Expression And Function Study Of Antibacterial Peptide In Marsupenaeus Japonicus

In invertebrates, innate immune system plays a rapid and effective role against microorganism, because of the absence of an adaptive immunity. AMPs (antimicrobial peptides) were regulated by innate immunity pathways, like Toll, IMD and JAK-STAT, as the effector molecules. And AMPs could be synthesized and excreted to kill microorganisms. In this study, functions of two kinds of AMPs (MjSWD and MjALFs) from Marsupenaeus japonicus were analyzed, and the results showed that they played important roles in shrimp immunity.1. Expression and function analyse of a single whey acidic protein domain (SWD)-containing protein in Marsupenaeus japonicusThe single whey acidic protein (WAP) domain-containing proteins (SWDs) in crustacean belong to type Ⅲ crustins and have anti-protease activities and/or antimicrobial activities. Their functions in vivo in crustacean immunity need to be clarify. In this study, a new single WAP domain-containing protein (SWD) was obtained from Marsupenaeus japonicus, designated as MjSWD. The full-length cDNA of MjSWD was 522bp.The open reading frame of MjSWD encoded a protein of 79 amino acids, with a 24 amino acid signal peptide and a WAP domain. Tissue distribution analysis revealed that MjSWD transcripts were generally expressed in all the tested tissues, including hemocytes, heart, hepatopancreas, gill, stomach and intestine. The time course expression of MjSWD was analyzed by quantitative real-time PCR, and the results exhibited that MjrSWD was upregulated after bacteria (Vibrio anguillarum, Staphylococcus aureus) and white spot syndrome virus (WSSV) challenge in gills and stomach of the shrimp. The purified recombinant protein of MjSWD could bind to several Gram-negative and Gram-positive bacteria though binding to microbial polysaccharides (peptidoglycan). MjSWD could inhibit the activity of subtilisin A and proteinase K and bacteria-secreted proteases. The results of natural infection with MjSWD-incubated bacteria showed that the inhibition of MjSWD against bacterial-secreted proteases was contributed to inhibiting bacteria invasion and dissemination in the shrimp. The MjSWD is, thus, involved in the shrimp antibacterial innate immunity.2. Functions analyse of a new group of anti-Iipopolysaccharide factors from Marsupenaeus japonic us in antibacterial responseAnti-lipopolysaccharide factors (ALFs) are a group of critical effector molecules with a broad spectrum of antimicrobial activities in crustaceans. Four groups of ALFs (A, B, C, and D) have been identified in peneaid shrimp. In the study, we identified a new group of ALFs (designated as MjALF-E) from Marsupenaeus japonicus. This new group (Group E) included MjALF-E1 and E2. MjALF-E1 was highly expressed in hemocytes, heart, and intestine, whereas E2 was highly expressed in gills, stomach, and intestine. Expressions of both MjALF-E1 and E2 were upregulated by bacterial challenge. Synthesized LPS-binding domain peptides of MjALF-E1 and E2 strongly bind to bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). The recombinant rMjALF-E2 showed relatively weak binding activity to LPS and PGN. Both synthesized peptides and rMjALF-E2 exhibited antimicrobial activity against Gram-negative bacteria, whereas rMjALF-E2 could promote the clearance of bacteria in vivo. After knockdown of MjALF-E2 and infection with Vibrio anguillarum, shrimp showed high and rapid mortality compared with GFPi shrimp. These results suggest that MjALF-Es serves a protective function against bacterial infection in shrimp.3. An anti-lipopolysaccharide factor (MjALF-D2) from Marsupenaeus japonicus has antiviral a activityAnti-lipopolysaccharide factors (ALFs) have a vital antimicrobial function against Gram-positive bacteria, Gram-negative bacteria. In recent years, ALFs were reported have antiviral function. However, the antiviral mechanism of ALF was not clear. There were nine isforms of ALFs obtained from our transcriptome sequencing of Marsupenaeus japonicus. Six of them were recombinantly expressed and purified. After incubation of rALFs and white spot syndrome virus (WSSV), the mixture of recombinant proteins and WSSV were injected into the shrimp and replication of WSSV was analyzed. The result showed that only MjALF-D2 exhibited activity against WSSV. MjALF-D2 expressed in all tested tissue except hepatopancreas, and had a high expression level in gills and stomach. After WSSV challenge, the expression of MjALF-D2 was upregulated in hemocytes of shrimp. In MjALF-D2-silenced shrimp, VP28, the important envelop protein of WSSV, expressed in a higher level than the dsGFP-injection shrimp. BThe activity of MjALF-D2 against bacteria and virus showed that MjALF-D2 played a critical role in...