Jingsheng Lun

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50-200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ▵OmpA and ▵OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.

The outer membrane protein, LamB (maltoporin), is a versatile vaccine candidate among the Vibrio species

Maltoporin (LamB) is a family of outer membrane proteins. There has been no report of immunological characteristics of LamB in the Vibrio species so far. In this study, lamB genes from eight Vibrio strains were cloned and sequenced. The bioinformatics analysis indicated that sequence similarities of LamB proteins were ranged from 46.7% to 81.1%. Further, the result showed that their antigenic epitopes were highly conserved implying that LamB might be a shared antigen among Vibrios. The Western blot of rabbit sera against recombinant LamB from V. alginolyticus ATCC 33787 with cell lysate of 18 Vibrio strains showed cross-recognition. Bands observed on cell lysate of Vibrio strains immunoblotted with the anti-LamB sera ranged between 40 and 49 kDa. The Whole-cell ELISA assay further confirmed that the antisera of recombinant LamB recognized the tested Vibrio strains indicating the surface-exposed of LamB. Finally, the cross-protective property of recombinant LamB was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in zebrafish. Recorded relative percent survival (RPS) of the vaccinated group varied from 54.1% to 77.8%, showing that zebrafish were protected from Vibrio infection after immunization with LamB protein. The cumulative evidences in this study suggested that LamB was a conserved antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of Vibriosis.

SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei.

In this study, we identified a variable region in the C‐terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288–2503 bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR‐SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus‐treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus‐resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C‐terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.

Evidences of SNPs in the variable region of hemocyanin Ig-like domain in shrimp Litopenaeus vannamei

Single nucleotide polymorphisms (SNPs) are the commonest mode of genetic variation in invertebrate immune-related genes. Hemocyanin presents in the hemolymph of both mollusks and arthropods and functions as an important antigen non-specific immune protein. But people know very little about its gene polymorphism so far. In current study, bioinformatics, molecular biology and environmental challenge approaches were used to identify the SNPs within hemocyanin Ig-like domain in shrimp Litopenaeus vannamei. A total of 11 SNPs were found in a variable region of Ig-like domain from L. vannamei hemocyanin large subunit (1258-1460 bp, HcLV1), 5 of which (1272, 1315, 1380, 1410 and 1450) were confirmed present in both genomic DNA and cDNA by clone sequencing. Furthermore, HcLV1 showed 3, 5 and 5 SSCP bands, respectively, in 16, 25 and 30 °C-treated shrimps, suggesting that the SSCP pattern of HcLV1 could be modulated by environmental stress. In addition, HcLV1 displayed two extra bands with different mobility when shrimps treated with Vibrio parahaemolyticus for 6-24 h, which was not observed in the control group. In conclusion, our data suggest that shrimp L. vannamei hemocyanin Ig-like domain possesses SNPs, which may be associated with environmental stress or pathogenic challenge.

Functional domains of Litopenaeus vannamei transglutaminase and their involvement in immunoregulation in shrimp

&NA; Shrimps, which mainly rely on their innate immune system to response to infectious pathogens, have clottable proteins as an important component of this system. While transglutaminases (TGase) are found in Litopenaeus vannamei and constitute part of the coagulation system, the specific immune‐related roles played by its functional domains in the immunoregulation of shrimp has not been well understood. In the present study, we report that the Ig‐like domain of L. vannamei transglutaminase (TGase‐C) is the main immune‐related domain among the three functional domains, as it had higher bacterial agglutinative activity against Vibrio parahaemolyticus and Streptococcus iniae. Using Co‐immunoprecipitation and LC‐MS/MS analysis, TGase‐C was shown to interact with 474 proteins, of which 52 proteins were annotated to L. vannamei. More than half of the L. vannamei annotated proteins have immune‐related functions, including apoptosis. Further analysis using pull‐down assay revealed that TGase‐C interacted with CAP‐3 (a homologue of caspase 3). In addition, siRNA‐mediated knockdown of LvTGase significantly (p < 0.01) increased the expression level of LvCAP‐3 coupled with a significant (p < 0.01) increase in caspase 3/7 activity, suggesting that probably LvTGase participates in shrimp immune response by modulating the activity of LvCAP‐3. These findings thus suggest the Ig‐like functional domain of L. vannameis transglutaminase is the domain that is involved in immunoregulation in shrimp. HighlightsThe Ig‐like region of Litopenaeus vannamei TGase (TGase‐C) is the main immune‐related functional domain.TGase‐C has bacteria agglutination activity and can interact with immune‐related proteins.TGase‐C modulates LvCAP‐3 expression to regulate apoptosis.

Molecular cloning and functional characterization of a homolog of the transcriptional regulator CSL in Litopenaeus vannamei

ABSTRACT The Notch signaling pathway transcriptional regulator, CSL (also called as CBF1, Suppressor of Hairless or Lag‐1 in different species, generally designated as CSL1), is not only associated with cell proliferation and differentiation but also involved in tumorigenesis, inflammation and immune regulation in vertebrates. We recently showed that Notch signaling was involved in the immune response of Litopenaeus vannamei shrimp. However, as an important transcriptional regulator of this pathway, whether or not shrimp CSL was also involved in immune response had not been explored. Here, we cloned and characterized the CSL gene in L. vannamei (LvCSL), which has a 2271 bp open reading frame (ORF) encoding a putative protein of 756 amino acids, and contains two conserved Lag1‐DNA bind as well as beta trefoil domains (BTD). LvCSL clustered with invertebrates in the phylogenetic tree and closely related to the RBP Jk X1 of Parasteatoda tepidariorum. The transcript level of LvCSL analyzed by quantitative polymerase chain reaction (qPCR) showed that LvCSL was widely expressed in all tissues tested, with induced levels observed in the hepatopancreas and hemocytes following immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), therefore, suggesting LvCSL involvement in shrimp immune response to pathogens. Besides, LvCSL knockdown decreased the expression of proliferation‐related genes (LvHey2 and LvAstakine), and attenuated the expression of immune‐related genes L. vannamei hypoxia inducible factor alpha (LvHIF‐&agr;), LvLectin and L. vannamei small subunit hemocyanin (LvHMCS) in shrimp hemocytes, as well as significantly decreased total hemocyte count. Moreover, high cumulative mortality was observed in LvCSL depleted shrimp challenged with V. parahaemoliticus. In conclusion, our present data strongly suggest that LvCSL is an important factor in shrimp, vital for shrimp survival and contributing to immune resistance to pathogens. HIGHLIGHTSWe found a novel CSL homolog in shrimp L.vannamei (LvCSL).It might regulate the expression of some immune related genes and the proliferation of hemocytes.The LvCSL could be involved in shrimp survival and resistance to pathogenic infection.

Acute Hepatopancreatic Necrosis Disease (AHPND) related microRNAs in Litopenaeus vannamei infected with AHPND-causing strain of Vibrio parahemolyticus

BackgroundAcute hepatopancreatic necrosis disease (AHPND) has emerged as a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. Given that several diseases and infections have been associated with microRNAs (miRNAs), we conducted a comparative transcriptomic analysis using the AHPND (VA) and non-AHPND (VN) strains of Vibrio parahemolyticus to identify miRNAs potentially involved in AHPND pathogenesis in Litopenaeus vannamei.ResultsA total of 83 miRNAs (47 upregulated and 36 downregulated) were significantly differentially expressed between the VA and VN challenged groups, while 222 target genes of these miRNAs were predicted. Functional enrichment analysis revealed that the miRNAs target genes were involved in multiple biological processes including metabolic pathways, amoebiasis, Vibrio cholerae infection etc. Finally, interaction network and qPCR (Real-time Quantitative PCR) analysis of 12 potential key AHPND-related miRNAs and their predicted target genes, revealed their possible involvement in modulating several immune-related processes in the pathogenesis of AHPND.ConclusionsWe have shown using comparative transcriptomic analysis, miRNAs and their target genes that are responsive to AHPND V. parahemolyticus infection in shrimp, therefore suggesting their possible role in defense response to AHPND V. parahemolyticus infection.