Walaiporn Charoensapsri

Prophenoloxidase system and its role in shrimp immune responses against major pathogens

The global shrimp industry still faces various serious disease-related problems that are mainly caused by pathogenic bacteria and viruses. Understanding the host defense mechanisms is likely to be beneficial in designing and implementing effective strategies to solve the current and future pathogen-related problems. Melanization, which is performed by phenoloxidase (PO) and controlled by the prophenoloxidase (proPO) activation cascade, plays an important role in the invertebrate immune system in allowing a rapid response to pathogen infection. The activation of the proPO system, by the specific recognition of microorganisms by pattern-recognition proteins (PRPs), triggers a serine proteinase cascade, eventually leading to the cleavage of the inactive proPO to the active PO that functions to produce the melanin and toxic reactive intermediates against invading pathogens. This review highlights the recent discoveries of the critical roles of the proPO system in the shrimp immune responses against major pathogens, and emphasizes the functional characterizations of four major groups of genes and proteins in the proPO cascade in penaeid shrimp, that is the PRPs, serine proteinases, proPO and inhibitors.

Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon.

Phenoloxidase (PO) plays an important role in arthropod melanization. Previously, a prophenoloxidase (PmproPO1) gene was cloned and characterized from the hemocytes of the black tiger shrimp, Penaeus monodon. In the present study, we report a novel proPO gene (PmproPO2) belonging to the proPO family identified from the P. monodon EST database (http://pmonodon.biotec.or.th). The full-length sequence of PmproPO2 consists of 2513bp encoding a predicted 689 amino acid residues with a calculated molecular mass and pI of 79.21kDa and 6.69, respectively. It is predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues and a thiol ester-like motif, sharing 67% amino acid sequence identity with PmproPO1. Tissue distribution analyses revealed that the two proPO genes are primarily expressed in the hemocyte. Gene silencing of either PmproPO1 or PmproPO2 or both by RNA interference (RNAi) resulted in a significant decrease in the respective endogenous proPO mRNA level in hemocytes and a reduction of total PO enzyme activity by 75, 73 and 88%, respectively. Experimental infection of P. monodon with the pathogenic bacterium, Vibrio harveyi, revealed that PmproPO silenced shrimps were more susceptible to bacterial infection than the control GFP injected shrimps, and suggesting that the two proPOs are important components in the shrimp immune defense.

Gene silencing of a prophenoloxidase activating enzyme in the shrimp, Penaeus monodon, increases susceptibility to Vibrio harveyi infection.

The prophenoloxidase (proPO) activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase (PO), is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases (clip-SPs). In this study, a cDNA encoding a proPO activating enzyme (PPAE) from the black tiger shrimp, Penaeus monodon, designated as PmPPAE1, was cloned and characterized. The full-length cDNA contains an open reading frame (ORF) of 1392bp encoding a predicted protein of 463 amino acids including an 18 amino acid signal peptide. The PmPPAE1 protein exhibits a characteristic sequence structure of clip-SPs consisting of the clip domain at the N-terminus and a SP domain at the C-terminus. Sequence analysis showed that PmPPAE1 exhibited the highest amino acid sequence similarity (70%) to a PPAE of the crayfish, Pacifastacus leniusculus. PmPPAE1 mRNA is abundantly expressed in hemocytes, and this is regulated after systemic Vibrio harveyi infection supporting that it is an immune-responsive gene. RNA interference-mediated suppression of PmPPAE1, performed by injection of double-stranded RNA (dsRNA) corresponding to the PmPPAE1 gene into shrimp, resulted in a significant reduction of PmPPAE1 but not other clip-SP and related gene transcript levels of P. monodon, suggesting gene-specific knockdown. RNAi-mediated silencing of PmPPAE1 gene significantly decreased the total PO activity (36.7%) in shrimp and additionally increased the mortality of V. harveyi infected shrimp, the latter of which correlated with an increase in the number of viable bacteria in the hemolymph. These results indicate that PmPPAE1 functions in the proPO system and is an important component in the shrimp immune system.

Pattern Recognition Protein Binds to Lipopolysaccharide and β-1,3-Glucan and Activates Shrimp Prophenoloxidase System

Background: LGBP is an important pattern recognition protein (PRP). Results: PmLGBP binds to β-1,3-glucan and LPS and could enhance the phenoloxidase (PO) activity. Knockdown shrimp showed decreased PO activity. Conclusion: PmLGBP functions as a PRP for LPS and β-1,3-glucan in the proPO system. Significance: PmLGBP is a PRP involved in the proPO system, exhibits LPS and β-1,3-glucan binding activity, and can activate the proPO system. The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and β-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 β-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 β-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 β-1,3-glucan in the shrimp proPO activating system.

PmPPAE2, a new class of crustacean prophenoloxidase (proPO)-activating enzyme and its role in PO activation.

The prophenoloxidase (proPO) activating system plays an important role in the defense against microbial invasion in invertebrates. In the present study, we report a second proPO-activating enzyme (designated PmPPAE2) from the hemocytes of the black tiger shrimp, Penaeus monodon. PmPPAE2 contained the structural features of the clip domain serine proteinase family and exhibited 51% amino acid sequence similarity to the insect Manduca sexta PAP-1. Amino acid sequence alignment with the available arthropod PPAE sequences demonstrated that PmPPAE2 is a new class of crustacean PPAE. Transcript expression analysis revealed that PmPPAE2 transcripts were mainly expressed in hemocytes. Double-stranded RNA-mediated suppression of PmPPAE2 transcript levels resulted in a significant decrease in the total hemolymph PO activity (41%) and also increased the shrimps susceptibility to Vibrio harveyi infection. Genomic organization analysis revealed that PmPPAE1 and PmPPAE2 are encoded by different genomic loci. The PmPPAE1 gene consists of ten exons and nine introns, whilst PmPPAE2 comprises of eight exons interrupted by seven introns. Analysis of the larval developmental stage expression of the four key genes in the shrimp proPO system (PmPPAE1, PmPPAE2, PmproPO1 and PmproPO2) revealed that PmPPAE1 and PmproPO2 transcripts were expressed in all larval stages (nauplius, protozoea, mysis and post-larvae), whilst PmPPAE2 and PmproPO1 transcripts were mainly presented in the late larval developmental stages (mysis and post-larvae). These results suggest that the PmPPAE2 functions as a shrimp PPAE and possibly mediates the activation of PmproPO1.

A clip domain serine proteinase plays a role in antibacterial defense but is not required for prophenoloxidase activation in shrimp.

The clip domain serine proteinases (clip-SPs) play critical roles in the signaling processes during embryonic development and in the innate immunity of invertebrates. In the present study, we identified a homolog of the clip-SP, designated as PmClipSP1, by searching the Penaeus monodon EST database (http://pmonodon.biotec.or.th), and using RACE-PCR to obtain the complete gene which contained a 1101bp open reading frame encoding 366 amino acids with a 25 amino acid signal peptide. The deduced PmClipSP1 protein sequence, which shares a predicted structural similarity to the clip-SPs of other arthropod species, appears to possess a clip domain at the N-terminus and an enzymatically active serine proteinase domain at the C-terminus. Tissue distribution analyses revealed that, at the transcript level, PmClipSP1 is mainly expressed in shrimp hemocytes, whilst temporal gene expression analyses showed that the hemocyte PmClipSP1 transcript levels were upregulated at 3h and downregulated at 6-48h following systemic Vibrio harveyi infection. RNAi-mediated silencing of the PmClipSP1 gene, by injection of double-stranded RNA (dsRNA) corresponding to the PmClipSP1 gene into shrimp, significantly reduced PmClipSP1 transcript levels, but neither significantly altered the other clip-SP and clip-SPH transcript levels nor reduced the total phenoloxidase (PO) enzyme activity in shrimp hemocytes, compared to the levels seen in the GFP dsRNA control, suggesting that PmClipSP1 is not involved in the proPO system. However, suppression of the PmClipSP1 gene led to a significant increase in the number of viable bacteria in the hemolymph (approximately 2.4-fold) and in the mortality rate (59%) of shrimp systemically infected with V. harveyi. These findings suggest that PmClipSP1 plays a role in the antibacterial defense mechanism of P. monodon shrimp.

Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection

Background: Melanization plays a major role in invertebrate defense. Results: Suppression of shrimp melanization increased the WSSV susceptibility. The viral protein, WSSV453, interferes the proPO system via PmPPAE2 inhibition. Conclusion: Shrimp melanization has an antiviral role. WSSV overcomes this by suppressing the host proPO proteinase cascade. Significance: The regulation of shrimp melanization during WSSV infection was first demonstrated. 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.

Shrimp humoral responses against pathogens: antimicrobial peptides and melanization

Diseases have caused tremendous economic losses and become the major problem threatening the sustainable development of shrimp aquaculture. The knowledge of host defense mechanisms against invading pathogens is essential for the implementation of efficient strategies to prevent disease outbreaks. Like other invertebrates, shrimp rely on the innate immune system to defend themselves against a range of microbes by recognizing and destroying them through cellular and humoral immune responses. Detection of microbial pathogens triggers the signal transduction pathways including the NF-κB signaling, Toll and Imd pathways, resulting in the activation of genes involved in host defense responses. In this review, we update the discovery of components of the Toll and Imd pathways in shrimp and their participation in the regulation of shrimp antimicrobial peptide (AMP) synthesis. We also focus on a recent progress on the two most powerful and the best-studied shrimp humoral responses: AMPs and melanization. Shrimp AMPs are mainly cationic peptides with sequence diversity which endues them the broad range of activities against microorganisms. Melanization, regulated by the prophenoloxidase activating cascade, also plays a crucial role in killing and sequestration of invading pathogens. The progress and emerging research on mechanisms and functional characterization of components of these two indispensable humoral responses in shrimp immunity are summarized and discussed. Interestingly, the pattern recognition protein (PRP) crosstalk is evidenced between the proPO activating cascade and the AMP synthesis pathways in shrimp, which enables the innate immune system to build up efficient immune responses.

An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates

An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca(2+) for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca(2+). The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100 mM Ca(2+) could become activated when the concentration of LPS was increased up to 3 mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.

Melanization reaction products of shrimp display antimicrobial properties against their major bacterial and fungal pathogens

Melanization is a rapid defense mechanism in invertebrates. The substrate specificity of phenoloxidases (POs) and the role of melanization reaction products were investigated in the black tiger shrimp, Penaeus monodon. Two PmPOs (PmproPO1 and PmproPO2) were found to display a substrate specificity towards monophenols and diphenols, and exhibit relatively weak activity against 5,6-dihydroxyindole (DHI). Systemic infection of the PmproPO1/2 co-silenced shrimp with the fungus, Fusarium solani, led to a significantly increased mortality, suggesting an important role of PmproPOs in shrimps defense against fungal infection. Using L-DOPA, dopamine or DHI as a substrate, the melanization reaction products exhibited in vitro antimicrobial activities towards Gram-negative bacteria (Vibrio harveyi and Vibrioparahaemolyticus) and Gram-positive bacteria (Bacillus subtilis), whereas the lower effect was detected against the fungus (F. solani). SEM analysis revealed the morphological changes and damage of cell membranes of V. harveyi and F. solani after treatment with shrimp melanization reaction products. Together, these findings demonstrate the crucial functions of the proPO system and the importance of melanization reaction products in the shrimps immune defense.