Characterization and PCR Detection Of Binary,Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VP_AHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VP_AHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24–48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JALL01000066.1","term_id":"576300946","term_text":"JALL01000066.1"}}JALL01000066.1) and similarity to known binary insecticidal toxins called ''Photorhabdus insect related'' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VP_AHPND isolates in the test set.     

Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp,Fenneropenaeus chinensis

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains

Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R₁, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gómez, I., Sánchez, J., Miranda, R., Bravo A., Soberón, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R₁ in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R₁. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R₁ is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R₁ was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.     

Analysis of Expression,Cellular Localization,and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)

Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei . LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrio alginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V . alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibrio penaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.     

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a ��Ping Pong�� Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R₁) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R₁ promotes cleavage of the amino-terminal end, including helix α-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7–12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R₁ fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a “ping pong” binding mechanism with both receptors is involved in toxin action.     

A thioredoxin response to the WSSV challenge on the Chinese white shrimp, Fenneropenaeus chinensis

Thioredoxin (TRX) is involved in cell redox homeostasis. In addition, it is responsible for maintaining proteins in their reduced state. In our study, a Fenneropenaeus chinensis thioredoxin (FcTRX) gene was identified from the Chinese white shrimp. The full length of FcTRX was 777 bp, including a 60 bp 5′ untranslated region (UTR), a 318 bp open reading frame (ORF) encoding a 105 amino acids protein, and a 399 bp 3′ UTR. FcTRX contained a TRX domain with a conserved motif of Cys-Gly-Pro-Cys (CGPC). No signal peptide was predicted by SMART analysis. The molecular mass and pI of FcTRX were 12 kDa and 4.62, respectively. FcTRX is a widely distributed gene, and its mRNA is detected in hemocytes, hearts, hepatopancreas, gills, stomach, and intestine from an unchallenged shrimp. The expression level of FcTRX was the highest in hepatopancreas, where it was down-regulated to the lowest level at 12 h white spot syndrome virus (WSSV) challenge. In the gills, it went up to the highest level at 6 h. Western blot showed that FcTRX protein in hepatopancreas challenged with WSSV was down-regulated from 2 h to 12 h and then restored to the level similar to that of unchallenged shrimp at 24 h. In the gills challenged with WSSV, the FcTRX protein was up-regulated from 6 h to 24 h. Our research indicated its possible role in the anti-WSSV innate immunity of shrimps.     

Bacillus thuringiensis toxin,Cry1C interacts with 128HLHFHLP134 region of aminopeptidase N of agricultural pest,Spodoptera litura

We modeled Cry1C toxin and its Aminopeptidase-N receptor and in silico docking analysis was performed. Further, we utilized biopanning against Cry1C followed by blocking assays and mutagenesis analysis to identify the binding epitope of SlAPN. We have identified a putative SlAPN binding region, APN-CRY (128HLHFHLP134). A derivative of SlAPN carrying the 128HLHFHLP134 region termed as binding region of APN (BR-APN) was cloned and its involvement in Cry1C binding and toxicity was checked. Cry1C-BR-APN binding was competed by synthetic peptides homologous to loop2 and loop3 of domain II but not by that of loopα. Additionally, alanines substitution of residues H128, H130, H132 and P134 affect the binding efficiency of receptor to Cry1C toxin (upto 4-fold lower affinity).These residues are also implicated in Cry1C toxicity as shown by the reduced ability to affect the mortality of Cry1C on S. litura larvae when toxin was preincubated with a fragment of the receptor.     

Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 μg/ml. ¹²⁵I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH₄Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.     

Effects of cadmium on respiratory burst,intracellular Ca2+ and DNA damage in the white shrimp Litopenaeus vannamei

Acute effects of heavy metal ions on shrimp have been an area of intense study worldwide. However, the molecular mechanism by which cadmium-induced injury occurs remains largely unclear, and methods for mitigating toxicity in vivo have rarely been reported. In this study, the changes in respiratory burst and intracellular free calcium in haemocytes of pacific white shrimp, Litopenaeus vannamei, after exposure to Cd²⁺ (CdCl₂) were examined using flow cytometry. Meanwhile, DNA damage and repair in haemocytes and hepatopancreas cells were studied using the comet assay. Respiratory burst generation, intracellular Ca²⁺ concentration ([Ca²⁺]i) and DNA damage in haemocytes and hepatopancreas cells all exhibited a dose-dependent increase and a time-dependent change after treatment with Cd²⁺ compared with controls. These results indicate that Cd can induce oxidative stress and DNA damage in the shrimp L. vannamei. Moreover, the results also demonstrate that these parameters can be used as sensitive indicators of exposure to this genotoxicant.     

Host response in the white shrimp, Penaeus setiferus, to infection by the larval trypanorhynchid cestode, Prochristianella penaei

White shrimp, Penaeus setiferus, infected by the plerocercoid larva of the trypanorhynchid cestode, Prochristianella penaei, respond to infections in the heptaopancreas by developing a progressively denser cyst, composed of hemocytes, fibroblasts, and collagenlike fibers, around the parasite. The pleocercoid is eventually destroyed and resorbed leaving a dense fibrous capsule in the hepatopancreas. Worms in the adjacent hemocoel are encapsulated by a thin host cyst and a less intense infiltration of cells and deposition of fibers, insufficient to destroy the pleroceroid. It is believed that the resorption process explains the drop in intensity of infection in juvenile white shrimp after a certain size is attained.     

First attempts to crystallize a non-homogeneous sample of thioredoxin from Litopenaeus vannamei: What to do when you have diffraction data of a protein that is not the target?

The importance of sample homogeneity and purity in protein crystallization is essential to obtain high-quality diffracting crystals. Here, in an attempt to determine the crystal structure of thioredoxin 1 from whiteleg shrimp Litopenaeus vannamei (LvTrx), we inadvertently crystallized the hexameric inorganic pyrophosphatase of Escherichia coli (E-PPase) from a non-homogeneous sample product during the initial over-expression steps and partial purification of LvTrx. The structure determination and identification of the crystallized protein were derived from several clues: the failures in the Molecular Replacement (MR) trials using LvTrx coordinates as a search model, the unit cell parameters and space group determination, and essentially by the use of the program BALBES. After using the previously deposited E-PPase structure (PDB entry 1mjw) as a search model and the correct space group assignation, the MR showed an E-PPase complexed with SO₄^?2 with small changes in the sulfate ion binding region when it compares to previously deposited E-PPases in the PDB. This work stresses the importance of protein purity to avoid the risk of crystallizing a contaminant protein or how pure need to be a protein sample in order to increase the possibility to obtain crystals, but also serves as a reminder that crystallization is by itself a purification process and how the program BALBES can be useful in the crystal structure determination of previously deposited structures in the PDB.     

Shewanella khirikhana sp. nov. – a shrimp pathogen isolated from a cultivation pond exhibiting early mortality syndrome

Early mortality syndrome (EMS) in cultivated shrimp is of complex aetiology. One of the causes is acute hepatopancreatic necrosis disease (AHPND) caused by unique Vibrio isolates that carry two Pir^vp toxin genes, but other causes of EMS remain mostly unexplained. Here, we describe the discovery of a Shewanella isolate TH2012^T from an EMS/AHPND outbreak pond and demonstrate its virulence for shrimp (the mean lethal concentration of 10⁵ colony-forming units per millilitre by immersion challenge) accompanied by distinctive histopathology, particularly of the ventral nerve cord and lymphoid organ but also including the digestive tract. On the basis of its complete genome sequence, multilocus phylogenetic trees, digital DNA–DNA hybridization analysis and differential phenotypic characteristics, we propose that Shewanella isolate TH2012^T represents a novel species, separated sufficiently from the type strains S. litorisediminis and S. amazonensis to justify naming it Shewanella khirikhana sp. nov. Analysis of the TH2012^T genome revealed no homologues of the Pir^vp toxin genes but revealed a number of other potential virulence factors. It constitutes the first Shewanella isolate reported to be pathogenic to shrimp.