Analysis of Two Lysozyme Genes and Antimicrobial Functions of Their Recombinant Proteins in Asian Seabass

Gui Hong Fu,Jun Hong Xia,Feng Liu,Gen Hua Yue,Zhi Yi Bai,Peng Liu,David R Booth

doi:10.1371/journal.pone.0079743

Gui Hong Fu, Jun Hong Xia + Show 5 more

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https://doi.org/10.1371/journal.pone.0079743

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Abstract

Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu50 and Asp67) and a “GSTDYGIFQINS” motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu71, Asp84, Asp95) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

Highlights

Lysozymes are the important antibacterial effector of the innate immunity in animals
In silico analyses revealed that the gtype lysozymes possesses a Goose Egg White Lysozyme (GEWL) domain containing three conserved catalytic residues Glu71, Asp84 and Asp95
Our results showed that high concentration (> 6.25 μg/ml mature peptide) of the c-type and g-type lysozymes could inhibit the growth of V. harveyi and P. damselae, and indicate that two recombinant lysozymes work against bacterial pathogens in a dosedependent manner (Figure 5)

Summary

Introduction

Lysozymes are the important antibacterial effector of the innate immunity in animals. They cleave the β-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and Nacetylglucosamine (NAG) in the peptidoglycan layer of bacterial cell walls, and [1]. The g-type and c-type lysozymes have been identified in some fish species, such as Senegalese sole (Solea senegalensis) [10], orange-spotted grouper (Epinephelus coioides) [11], large yellow croaker (Larimichthys crocea) [12], grass carp (Ctenopharyngodon idellus) [13], Japanese flounder (Paralichthys olivaceus) [14], turbot (Scophthalmus maximus) [15], rainbow trout (Oncorhynchus mykiss) [16] and common carp (Cyprinus carpio L.) [17]. The recombinant proteins of two types of lysozymes purified from Escherichia coli show antimicrobial activity against Vibrio anguillarum [14] in Japanese flounder, Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus, in gastropod Oncomelania hupensis [18], V. anguillarum or M lysodeikticus in Mediterranean mussel [19], V. splendidus and V.parahaemolyticus in Chlamys farreri [8], as well as Vibrio alginolyticus and Vibrio parahemolyticus in shrimp [20]

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