Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappaB and Sp1 of a medaka beta-defensin

Dev Comp Immunol. 2009 Apr;33(4):624-37. doi: 10.1016/j.dci.2008.11.006. Epub 2008 Dec 11.

Abstract

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (ORF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fold up-regulation of the beta-defensin within first 12h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair (bp) sequence (+26 to -73) and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappaB) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappaB and Sp1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Line
  • Gram-Negative Bacteria / drug effects
  • Gram-Negative Bacteria / immunology*
  • Lipopolysaccharides / immunology
  • Molecular Sequence Data
  • Mutation / genetics
  • NF-kappa B / immunology
  • NF-kappa B / metabolism*
  • Oryzias / immunology*
  • Promoter Regions, Genetic
  • Sequence Alignment
  • Sp1 Transcription Factor / immunology
  • Sp1 Transcription Factor / metabolism*
  • beta-Defensins / genetics
  • beta-Defensins / immunology*
  • beta-Defensins / pharmacology

Substances

  • Lipopolysaccharides
  • NF-kappa B
  • Sp1 Transcription Factor
  • beta-Defensins

Associated data

  • GENBANK/EU676010