A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade.

Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.

Anti-melanization mechanism of the white spot syndrome viral protein, WSSV453, via interaction with shrimp proPO-activating enzyme, PmproPPAE2.

Inhibition of the host melanization reaction, activated by the prophenoloxidase activating (proPO) system, is one of the crucial evasion strategies of pathogens. Recently, the shrimp pathogen, white spot syndrome virus (WSSV), was found to inhibit melanization in the shrimp Penaeus monodon. The viral protein WSSV453 was previously shown to interact with PO-activating enzyme 2 (PmPPAE2) and reported to be involved in suppressing the shrimp melanization response after WSSV infection. Here, we characterized how WSSV453 inhibits melanization. WSSV453 is a non-structural viral protein, which was first detected in shrimp haemocytes at 6 hours post-infection (hpi) by WSSV and in shrimp plasma at 24 hpi. We produced recombinant proteins for three components of the P. monodon proPO system: PmproPPAE2, PmproPO1 and PmproPO2. Functional assays showed that active PmPPAE2 processed PmproPO1 and 2 to produce functional PO. Incubation of WSSV453 with PmproPPAE2 dose-dependently reduced PmPPAE2 activity toward PmPO1 or PmPO2. In contrast, WSSV453 had no effect on activated PmPPAE2. The addition of active PmPPAE2 to WSSV-infected shrimp plasma at day 2 post-infection also rescued PO activity. Taken together, these results indicate that the anti-melanization activity of WSSV is due to WSSV453, which interacts with PmproPPAE2 and interferes with its activation to active PmPPAE2.

Suppression of shrimp melanization during white spot syndrome virus infection.

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.

A serine proteinase PmClipSP2 contributes to prophenoloxidase system and plays a protective role in shrimp defense by scavenging lipopolysaccharide.

Serine proteinases (SPs) participate in various biological processes and play vital role in immunity. In this study, we investigated the function of PmClipSP2 from shrimp Penaeus monodon in defense against bacterial infection. PmClipSP2 was identified as a clip-domain SP and its mRNA increased in response to infection with Vibrio harveyi. PmClipSP2-knockdown shrimp displayed a significantly reduced phenoloxidase (PO) activity and increased susceptibility to V. harveyi infection. Injection of LPS and/or ß-1,3-glucan induced a dose-dependent mortality and a significant decrease in the number of total hemocytes, with clear morphological changes in the hemocyte surface, of the PmClipSP2-knockdown shrimp. Recombinant PmClipSP2 was shown to bind to LPS and ß-1,3-glucan and to activate PO activity. These results reveal that PmClipSP2 acts as a pattern-recognition protein, binding to microbial polysaccharides and likely activating the proPO system, whilst it may play an essential role in the hemocyte homeostasis by scavenging LPS and neutralizing its toxicity.

Pattern recognition protein binds to lipopolysaccharide and ß-1,3-glucan and activates shrimp prophenoloxidase system.

The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to ß-1,3-glucan and LPS with a dissociation constant of 6.86 × 10(-7) M and 3.55 × 10(-7) M, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or ß-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and ß-1,3-glucan in the shrimp proPO activating system.