Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappaB and Sp1 of a medaka beta-defensin.
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.
Zhao
Jiu-Gang
JG
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of Chinese Academy of Sciences, 7# Donghu South Road, Wuhan, Hubei 430072, China.
Zhou
Li
L
Jin
Jun-Yan
JY
Zhao
Zhe
Z
Lan
Jing
J
Zhang
Yi-Bin
YB
Zhang
Qi-Ya
QY
Gui
Jian-Fang
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism*; D009990:Oryzias / Q000276:immunology*; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000276:immunology / Q000378:metabolism*; D023083:beta-Defensins / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology
D008070:Lipopolysaccharides; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D023083:beta-Defensins