Cloning and Expression of β-Defensin from Soiny Mullet (Liza haematocheila), with Insights of its Antibacterial Mechanism

• Published in: PloS one Vol. 11; no. 6; p. e0157544
• Main Authors: Qi, Zhitao, Xu, Wei, Meng, Fancui, Zhang, Qihuan, Chen, Chenglung, Shao, Rong
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20.06.2016; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Amino acids; Animals; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - metabolism; Antiinfectives and antibacterials; Antimicrobial agents; beta-Defensins - chemistry; beta-Defensins - genetics; beta-Defensins - metabolism; Biochemistry; Biology and Life Sciences; Chemical bonds; Cloning; Cloning, Molecular; Computer simulation; Conformation; Conserved sequence; Defensins; Disulfide bonds; Electrostatic properties; Fish; Gene Expression Regulation; Hydrogen; Hydrogen bonds; Immunity; Infections; Innate immunity; Laboratories; Lipids; Medicine and Health Sciences; Membranes; Nucleotide sequence; Oncorhynchus mykiss; Organ Specificity - genetics; Organs; Peptides; Phospholipids; Phylogeny; Physical sciences; Protein Multimerization; Proteins; Research and Analysis Methods; Residues; Sequence Alignment; Smegmamorpha - genetics; Smegmamorpha - microbiology; Spleen; Strands; Streptococcal Infections - pathology; Streptococcus - physiology; Streptococcus infections; Water - metabolism; Wetlands; Zebrafish; United States > US; China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0157544

Beta-defensins are important part of innate immunity of fish, which are the first defense line against invading pathogens. In this study, the β-defensin (Lhβ-defensin) gene was cloned from spleen tissue of soiny mullet (Liza haematocheila). Lhβ-defensin cDNA was 747 bp in length, encoding 63 amino acids. Sequence alignment revealed that Lhβ-defensin contained six conserved cysteine residues and shared 97.5% sequence identities with grouper (Epinephelus coioides) β-defensin. Realtime PCR revealed that Lhβ-defensin was highest expressed in the immune related organs, such as spleen, kidney and gut of healthy fish. Following Streptococcus dysgalactiae infection, Lhβ-defensin was up-regulated in immune related organs, e.g. 17.6-fold in spleen and 10.87-fold in gut at 24 h post infection (hpi). Lhβ-defensin possessed a monomeric structure of a three-stranded anti-parallel β-sheet and an α-helix stabilized by three disulfide bonds formed by Cys30-Cys58, Cys36-Cys52, and Cys40-Cys59. In addition to the experimental work, computer simulation was also carried out to determine the possible conformation of β-defensin and its interaction with palmitoyloleoylphosphatidylglycerol (POPG), a model of bacteria membrane. The Lhβ-defensin was found to form dimeric structure stabilized by the van der Waals contacts of Leu35 and Cys37 in two anti-parallel β1-strands and the cation-π interaction between Tyr32 and Arg54 respectively in the two β1-strands. The most important interactions between β-defensin and membrane are the electrostatic interactions between Arg residues in β-defensin and head group of POPG bilayer as well as hydrogen bond interactions between them. Our results were useful for further understanding the potential mechanism of antimicrobial property of fish β-defensins.