A novel ML domain-containing protein (SpMD2) functions as a potential LPS receptor involved in anti-Vibrio immune response.

The myeloid differentiation protein 2 (MD2)-related lipid-recognition (ML) proteins display diverse biological functions in host immunity and lipid metabolism by interacting with different lipids. Human MD2, an indispensable accessory protein in TLR4 signaling pathway, specifically recognizes lipopolysaccharides (LPS), thereby leading to the activation of TLR4 signaling pathway to produce many effectors that participate in inflammatory and immuneresponses against Gram-negative bacteria. Toll and immune deficiency (IMD) pathways are first characterized in Drosophila and are reportedly present in crustaceans, but the recognition and activation mechanism of these signaling pathways in crustaceans remains unclear. In the present study, a novel ML protein was characterized in mud crab (Scylla paramamosain) and designated as SpMD2. The complete SpMD2 cDNA sequence is 1114 bp long with a 465 bp open reading frame; it encodes a protein that contains 154 amino acids (aa). In the deduced protein, a signal peptide (1-21 aa residues) and a ML domain (43-151 aa residues) were predicted. SpMD2 shared a similar three-dimensional structure and a close evolutionary relationship with human MD2. SpMD2 was highly expressed in gills, hemocytes, intestine, and hepatopancreas and was upregulated in gills and hemocytes after challenges with bacteria, thereby suggesting its involvement in antibacterial defense. Western blot assay showed that SpMD2 possesses strong binding activities to different bacteria and two fungi. ELISA demonstrated that SpMD2 exhibits binding abilities to LPS, lipid A, peptidoglycan (PGN), and lipoteichoic acid (LTA). Its binding ability to LPS and lipid A were stronger than to PGN or LTA, implying that SpMD2 was an important LPS-binding protein in mud crab. Bacterial clearance assay revealed that the pre-incubation of Vibrio parahemolyticus with SpMD2 facilitates bacterial clearance in vivo and that knockdown of SpMD2 dramatically suppresses the bacterial clearance and decreases the expression of several antimicrobial peptides (AMPs). Furthermore, SpMD2 overexpression could enhance the promoter activity of SpALF2. These results revealed that SpMD2 affects bacterial clearance by regulating AMPs. Thus, by binding to LPS and by regulating AMPs, SpMD2 may function as a potential receptor, which is involved in the recognition and activation of a certain immune signaling pathway against Gram-negative bacteria. This study provides new insights into the diverse functions of ML proteins and into the antibacterial mechanisms of crustaceans.

Complete mitochondrial genome of the deep pugnose ponyfish Secutor ruconius (Perciformes: Leiognathidae) in the East China Sea.

The complete mitochondrial genome sequence of Secutor ruconius is firstly described in this article. The total length of mitogenome is16,465 bp. It contains 13 protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes. The overall base composition of H-strand is 31.58% A, 28.65% C, 25.23% T, and 14.53% G, with an A + T bias of 56.81%. The phylogenetic analysis result showed that the S. ruconius, Zebrasoma flavescens, and Pristipomoides multidens were closely related.

Complete mitochondrial genome of the glowbelly Acropoma japonicum (Perciformes: Acropomatidae) in the East China Sea.

The complete mitochondrial genome sequence of Acropoma japonicum is first described in this article. The total length of mitogenome is16,973 bp. It contains 13 protein-coding genes, 23 tRNA genes, and 2 ribosomal RNA genes. The overall base composition of H-strand is 27.64% A, 29.49% C, 26.84% T, and 16.03% G, with an A＋T bias of 54.48%. The phylogenetic analysis result showed that the A. japonicum and Lutjanus peru had a close relationship.

Complete mitochondrial genome of the skinnycheek lantern fish Benthosema pterotum (Perciformes: Myctophidae) in the East China Sea.

The complete mitochondrial genome sequence of Benthosema pterotum is first described in this article. The total length of mitogenome is 18,052 bp. It contains 13 protein-coding genes, 22 tRNA genes, and two ribosomal RNA genes. The overall base composition of H-strand is 27.83% A, 30.88% C, 25.61% T, and 15.69% G, with an A＋T bias of 53.43%. The phylogenetic analysis result showed that the B. pterotum and Electrona carlsbergi were close relationship.

Characterization of TLR5 and TLR9 from silver pomfret (Pampus argenteus) and expression profiling in response to bacterial components.

Toll like receptor (TLR) 5 and 9 are important members of the TLR family that play key roles in innate immunity in all vertebrates. In this study, paTLR5 and paTLR9 were identified in silver pomfret (Pampus argenteus), a marine teleost of great economic value. Open reading frames (ORFs) of paTLR5 and paTLR9 are 2646 and 3225 bp, encoding polypeptides of 881 and 1074 amino acids, respectively. Sequence analysis revealed several conserved characteristic features, including signal peptides, leucine-rich repeat (LRR) motifs, and a Toll/interleukin-I receptor (TIR) domain. Sequence, phylogenetic and synteny analysis revealed high sequence identity with counterparts in other teleosts, confirming their correct nomenclature and conservation during evolution. Quantitative real-time PCR revealed that the that both TLRs were ubiquitously expressed in all investigated tissues, most abundantly in liver, kidney, spleen, intestine and gill, but lower in muscle and skin. In vitro immunostimulation experiments revealed that Aeromonas hydrophila lipopolysaccharide (LPS) and Vibrio anguillarum flagellin induced higher levels of paTLR9 and paTLR5 mRNA expression in isolated fish intestinal epithelial cells (FIECs) than Lactobacillus plantarum lipoteichoic acid (LTA), but all increased the secretion of IL-6 and TNF-α and induced cell apoptosis and necrosis. Together, these results indicate that paTLR5 and paTLR9 may function in the response to bacterial pathogens. Our findings enhance our understanding of the function of TLRs in the innate immune system of silver pomfret and other teleosts.