Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera

Abstract

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

CLIPB10 is a Terminal Protease in the Regulatory Network That Controls Melanization in the African Malaria Mosquito Anopheles gambiae

“…Studies in other model insects have shown that PAP activation cleavage is mediated by cSPs belonging to the CLIPC family, which in turn are proteolytically activated by an upstream modular serine protease (ModSP). Examples of such proPO activation cascades have been described in several model organisms, including M. sexta ( Gorman et al, 2007 ; Wang and Jiang, 2007 ), Tenebrio molitor ( Kan et al, 2008 ; Jiang et al, 2009 ), and most recently Helicoverpa armigera ( Wang Q. et al, 2020 ). While currently no data exist that show activation cleavage of either CLIPB9 or CLIPB10 by an endogenous CLIPC, recent genetic evidence suggests that CLIPC9 is required for humoral melanization of parasites as well as tissue melanization ( Sousa et al, 2020 ).…”

Advances in the Arms Race Between Silkworm and Baculovirus

“…It consists of a cascade of clip-domain serine proteases (cSPs) that converts zymogen prophenoloxidase (PPO) into active phenoloxidase (PO), which is negatively regulated by serpins. PO catalyzes melanin formation to encapsulate and kill invading pathogens ( 50 , 51 ). Baculovirus infection is efficiently blocked by the PPO activation cascade ( 50 ).…”

“…PO catalyzes melanin formation to encapsulate and kill invading pathogens ( 50 , 51 ). Baculovirus infection is efficiently blocked by the PPO activation cascade ( 50 ). Bmserpin2 knockdown increases PO activity and decreases viral DNA content in silkworm haemolymph infection with BmNPV ( 52 ).…”

“…Baculoviruses can suppress host melanization so that they can proliferate. Several SPs (serine proteases) and their homologs are upregulated in response to bacterial or fungal challenge but downregulated in response to baculovirus infection ( 50 , 51 ). For example, when serpins 5 and 9 are induced by HearNPV in Helicoverpa armigera , they inhibit SPs and melanization and promote viral infection ( 51 ).…”

Insights Into the Antiviral Pathways of the Silkworm Bombyx mori

“…PO catalyzes the oxidation of phenols to form quinones and melanin, wherein the rapid polymerization of melanin at infection sites can kill and immobilize microbes ( 61 – 63 ) ( Figure 1D ). The melanization can kill baculovirus in vitro ( 64 , 65 ). However, the PPO pathway is negatively regulated by serpins, and baculovirus can induce serpins to suppress the melanization response of host insects for survival ( 57 , 64 ).…”