The immune function of a novel crustin with an atypical WAP domain in regulating intestinal microbiota homeostasis in Litopenaeus vannamei.

Crustins are a family of antimicrobial peptides (AMP) with multiple functions, including antimicrobial activity, capability of protease inhibition, phagocytosis promotion, and wound healing in crustaceans. Till present, several members of crustins have been identified and their activities were studied. However, there are still less investigations on how they play functions in vivo. Here, we identified a novel crustin with an atypical WAP domain, LvCrustin Ⅰ-1, which is mainly distributed in tissues, including intestine, gill, epidermis and stomach of the shrimp Litopenaeus vannamei. The expression level of LvCrustin Ⅰ-1 was significantly up-regulated at 3 h, 6 h, 12 h, and 24 h after Vibrio parahaemolyticus infection. Knockdown of LvCrustin Ⅰ-1 with dsRNA resulted in a significant increase of the bacteria number in hepatopancreas of shrimp upon V. parahaemolyticus infection, showing that LvCrustin Ⅰ-1 participated in pathogen infection process. Recombinant LvCrustin Ⅰ-1 protein showed microorganism-binding activity rather than antibacterial activity against tested bacteria. Furthermore, significant difference existed between the intestinal microbiota in shrimp before and after LvCrustin Ⅰ-1 knockdown based on the result of alpha and NMDS analyses. Knockdown of LvCrustin Ⅰ-1 increased the proportion of Demequina, Nautella, Propionibacterium, Anaerospora and decreased the proportion of Bacteroidia and Vibrio. These data suggest that LvCrustin Ⅰ-1 might perform its immunological function through modulation of the intestinal microbiota homeostasis rather than direct inhibition of bacterial growth in shrimp.

Lv X ，Li S ，Yu Y ，Xiang J ，Li F ... -  《-》

Characterization of a gill-abundant crustin with microbiota modulating function in Litopenaeus vannamei.

Gills as the respiratory tissue of crustacean encounter various threats in the environment. The immune defense in gills is essential to the health of shrimp. In the present study, we identified a novel gill-abundant crustin, LvCrustin Ⅱ-1, from the shrimp Litopenaeus vannamei. The full-length open reading frame of LvCrustin Ⅱ-1 is 522 bp, which encodes 173 amino acid residues. The amino acid sequence of LvCrustin Ⅱ-1 contained a signal peptide, a glycine rich region, a cysteine rich region and a whey acidic protein (WAP) domain. The expression level of LvCrustin Ⅱ-1 was significantly up-regulated at different time points after Vibrio parahaemolyticus immersion. When LvCrustin Ⅱ-1 was silenced by dsRNA interference, the count of bacteria colonies increased significantly in the hepatopancreas of shrimp after V. parahaemolyticus immersion, which indicated that the infection progress of pathogenic bacteria was accelerated after LvCrustin Ⅱ-1 knockdown. Compared with the microbiota of seawater, the lower proportion of aquatic bacteria and higher proportion of symbiont in the gills microbiota of shrimp indicated the bacterial colonization was modulated by the host. Knockdown of LvCrustin Ⅱ-1 changed the proportion of some potential pathogens and aquatic bacteria, which supported the idea that the new identified crustin in the gills played important roles in modulation of the microbiota community in shrimp. The present data provided new insights into the multiple functions of crustin in the immunity of shrimp.

Lv X ，Li S ，Yu Y ，Zhang X ，Li F ... -  《-》

Crustin Defense against Vibrio parahaemolyticus Infection by Regulating Intestinal Microbial Balance in Litopenaeus vannamei.

Lv X ，Li S ，Yu Y ，Zhang X ，Li F ... -  《Marine Drugs》

Newly identified type II crustin (SpCrus2) in Scylla paramamosain contains a distinct cysteine distribution pattern exhibiting broad antimicrobial activity.

Type II crustins are the most abundant type of crustins in shrimps that exhibit remarkable sequence diversities and broad antibacterial activities. This study characterized a novel type II crustin, SpCrus2, in the mud crab Scylla paramamosain. The SpCrus2 cDNA sequence is 620-bp long with a 495-bp open reading frame encoding a 164-amino acid protein. In the deduced protein, a 17-amino acid signal peptide, a glycine-rich hydrophobic region (GRR), and a cysteine-rich region (CRR) containing a whey acidic protein domain were predicted. SpCrus2 shares high similarity with most type II crustins (types IIa and IIb crustins) in shrimps but has a novel distribution pattern of cysteine residues that is distinct from most crustins. SpCrus2 and PlCrus3 from Pacifastacus leniusculus share high similarity and the same distribution pattern of cysteine residues. Thus, we proposed them as type IIc crustins. SpCrus2 is mainly distributed in the gills and can be up-regulated through Vibrio parahemolyticus or Staphylococcus aureus challenge. To investigate the biological functions of SpCrus2 and the underlying mechanisms, SpCrus2, GRR, CRR, and the mutant of CRR (CRR-M, the cysteine distribution pattern is mutated into that in most conventional crustins) were all overexpressed and purified. SpCrus2 GRR itself, as a glycine-rich amphiphilic peptide, exhibited evident antibacterial ability against Gram-negative bacteria, whereas CRR possessed potent antibacterial activity against Gram-positive bacteria. Either GRR or CRR exhibited weaker antibacterial activity than the whole protein of SpCrus2, indicating that GRR and CRR synergized to exert their potential antibacterial functions. In addition, CRR exhibited slightly stronger antimicrobial activity than CRR-M, suggesting that SpCrus2 containing this novel cysteine distribution pattern may exhibit stronger antimicrobial activity than most type II crustins with the conventional distribution pattern of cysteine residues. The likely antimicrobial ability of SpCrus2 may result from its microbial polysaccharide-binding and agglutination activities. Overall, this study characterized the first type II crustin in crabs and provided new insights into understanding the sequence and functional diversity of crustins and their immune functions in crustaceans.

Wang H ，Zhang JX ，Wang Y ，Fang WH ，Wang Y ，Zhou JF ，Zhao S ，Li XC ... -  《-》

Molecular cloning, expression, promoter analysis and functional characterization of a new Crustin from Litopenaeus vannamei.

Antimicrobial peptides (AMPs) are the most important players in the innate immune system, providing a principal first-line of defense against the invading pathogens. Crustin, a type of whey acidic protein (WAP) domain-containing and cationic cysteine-rich AMP, can function in a protease inhibition or an effector molecule manner. In the present study, a new Crustin was cloned and identified from Pacific white shrimp Litopenaeus vannamei and designated as LvCrustinA. The full-length cDNA of LvCrustinA was 687 bp, with a 519 bp open reading frame (ORF) that encoded a peptide of 172 amino acids. Domain analysis indicated that LvCrustinA contained a Glycine-rich region in the N-terminal and a single WAP domain within eight cysteines in the C-terminal. The 5' upstream regulatory sequence of 1249 bp (promoter) was obtained using a genome walking method, and it contained several conserved transcription factors binding motifs including NF-κB, AP-1 and STAT (Signal transducers and activators of transcription). Dual-reporter assay showed that NF-κB transcription factors LvDorsal and LvRelish, and AP-1 transcription factor Lvc-Jun could up-regulate the promoter activity of LvCrustinA, suggesting that NF-κB and JNK-c-Jun pathways could be involved in regulating the expression of LvCrustinA. Moreover, LvCrustinA was abundantly expressed in immune related tissues such as gill, hemocyte and epithelium, and its expression was up-regulated in response to Vibrio parahaemolyticus and White spot syndrome virus (WSSV) challenges in gill tissue, suggesting that LvCrustinA could be involved in the host defense against bacterial and viral infection. Additionally, RNAi mediated knockdown of LvCrustinA resulted in shrimps with the higher cumulative mortality during V. parahaemolyticus and WSSV infection. Taken together, these results provided some insight into the expression and transcriptional regulatory role of LvCrustinA, and its defensive role against pathogenic infection.

Li M ，Ma C ，Li H ，Peng J ，Zeng D ，Chen X ，Li C ... -  《-》