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The PirB toxin protein from Vibrio parahaemolyticus induces apoptosis in hemocytes of Penaeus vannamei.

ABSTRACT: Acute hepatopancreatic necrosis disease (AHPND) is a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. The Pir toxin proteins secreted by a unique strain of Vibrio parahaemolyticus play an essential role in the pathogenesis of AHPND. At present, most studies on the effects of Pir toxin proteins in shrimp focus on digestive tissues or organs such as hepatopancreas, stomach, etc., with none on the immune organs. In the present study, two recombinant Pir toxin proteins (rPirA and rPirB) of V. parahaemolyticus were expressed with rPirB shown to enter shrimp hemocytes. Employing pull-down and LC-MS/MS analysis, GST-rPirB was found to interact with 13 proteins in hemocytes, including histone H3 and histone H4 and among which histone H4 had the highest protein score. Further analysis using GST pull-down and Far-Western blot analysis revealed that rPirB could interact with histone H4. In addition, using the purified nucleosome protein from Drosophila S2 cells, it was found that PirB protein could specifically bind to histones. When flow cytometry was applied, it was observed that the interaction between PirB and histones in shrimp hemocytes induces apoptosis, which results in the dephosphorylation of Serine 10 in histone H3. Collectively, the current study shows that in addition to its effect on the digestive tract of shrimp, the PirB toxin protein interacts with histones to affect the phosphorylation of histone H3-S10, thereby inducing apoptosis.

SUBMITTER: Zheng Z

PROVIDER: S-EPMC7834086 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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The PirB toxin protein from Vibrio parahaemolyticus induces apoptosis in hemocytes of Penaeus vannamei.

Zheng Zhou Z Li Ruiwei R Aweya Jude Juventus JJ Yao Defu D Wang Fan F Li Shengkang S Tuan Tran Ngoc TN Zhang Yueling Y

Virulence 20211201 1

Acute hepatopancreatic necrosis disease (AHPND) is a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. The Pir toxin proteins secreted by a unique strain of Vibrio parahaemolyticus play an essential role in the pathogenesis of AHPND. At present, most studies on the effects of Pir toxin proteins in shrimp focus on digestive tissues or organs such as hepatopancreas, stomach, etc., with none on the immune organs. In th ...[more]

PMID: 33487106

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Project description:Astaxanthin (AST), a super antioxidant with coloring and medical properties, renders it a beneficial feed additive for shrimp. This study conducted a white shrimp feeding trial of 3S, 3'S isoform AST, which was derived from metabolic-engineered Kluyveromyces marxianus fermented broth (TB) and its extract (TE) compared to sources from two chemically synthetic ASTs (Carophyll Pink [CP] and Lucantin Pink [LP]), which contain 3S, 3'S, 3R, 3'S (3S, 3'R) and 3R, 3'R isoforms ratio of 1:2:1. The effects on red coloration, immune parameters and resistance to Vibrio infection were evaluated. Four AST sources were incorporated into the diets at concentrations of 0 (control), 100 mg kg-1 (TB100, TE100, CP100, and LP100), and 200 mg kg-1 (TB200, TE200, CP200, and LP200). Results revealed that in week 4, shrimps that received AST-supplemented feeds, especially TB100, TB200, and TE200, significantly increased redness (a*) values. Immune responses including phagocytosis activity, superoxide-anion production, phenoloxidase activity, and immune-related genes were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Generally, shrimps that received AST-supplemented feeds exhibited higher immune responses on days 7 and 14 than the control feed. Gene expression levels of superoxide dismutase and glutathione peroxidase were significantly upregulated on days 7 and 14 in shrimps that received AST-supplemented feeds, while genes of penaeidins, antilipopolysaccharide factor, and lysozyme were upregulated on days 4, 7, and 14, especially received TB200 and TE200. Furthermore, shrimps that received TB100, TE100, CP100, and LP100 7 days were then challenged with Vibrio parahaemolyticus and the result demonstrated higher survival rates especially TB100 at 168 h than the control feed. In conclusion, incorporating AST into the diets enhanced shrimp red coloration, immune parameters, and resistance against V. parahaemolyticus infection. The K. marxianus-derived AST exhibited higher performance than did chemical AST to be a potential feed additive in shrimp aquaculture.

Project description:BackgroundViral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time.ResultsThe two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR.ConclusionsThis work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14-3-3 protein epsilon. The information is useful for understanding the immune system of shrimp against pathogenic bacteria.

Project description:Vibrio parahaemolyticus, the causative agent of Acute hepatopancreatic necrosis disease (AHPND), was discovered in 2013 as a unique isolate that produces toxins and kills penaeid shrimps in devasting nature in Bangladesh and causes severe economic losses. This research aimed to understand the expressions of immune genes in different stages of the host species, Penaeus monodon, against virulence and toxin genes upon being challenged with V. parahaemolyticus. Healthy post-larvae (PL) samples were collected from southwestern of Bangladesh from July 2021 to August 2022. The tryptic soy agar with 1.5% sodium chloride (NaCl) was used to inoculate the cells of V. parahaemolyticus, and the tryptic soy broth (TSB) with 1.5% NaCl was used to transfer the colonies. The spectrophotometry measured bacteria density. PCR, qPCR, SDS-PAGE, and Western blot measured gene expression and survivability after the immersion challenge. The 1 × 105CFU/mL of V. parahaemolyticus was used for 144 h.p.i (hours post-infection) challenge to six stages of post-larvae (PL) of P. monodon (PL20, PL25, PL30, PL35, PL40, and PL45), PL30 and PL35 showed 100% mortality by day 72 (h.p.i.) after exposure that indicated most vulnerable to V. parahaemolyticus. The expression of immune and toxic genes was confirmed by qPCR. The immune genes toll-like receptors (TLR), prophenoloxidase (ProPO), lysozyme (lyso), and penaeidin (PEN) of PL20 and PL25 of P. monodon were expressed robustly up-trends. PL30 and PL35 showed the lowest gene expression at the end of 72 (h.p.i.). At the end of the 144 (h.p.i.) exposure, the immune genes TLR, ProPO, lyso, and PEN expressed highest in PL45 than other post-larvae stages of P. monodon. The toxic genes (pirA, ToxR, ToxA, ToxB, tlh, tdh, and trh) in PL30 and PL35 of P. monodon after exposure of V. parahaemolyticus were expressed highest at the end of the 72 (h.p.i.). The lowest toxic genes expressions were revealed in PL20 and PL45 at the end of the 144 (h.p.i.). The SDS-PAGE analysis of proteins from the bacterium revealed identical protein profiles with toxic genes, and those toxins were further confirmed by Western blot. The 20 kDa, 78 kDa (ToxR), 20 kDa, 25 kDa (ToxA), 25 kDa (ToxB), 20 kDa, 27 kDa, 75 kDa (tdh), and 20 kDa, 27 kDa, 75 kDa, and 78 kDa (trh) proteins were strong responses in Western blot, indicating the crucial involvement of these immune-related genes in the defense and recovery of the first-line defense mechanisms during V. parahaemolyticus infection to shrimp. The all-toxic genes showed a unique homology and those derived from the common ancestor compared with V. parahaemolyticus (NCBI accession no. AP014859.1). All clades were derived with different traits with very low genetic distance, where the overall mean distance was 3.18 and showed a very uniform and homogenous pattern among the lineages. The V. parahaemolyticus infection process in different PL stages in P. monodon revealed novel insights into the immune responses. The responses may lead to the subsequent production of a DNA vaccine, enhancing shrimp health management to minimize the economic losses due to AHPND experiencing an outbreak of early mortality syndrome (EMS) toward sustainable production P. monodon (shrimp).

Dataset Information

The PirB toxin protein from Vibrio parahaemolyticus induces apoptosis in hemocytes of Penaeus vannamei.

Publications

The PirB toxin protein from <i>Vibrio parahaemolyticus</i> induces apoptosis in hemocytes of <i>Penaeus vannamei</i>.

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