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

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Highlights

Abstract

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 shrimp’s 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 Vibrio parahaemolyticus) 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 shrimp’s immune defense.

Introduction

Phenoloxidase (PO)-mediated melanization is a conserved non-self defense response involved in the synthesis of a melanin coat, and several cytotoxic effectors, such as reactive intermediates of oxygen or nitrogen molecules, to combat foreign microbes in invertebrates (Amparyup et al., 2013a, Beutler, 2004, Cerenius and Söderhäll, 2004, Cerenius et al., 2008, Janeway and Medzhitov, 2002, Kanost and Gorman, 2008). Uncontrolled or systemic melanogenesis is thought to be deleterious to the host cells because excess or prolonged levels of the cytotoxic substances can lead to host tissue damage and cell death. Melanin synthesis should, therefore, be very site-specific and tightly regulated to minimize the damage of an aberrant process (Cerenius et al., 2008, Nappi and Christensen, 2005).

The melanization cascade is initiated by the specific recognition the pathogen-associated molecular patterns, including peptidoglycan from Gram-positive bacteria, lipopolysaccharide (LPS) from Gram-negative bacteria and β-1,3-glucan from fungi, by host pattern recognition proteins. This is followed by the subsequent sequential activation of the clip-domain serine proteinases (clip-SPs) and their homologues, which in turn activates the terminal clip-SP, proPO-activating enzymes (PPAEs), and leads to the specific conversion of the proPO precursor to the functionally active PO (Söderhäll et al., 2013). Active PO then oxidizes the mono- and di-phenolic substances to quinone precursors that are required for the synthesis of melanin at the wound site or around the foreign invaders (Cerenius and Söderhäll, 2004, Nappi and Christensen, 2005).

PO is a multimeric type III copper protein that contains two copper atoms co-ordinated by six histidine residues and participates in multiple steps of melanin formation (Nappi and Christensen, 2005, Sugumaran, 2002). Arthropod POs have been classified into two types based on their substrate specificity (Barrett and Trevella, 1989, Decker et al., 2007). The first type, the tyrosinase-POs catalyze the o-hydroxylation of monophenols (monophenolase or cresolase activity) and the subsequent oxidation of o-diphenols to the reactive intermediate o-quinones (o-diphenolase or catecholase activity), whilst the second type, the catecholoxidase-POs contain only a diphenolase activity and so catalyze only the oxidation of diphenolic substrates. During melanization, PO converts tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA) by its monophenolase activity. DOPA in turn is oxidized to dopaquinone by the diphenolase activity of PO or to dopamine by DOPA decarboxylase (DDC). Dopaquinone is converted to dopachrome non-enzymatically and further decarboxylated to form 5,6-dihydroxyindole (DHI) using the dopachrome conversion enzyme (Bai et al., 1996, Shelby et al., 2000, Sugumaran, 1996). Ultimately, dopamine and its oxidation product (DHI) are converted to melanin either by PO activity or via a series of intermediate steps involving both enzymatic and non-enzymatic reactions (Fang et al., 2002, Shelby et al., 2000, Sritunyalucksana and Söderhäll, 2000).

In invertebrates, the melanization reaction, proPO activation and DHI production play critical roles in the defense against microbial infections (Amparyup et al., 2009, Amparyup et al., 2013a, Amparyup et al., 2013b, Binggeli et al., 2014, Cerenius et al., 2008, Cerenius et al., 2010, Charoensapsri et al., 2009, Charoensapsri et al., 2011, Eleftherianos et al., 2007; Fagutao et al., 2009; Kan et al., 2008, Liu et al., 2007, Nappi and Christensen, 2005, Yassine et al., 2012, Zhao et al., 2007, Zhao et al., 2011). Several reactive compounds generated during melanization have been reported to be toxic to pathogens. In Manduca sexta, the melanization reaction products generated from dopamine and DHI are much more efficiently aggregated and killed bacterial cells than that of l-DOPA (Zhao et al., 2007). In Tenebrio molitor, the melanization complex assembled from PO and serine proteinase homologue (SPH) 1 has been reported to possess strong bactericidal effect and induced melanin synthesis deposition on the surface of bacteria (Kan et al., 2008). In addition, DHI and its spontaneous oxidation products also shown to exhibit a strong toxicity against various fungi, baculoviruses, bacteriophages and parasitic wasps (Zhao et al., 2007, Zhao et al., 2011). In the crustacean Pacifastacus leniusculus, it is also evident that the melanization reaction products generated during an in vitro PO activation exhibited an antibacterial effect against selected bacteria in which a strong antibacterial activity was observed when dopamine was used as substrate, as compared with l-DOPA, and this effect was abolished by the PO inhibitor, phenylthiourea (PTU) (Cerenius et al., 2010). These findings collectively indicated that the potential roles of reactive intermediates generated during melanization include their toxicity towards, and killing of, invading microorganisms (Cerenius et al., 2010, Kan et al., 2008, Zhao et al., 2007, Zhao et al., 2011).

In the black tiger shrimp, Penaeus monodon, several genes implicated in the proPO system, including two proPOs (PmproPO1 and PmproPO2), two PPAEs (PmPPAE1 and PmPPAE2), a LPS- and β-1,3-glucan binding protein (PmLGBP) and a clip-SP (PmClipSP2), have been identified and their important functions in the shrimp’s defense against Vibrio harveyi infection have been characterized (Amparyup et al., 2009, Amparyup et al., 2012, Amparyup et al., 2013b, Charoensapsri et al., 2009, Charoensapsri et al., 2011). In this study, the substrate specificity of the two PmproPO proteins in P. monodon shrimp was investigated using RNAi-mediated gene silencing of the PmproPO1 and PmproPO2 genes (and so likely represents the substrate specificity of PmPO1 and PmPO2). Fusarium solani is one of the fungal pathogens caused disease problem in shrimp aquaculture. Here, the crucial role of the two PmproPOs in the host defense against infection with the filamentous fungus F. solani was subsequently elucidated. The in vitro potential effects of the reactive compounds generated from the available phenolic substrates, l-DOPA, dopamine and DHI, during the melanization process in contributing to the killing of selected model pathogenic Gram-negative bacteria, Gram-positive bacteria and a fungus was demonstrated.

Section snippets

Animals

Specific pathogen-free (SPF) P. monodon shrimp (∼5 g wet weight) were obtained from the Shrimp Genetic Improvement Center, BIOTEC, Thailand. Prior to the experiments, all shrimp were acclimatized in aerated seawater (20 ppt salinity) under laboratory conditions for at least 7 d.

Chemicals

Dopamine, l-tyrosine, LPS from Escherichia coli 0111:B4 and laminarin (β-glucan) from Laminaria digitata were purchased from Sigma–Aldrich, l-DOPA was from Fluka and DHI was from Santa Cruz Biotechnology. All general

PmproPO1 and PmproPO2 both exhibit substrate specificity toward monophenol and diphenols

PO catalyzes the key step of melanin biosynthesis by oxidation of mono- and di-phenol substrates. In order to assess the substrate specificity of the two known POs in P. monodon shrimp, the gene transcripts of their precursors (PmproPO1 and PmproPO2) were independently silenced by dsRNA-mediated gene knockdown. As shown in Fig. 1A, sq-RT-PCR analysis revealed that PmproPO1- and PmproPO2- dsRNAs significantly suppressed the expression of PmproPO1 and PmproPO2 transcripts, respectively, and no

Discussion

Melanin biosynthesis in invertebrates is achieved by the activation cascade of the proPO system (Amparyup et al., 2013a, Cerenius and Söderhäll, 2004, Cerenius et al., 2008, Kanost and Gorman, 2008). In P. monodon, several proPO system associated genes, including two proPOs (PmproPO1 and PmproPO2), two PPAEs (PmPPAE1 and PmPPAE2) and a clip-SP (PmClipSP2), have been identified and characterized as important components that not only play key roles in the shrimp proPO system, but are also crucial

Acknowledgments

This work was supported by research grants from the Thailand Research Fund to A.T. (TRF Senior Scholar No. RTA5580008), the Thailand Research Fund and National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency to P.A. (Grant No. RSA5580046), JST/JICA, SATREPS, and the Visiting Fellowship under the JSPS core-University Program. W.C. is supported by a postdoctoral fellowship from the Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University.

References (43)

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1

These two authors contributed equally to this work, and share the first authorship.

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