White Spot Syndrome Virus-Induced Shrimp miR-315 Attenuates Prophenoloxidase Activation via PPAE3 Gene Suppression

MicroRNAs (miRNAs), the small non-coding RNAs, play a pivotal role in post-transcriptional gene regulation in various cellular processes. However, the miRNA function in shrimp antiviral response is not clearly understood. This research aims to uncover the function of pmo-miR-315, a white spot syndrome virus (WSSV)-responsive miRNAs identified from Penaeus monodon hemocytes during WSSV infection. The expression of the predicted pmo-miR-315 target mRNA, a novel PmPPAE gene called PmPPAE3, was negatively correlated with that of the pmo-miR-315. Furthermore, the luciferase assay indicated that the pmo-miR-315 directly interacted with the target site in PmPPAE3 suggesting the regulatory role of pmo-miR-315 on PmPPAE3 gene expression. Introducing the pmo-miR-315 into the WSSV-infected shrimp caused the reduction of the PmPPAE3 transcript level and, hence, the PO activity activated by the PmPPAE3 whereas the WSSV copy number in the shrimp hemocytes was increased. Taken together, our findings state a crucial role of pmo-miR-315 in attenuating proPO activation via PPAE3 gene suppression and facilitating the WSSV propagation in shrimp WSSV infection

Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

BACKGROUND: Viral 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. RESULTS: The 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. CONCLUSIONS: This 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

Shrimp alpha-2-macroglobulin prevents the bacterial escape by inhibiting fibrinolysis of blood clots.

Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis

Prx4 acts as DAMP in shrimp, enhancing bacterial resistance via the toll pathway and prophenoloxidase activation

Summary: Peroxiredoxin (Prx), an antioxidant enzyme family, has been identified as immune modulating damage-associated molecular patterns (DAMPs) in mammals but not in shrimp. Acute non-lethal heat shock (NLHS) that enhances shrimp Penaeus vannamei resistance to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND). Among the five P. vannamei Prxs (LvPrx) isoforms, LvPrx4, the most abundant in unchallenged shrimp hemocytes that was upregulated in hemocytes following NLHS treatment, is of great interest. The escalation of the LvPrx4 monomer in hemolymph of NLHS treated shrimp indicates that it probably acts as DAMP. This study revealed that pre-challenge with rLvPrx4 could prolong VPAHPND-infected shrimp survival, increase prophenoloxidase (proPO) activity and promote Toll pathway-related genes expression mediated by Toll-like receptor (TLR) 1 and 2. The presented findings elucidated the molecular mechanism of LvPrx4 monomer as DAMP in NLHS-induced VPAHPND resistance by inducing the TLR1/2 signaling pathway and the proPO activating system

DNA sequences of the primers used in experiments.

<p>DNA sequences of the primers used in experiments.</p

Co-localization of A2M and CPs in hemocytes and extracellular fibrils of blood clots.

<p>The immunocytology and confocal microscopy were performed. Anti-r<i>Pm</i>A2M and anti-rCP Abs were visualized by Alexa Fluor® 568 and Alexa Fluor® 647 conjugated secondary Abs and show up as green and blue colors for A2M and CP, respectively, or cyan for colocalized in the merged field. Red arrowheads demonstrate fibril clots and the scale bar corresponds to 50 µm. Images shown are representative of at least 3 such fields of view per sample.</p