title: Characterization and expression analysis of a goose-type lysozyme from the rock bream Oplegnathus fasciatus, and antimicrobial activity of its recombinant protein.
abstract: Lysozyme (muramidase) represents an important defense molecule of the fish innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of β-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of a g-type lysozyme (RBgLyz) was identified in the Oplegnathus fasciatus rock bream fish genome by means of multi-tissue normalized cDNA pyrosequencing using Roche 454 GS-FLX™ technology. RBgLyz is composed of 669 bp, with a 567 bp open reading frame that encodes 188 amino acids. Protein motif searches indicated that RBgLyz contains the soluble lytic transglycosylase domain involved in maintaining cell wall integrity. Furthermore, RBgLyz shares significant identity (81.4%) with Chinese perch Siniperca chuatsi. Quantitative real-time RT-PCR analysis results showed that RBgLyz transcripts are constitutively expressed in various tissues from healthy rock breams. In order to determine RBgLyz function in immunity, its expression was analyzed in head kidney following exposure to known immune stimulants or pathogens. RBgLyz transcripts were significantly up-regulated in response to challenge with lipopolysaccharide (LPS) and Edwardsiella tarda, as compared to non-injected control fish. Polyinosinic:polycytidylic acid (poly I:C) dsRNA stimulated a moderate expression of RBgLyz, as did Streptococcus iniae but to a lesser extent. There were no specific time-dependent effects on RBgLyz mRNA expression observed in response to rock bream iridovirus (RBIV) infection. Taken together, the gene expression results indicated that g-type lysozyme plays a role in the innate immune response to LPS, poly I:C, E. tarda and S. iniae in rock bream. Thus, we generated recombinant RBgLyz in an Escherichia coli expression system and characterized its antimicrobial activity. Our results indicated that recombinant RBgLyz had lytic activity against Gram-negative Vibrio salmonicida, Gram-positive Listeria monocytogenes, S. iniae and Micrococcus lysodeikticus. In addition, observations by scanning electron microscope (SEM) confirmed that the cell morphology of M. lysodeikticus was altered in the presence of recombinant RBgLyz.
authors.affiliation: Department of Life Sciences, College of Natural Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
authors.forename: Ilson
authors.initials: I
authors.lastname: Whang
authors.forename: Youngdeuk
authors.initials: Y
authors.lastname: Lee
authors.forename: Sukkyoung
authors.initials: S
authors.lastname: Lee
authors.forename: Myung-Joo
authors.initials: MJ
authors.lastname: Oh
authors.forename: Sung-Ju
authors.initials: SJ
authors.lastname: Jung
authors.forename: Cheol Young
authors.initials: CY
authors.lastname: Choi
authors.forename: Woo Song
authors.initials: WS
authors.lastname: Lee
authors.forename: Hyung Soo
authors.initials: HS
authors.lastname: Kim
authors.forename: Se-Jae
authors.initials: SJ
authors.lastname: Kim
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D010473:Perciformes / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment
chemical_list: D000276:Adjuvants, Immunologic; D000890:Anti-Infective Agents; D011994:Recombinant Proteins; D009113:Muramidase