Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies

Abstract

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila

“…Because Psh and Hayan redundantly regulate Toll signaling, as characterized by genetic analysis ( 6 ), our result that two Hayan isoforms did not activate SPE ( Fig. 5 ) suggests the existence of unidentified protease(s) downstream of Hayan that can process proSpz ( 22 , 23 ). We identified four other proSpz activators, namely, Sp7, MP1, Ser7, and Grass ( Fig.…”

“…As the protease acting between ModSP and Grass is unknown ( 16 ), we speculate that cSP48 may play the same role as Ms-HP21 ( 11 , 37 ), linking ModSP (an ortholog of Ms-HP14) and Grass (an ortholog of Ms-HP5). In the case of MP1, although it formed a cluster with Dm-SPE, Ms-HP8, and Tm-SPE, and expression of the MP1 catalytic domain only led to constitutive melanization ( 20 ) and cleavage of Spz ( 22 , 23 ), MP1 null mutants failed to show a clear role in either the melanization response or Toll pathway activity ( 6 ). Perhaps, MP1 only contributes to the immune responses and its redundant role needs clarification by a biochemical analysis using full-length protein.…”

“…Although there is genetic evidence suggesting that Psh and Hayan redundantly regulate the Toll pathway ( 6 ), our experiments indicate that Psh and Hayan (-PA and -PB) may regulate activation of the Toll pathway differently. Given that there could be other proteases that may have the ability to cleave proSpz ( 6 , 22 , 23 ), we extended our investigation to explore the cleavage activation of proMP1 and proSer7, close paralogs of SPE. The 50-kDa proMP1 can be cleaved by Pr1A-generated active Psh, Hayan-PA, and Hayan-PB ( Fig.…”

“…Functional redundancy, as exemplified by the case of Psh and Hayan, leads to complexity of the serine protease cascades and limits the resolving power of genetic tools in elucidating mechanisms underlying the innate immune responses. Similarly, genetic evidence suggests the existence of another SPE-like protease capable of cleaving proSpz ( 6 , 22 , 23 ). Because SPE has as many as eight close paralogs (Easter, MP1, Ser7, cSP14, cSP16, cSP38, cSP61, and cSP229) ( 8 ), putative functional overlaps may make it difficult, if not impossible, to resolve this complicated step solely by genetic approach.…”

Innate immunity in Aedes mosquitoes: from pathogen resistance to shaping the microbiota

“…In the Drosophila model, the Imd pathway is activated by the binding of the PGRP-LC receptor to DAP-type peptidoglycan [77,78] (characteristic of Gramnegative bacteria) while the Toll pathway is specifically activated by Lys-type peptidoglycan [79] (characteristic of Gram-positive bacteria) and β-glucan [80] (characteristic of fungi). Toll signalling is also more generally activated by danger signals associated with microbial virulence and host damage [81][82][83][84]. To our knowledge, however, none of these triggers (DAP-PGN, Lys-PGN, β-glucan, danger signals) have been specifically validated in connection with either pathway in any mosquito species.…”