A Venom Serpin Splicing Isoform of the Endoparasitoid Wasp Pteromalus puparum Suppresses Host Prophenoloxidase Cascade by Forming Complexes with Host Hemolymph Proteinases

Abstract

Edited by Charles E. SamuelTo ensure successful parasitism, parasitoid wasps inject venom along with their eggs into their hosts. The venom serves to suppress host immune responses, including melanization. Venom from Pteromalus puparum, a pupal endoparasitoid, inhibits melanization of host hemolymph in vitro in a dose-dependent manner. Using assay-guided fractionation, a serpin splicing isoform with phenoloxidase inhibitory activity was identified as P. puparum serpin-1, venom isoform (PpS1V). This serpin gene has 16 predicted splicing isoforms that differ only in the C-terminal region. RT-PCR results show that the specific serpin isoform is differentially expressed in the venom gland. Recombinant PpS1V (rPpS1V) suppresses host prophenoloxidase (PPO) activation rather than inhibiting the phenoloxidase directly. Pulldown assays show that PpS1V forms complexes with two host hemolymph proteins, here named Pieris rapae hemolymph proteinase 8 (PrHP8) and P. rapae prophenoloxidase-activating proteinase 1 (PrPAP1), based on gene sequence blasting and phylogenetic analysis. The role of rPrPAP1 in the PPO activation cascade and its interaction with rPpS1V were confirmed. The stoichiometry of inhibition of PrPAP1 by PpS1V is 2.3. PpS1V also inhibits PPO activation in a non-natural host, Ostrinia furnacalis, through forming a complex with O. furnacalis serine protease 13 (OfSP13), an ortholog to PrPAP1. Our results identify a venom-enriched serpin isoform in P. puparum that inhibits host PPO activation, probably by forming a complex with host hemolymph proteinase PrPAP1.

Superoxide dismutase from venom of the ectoparasitoid Scleroderma guani inhibits melanization of hemolymph

“…The investigation on insect PPO for over a century reveals that the PPO cascade can be activated by endogenous serine proteases (SPs), exogenous proteases secreted by pathogens, and various compounds such as ethanol and cetylpyridinium chloride, which is mediated by serine protease homologs (SPHs) of the clip‐domain family that lack protease activity, and a family of serine proteinase inhibitors, serpins (Kausar et al, ; Lu et al, ; K. Wu et al, ; Yang et al, ). SPHs and serpins identified from venoms of parasitoid can inhibit the melanization of host hemolymph (Colinet et al, ; Yan et al, ; G. Zhang, Lu, Jiang, & Asgari, ). Regarding to their action mechanisms, it has been asserted that inhibition of melanization by an a SPH venom protein from C. rubecula is through competing with host SPHs for the binding of PPO (Thomas & Asgari, ; G. Zhang et al, ).…”

“…Regarding to their action mechanisms, it has been asserted that inhibition of melanization by an a SPH venom protein from C. rubecula is through competing with host SPHs for the binding of PPO (Thomas & Asgari, ; G. Zhang et al, ). Moreover, a venom serpin from Pteromalus puparum has been found to inhibit host PPO activation by forming a complex with host hemolymph PPO‐activating proteinase (Yan et al, ). It could propose that venom SPH and serpin target PPO activation pathway to display inhibitory effect on melanization.…”

“…As suggested by Colinet et al (), host melanization pathway would be interfered by SOD3 through dismutation of the superoxide anion, implying a unique mechanism of disturbing PPO cascade. The action mechanism of venom SOD3 involved in disrupting the PPO activation pathway might be different from that of venom SPH and serpin of parasitoids (Colinet et al, ; Yan et al, ; G. Zhang et al, ). The underlying mechanisms deserve to be unraveled in future, which will shed insights into the melanin synthesis and PPO cascade.…”

“…Wu et al, 2018;Yang et al, 2018). SPHs and serpins identified from venoms of parasitoid can inhibit the melanization of host hemolymph (Colinet et al, 2009;Yan et al, 2017;G. Zhang, Lu, Jiang, & Asgari, 2004).…”

“…Zhang et al, 2004). Moreover, a venom serpin from Pteromalus puparum has been found to inhibit host PPO activation by forming a complex with host hemolymph PPO-activating proteinase (Yan et al, 2017). It could propose that venom SPH and serpin target PPO activation pathway to display inhibitory effect on melanization.…”