Shrimp Serine Proteinase Homologues PmMasSPH-1 and -2 Play a Role in the Activation of the Prophenoloxidase System

Abstract

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.

A shrimp pacifastin light chain-like inhibitor: Molecular identification and role in the control of the prophenoloxidase system

“…This result is in accord with previous observations in the crayfish P. leniusculus, which revealed that pacifastin transcripts were present during the middle phase of crayfish embryo development (Zhang et al, 2010). In the shrimp P. monodon, the relationship of the expression profiles of proPO, PPAE, and SPH genes during larval development stages has been investigated (Charoensapsri et al, 2011;Jearaphunt et al, 2015). PmPPAE1, PmproPO2, PmMasSPH1, and PmMasSPH2 transcripts were detected during all of the larval stages, whereas PmPPAE2 and PmproPO1 transcripts were primarily expressed during the late stages of larval development (mysis and postlarvae).…”

“…Previously, we showed that disruption of PmproPO gene expression could affected the expression of a terminal serine proteinase (PmPPAE2) and a pattern recognition protein (PmLGBP) (Amparyup et al, 2012). Likewise, suppression of PmMasSPH1 also reduced the expression of PmPPAE2 transcripts (Jearaphunt et al, 2015).…”

“…Previously, we identified and characterized the function of several proPO system components as well as their important roles in pathogen defense in the shrimp P. monodon (Amparyup et al 2009(Amparyup et al , 2012(Amparyup et al , 2013bCharoensapsri et al, 2009Charoensapsri et al, , 2011Charoensapsri et al, , 2014Jearaphunt et al, 2015). Two proPOs (PmproPO1 and PmproPO2) and two PPAEs (PmPPAE1 and PmPPAE2) were found to be essential for the proPO system and for immune defense against Vibrio harveyi and Fusarium solani infections (Amparyup et al, 2009;Charoensapsri et al, 2009Charoensapsri et al, , 2011Charoensapsri et al, , 2014).…”

“…An LPS and β-1,3-glucan binding protein (PmLGBP), a clip-SP 6 (PmClipSP2), and two masquerade-like serine proteinase homologues (PmMasSPH1 and PmMasSPH2) were found to be required for pathogen recognition. PmLGBP, PmClipSP2, and PmMasSPH1 also participated in proPO system activation (Amparyup et al, 2007(Amparyup et al, , 2012(Amparyup et al, , 2013bJearaphunt et al, 2015;Jitvaropas et al, 2009). A homologue of clip-SP, PmClipSP1, was also identified; however, it does not appear to have a function in the shrimp proPO system (Amparyup et al, 2010).…”

The serine protease homolog CLIPA14 modulates the intensity of the immune response in the mosquito Anopheles gambiae

“…The roles of clip domain-containing SPHs characterized in insects ( 15 – 17 ) and crustaceans ( 46 ) have been almost exclusively restricted to the positive regulation of the PPO cascade where they seem to act as cofactors for the proper cleavage and activation of PPO by PAPs. Although the mosquito CLIPA8 ( 22 ) is thought to fall within this functional category of SPHs, CLIPA2 ( 30 ) negatively regulates PPO activation indirectly by fine-tuning the level of activation of TEP1 in a yet undefined mechanism ( 7 , 24 , 26 , 47 ).…”