Shike et al (2002) showed that the LEAP-1 gene is expressed strongly in the liver but at lower levels in other tissues of bacterially challenged fish. With the recent discovery of LEAP-2 in mammals (Krause et al, 2003) and birds (Lynn et al, 2003), we have searched the fish EST databases to see if this molecule also exists in bony fish, especially salmonids.…”

NK-lysin of channel catfish: Gene triplication, sequence variation, and expression analysis

“…Over 800 AMP sequences have been deposited in the Antimicrobial Peptide Database (http://bbcm1.univ.trieste.it/∼tossi/pag1.htm). Increasing numbers of antimicrobial peptides have been identified from teleost fishes in recent years (e.g., Cole et al, 1997;Douglas et al, 2001Douglas et al, , 2003aLauth et al, 2002;Shike et al, 2002Shike et al, , 2004Noga and Silphaduang, 2003;Zhang et al, 2004;Bao et al, 2005, in press;Xu et al, 2005). As the largest vertebrate group containing over 23,000 species (Helfman et al, 1997), teleosts should be a rich source of antimicrobial peptides.…”