Hemocyanin-derived phenoloxidase reaction products display anti-infective properties

Hemocyanin is a multi-functional protein located in the hemolymph (blood) of certain arthropods and molluscs. In addition to its well-defined role in oxygen transport, hemocyanin can be converted into a phenoloxidase-like enzyme. Herein, we tested the antimicrobial properties of horseshoe crab (Limulus polyphemus) hemocyanin-derived phenoloxidase reaction products using broad ranges of phenolic substrates (e.g. L-DOPA) and microbial targets (Gram-positive/negative bacteria, yeast). The enzyme-catalysed turnover of several substrates generated (by)products that reduced significantly the number of colony forming units. Microbicidal effects of hemocyanin-derived phenoloxidase were thwarted by the inhibitor phenylthiourea. Data presentedhere further support a role for hemocyanin in invertebrate innate immunity

Shrimp serine proteinase homologues PmMasSPH-1 and -2 play a role in the activation of the prophenoloxidase system.

Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system

Expression of <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 transcripts at various stages of shrimp larval development.

<p>Expression profiles of <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 were examined at four larval stages including nauplius 3 (N3), protozoea 2 (Z2), mysis 2 (M2) and post-larvae 15 (PL15) by semi-quantitative RT-PCR. The elongation factor 1-α (EF1-α) served as an internal control. Each lane represents the result of individual shrimp (n = 3).</p

Increase in bacterial load in hemolymph from <i>Pm</i>MasSPHs silenced shrimp.

<p>Shrimp were injected twice with dsRNA specific to <i>Pm</i>MasSPH1 (A) or <i>Pm</i>MasSPH2 (B). At the second dsRNA injection, shrimp were also injected with <i>V</i>. <i>harveyi</i> (2×10⁵ CFU/shrimp). Control groups given injections of GFP dsRNA or NaCl. The number of viable bacteria in the knockdown shrimp hemolymph is shown as the bacterial CFUs 6 h after challenge. The data are shown as the mean ± standard deviation derived from three independent experiments. Means with lower case letters above each bar indicate significant differences (<i>p</i> < 0.05).</p

Enhancement of PGN-triggered hemolymph PO activity by r<i>Pm</i>MasSPH1 and r<i>Pm</i>PPAE2 <i>in vitro</i>.

<p>The total PO activity of shrimp hemolymph (HL) alone or mixed with <i>B</i>. <i>subtilis</i> PGN, r<i>Pm</i>MasSPH1 and r<i>Pm</i>PPAE2 was determined. BSA served as a protein control. Each bar represents the PO activity as the mean ± standard deviation from three replicates. The percentages of each treatment are relative to the PO activity of HL alone.</p

The phylogenetic relationship between serine proteinase domains from <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 and other serine proteinases.

<p>The deduced amino acid sequences of SP-domains from various clip-SPH species were used to generate a phylogenetic tree by the neighbor-joining method. Percent bootstrap values (1000 replicates) are shown at each branch point. <i>Pm</i>MasSPH1 (ABE03741); <i>Pm</i>MasSPH2 (ACP19560); <i>Pm</i>SP, <i>P</i>. <i>monodon</i> serine proteinase (ABW87872); <i>Pm</i>SPL, <i>P</i>. <i>monodon</i> serine proteinase-like protein (ABD62888); <i>Pm</i>SPL3 (ABO33174); <i>Pm</i>Mas, <i>P</i>. <i>monodon</i> mas-like protein (AAT42131); <i>Fc</i>PPAF, <i>Fenneropenaeus chinensis</i> prophenoloxidase activating factor (AFW98986); <i>Fc</i>Mas (AFW98983); <i>Lv</i>PPAF, <i>Litopenaeus vannamei</i> PPAF (AFW98993); <i>Lv</i>SP (AY368151); <i>Lv</i>Mas (AFW98990); <i>Pl</i>SPH1, <i>P</i>. <i>leniusculus</i> SPH1 (AAX55746); <i>Pl</i>SPH2 (ACB41379); <i>Ms</i>SPH3, <i>Manduca sexta</i> SPH3 (AF413067); <i>Hd</i>PPAFII, <i>Holotrichia diomphalia</i> PPAFII (CAC12665); <i>Mj</i>SPH, <i>Marsupenaeus japonicus</i> SPH (AB161692); <i>Nv</i>SPH21, <i>Nasonia vitripennis</i> SPH21 (NP_001155060); <i>Cq</i>SP, <i>Culexquin quefasciatus</i> SP (XP_001868413); <i>Aa</i>SP, <i>Aedes aegypti</i> SP (XP_001655705); <i>Pt</i>PPAF, <i>Portunus trituberculatus</i> PPAF (ACN87221); <i>Cs</i>PAF, <i>Callinectes sapidus</i> PAF (AAS60227); <i>Es</i>PPAF, <i>Eriocheir sinensis</i> PPAF (ACU65942); <i>Es</i>Trypsin-likeSP (ACT78700); <i>Ls</i>ST, <i>Lucilia sericata</i> salivary trypsin (AEX33291); <i>Cp</i>PPAF, <i>Cancer pagurus</i> PPAF (CCE46009); <i>Pp</i>SP, <i>Papilio polytes</i> SP(BAM19108); <i>Ha</i>SPL1, <i>Helicoverpa armigera</i> SPL1 (ACI32835); <i>Tm</i>PPAF, <i>Tenebrio molitor</i> PPAF (CAC12696); <i>St</i>SPL, <i>Scylla tranquebarica</i> SPL (ADN44616); <i>Lo</i>PPAF1, <i>Lonomia obliqua</i> PPAF1 (AAV91458); <i>Px</i>SP, <i>Papilio xuthus</i> SP (BAM17901); <i>Pr</i>MSPH, <i>Pieris rapae</i> MSPH (ACZ68116); <i>Tc</i>SP4, <i>Tribolium castaneum</i> SP4 (EEZ99183); <i>Dm</i>Mas, <i>Drosophila melanogaster</i> Mas (AAC46512); <i>Sp</i>-SPH, <i>S</i>. <i>paramamosain</i> SPH (ADG83846).</p

A multiple amino acid sequence alignment of <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 with other arthropod SPHs.

<p>The amino acid sequence of <i>Penaeus monodon Pm</i>MasSPH1 (ABE03741), <i>Pm</i>MasSPH2 (ACP19560), <i>Pacifastacus leniusculus Pl</i>SPH1 (AAX55746), <i>Pl</i>SPH2 (ACB41379), <i>Scylla paramamosain Sp</i>-SPH (ADG83846) and <i>Holotrichia diomphalia Hd</i>PPAFII (CAC12665) were collectively compared. The predicted signal peptides are in bold and underlined. The dash line indicates the glycine-rich domain of <i>Pm</i>MasSPH1. The conserved clip-domains are indicated below the line. The light-grey highlight indicates the cysteine residues in the clip-domains. The black box shows the conserved serine proteinase-like domain with the grey highlight indicating the catalytic triad (His, Asp, and Gly residues) in the domain.</p