Xuechun Li's research while affiliated with China Academy of Chinese Medical Sciences and other places

The nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play important roles in innate immunity. Previously, we identified an NLR-like gene, LvNLRPL1, and found that it participated in Vibrio infection and regulated hemocytes apoptosis in the Pacific whiteleg shrimp Litopenaeus vannamei. However, it is still unclear whether other NLR-like genes exist in shrimp and how they function during virus infection. In the present study, a novel NLR-like gene (LvNLRPL2) was identified and functionally characterized in L. vannamei. LvNLRPL2 was highly expressed in hemocytes and responsive to both Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infection. Knockdown of LvNLRPL2 could markedly increase the proliferation of Vibrio and the mortality of shrimp infected with V. parahaemolyticus, whereas inhibit in vivo WSSV propagation in shrimp, indicating its distinct roles during Vibrio and WSSV infection. After LvNLRPL2 knockdown, the apoptotic rate of hemocytes increased, and the expression levels of LvCaspase 2, 3 and 5 were significantly up-regulated. In addition, LvNLRPL2 could form a hetero-dimer with LvNLRPL1 through their NACHT domains. These results suggest that LvNLRPL2 might resist bacterial infection while promote WSSV propagation by forming hetero-dimer with LvNLRPL1 and then inhibiting apoptosis of hemocytes. These data will be helpful for understanding the function of NLR-like genes and their regulation mechanisms in crustaceans.

The TRIpartite Motif (TRIM) proteins play key roles in cell differentiation, apoptosis, development, autophagy, and innate immunity in vertebrates. In the present study, a novel TRIM9 homolog (designated as LvTRIM9-1) specifically expressed in the lymphoid organ of shrimp was identified from the Pacific whiteleg shrimp Litopenaeus vannamei. Its deduced amino acid sequence possesses the typical features of TRIM proteins, including a RING domain, two B-boxes, a coiled-coil domain, a FN3 domain, and a SPRY domain. The transcripts of LvTRIM9-1 were mainly located in the lymphoid tubules of the lymphoid organ. Knockdown of LvTRIM9-1 could apparently inhibit the transcriptions of some genes from white spot syndrome virus (WSSV) and reduce the viral propagation in the lymphoid organ. Overexpression of LvTRIM9-1 in mammalian cells could activate the promoter activity of NF-κB, and an in vivo experiment in shrimp showed that knockdown of LvTRIM9-1 reduced the expression of LvRelish in the lymphoid organ. Yeast two-hybridization and co-immunoprecipitation (Co-IP) assays confirmed that LvTRIM9-1 could directly interact with LvIMD, a key component of the IMD pathway, through its SPRY domain. These data suggest that LvTRIM9-1 could activate the IMD pathway in shrimp via interaction with LvIMD. This is the first evidence to show the regulation of a TRIM9 protein on the IMD pathway through its direct interaction with IMD, which will enrich our knowledge on the role of TRIM proteins in innate immunity of invertebrates.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

NOD-like receptors (NLRs) are a kind of pattern recognition receptors, which are vital for detection on pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and then trigger downstream immune responses in vertebrates. Although many NLR like genes have been identified in invertebrates in recent years, knowledge about their immune functions is still very limited. In the present study, a NLR like gene, designated as LvNLRPL1, was identified in Litopenaeus vannamei. It was widely expressed in multiple tissues and responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvNLRPL1 could accelerate the proliferation of Vibrio in the target tissue hepatopancreas and increase the mortality rate of shrimp after Vibrio infection. Meanwhile, knockdown of LvNLRPL1 also up-regulated the expression of Caspase 2, 3 and 5 in hemocytes, which caused apoptosis of more hemocytes. These results indicated that LvNLRPL1 played important immune functions in shrimp during Vibrio infection through regulating the apoptosis of hemocytes in shrimp. To our knowledge, this is the first time to reveal the immune function of a NLR like gene in crustaceans.

As the most productive crustacean species in aquaculture, Litopenaeus vannamei is seriously threatened by white spot syndrome virus (WSSV), which has caused huge economic damage in the past decades. Shrimp cuticle proteins are the important components in the frontier target tissues, including cuticle and the chitinous lining of the digestive tract. In present study, a novel cuticle protein gene, named LvCPAP1, was isolated and demonstrated to play an important role in WSSV infection. The deduced amino acid sequence of LvCPAP1 contained a signal peptide and a conserved chitin-binding domain type 2 (ChBD2). Tissue distribution analysis revealed that LvCPAP1 was predominantly expressed in epidermis and stomach. The transcription levels of LvCPAP1 in epidermis and stomach were significantly regulated upon WSSV challenge. DsRNA silencing of LvCPAP1 decreased the in vivo WSSV copy numbers and the death rate of shrimp after WSSV infection, indicating that LvCPAP1 might facilitate WSSV invasion. In addition, the interaction between LvCPAP1 and the major envelop protein VP24 of WSSV was revealed by yeast two-hybrid system and further confirmed by dot blot and pull-down assays. The present study implied that cuticle protein LvCPAP1 might favor the entry process of WSSV, which provided new clues for understanding the role of cuticle proteins during virus infection.