Spätzle processing enzyme is required to activate dorsal switch protein 1 induced Toll immune signalling pathway in Tenebrio molitor

Abstract

Dorsal switch protein 1 (DSP1) acts as a damage-associated molecular pattern (DAMP) molecule to activate immune responses in Tenebrio molitor. From a previous study in Spodoptera exigua, we found that DSP1 activates Toll immune signalling pathway to induce immune responses by melanisation, PLA2 activity and AMP synthesis. However, the target site of DSP1 in this pathway remains unknown. The objective of this study was to determine the role of spätzle processing enzyme in the DSP1 induced toll immune signalling pathway. To address this, we analyzed spätzle processing enzyme (Tm-SPE) of the three-step serine protease cascade of T. molitor Toll pathway. Tm-SPE expressed in all developmental stages and larval tissues. Upon immune challenge, its expression levels were upregulated but significantly reduced after RNA interference (RNAi). In addition, the induction of immune responses upon immune challenge or recombinant DSP1 injection was significantly increased. Loss of function using RNA interference revealed that the Tm-SPE is involved in connecting DSP1 induced immune responses like hemocyte nodule formation, phenoloxidase (PO) activity, phospholipase A2 (PLA2) activity and antimicrobial peptide (AMP) synthesis. These suggest that Tm-SPE controls the DSP1 induced activation of Toll immune signalling pathway required for both cellular and humoral immune responses. However, to confirm the target molecule of DSP1 in three-step proteolytic cascade, we have to check other upstream serine proteases like Spatzle activating enzyme (SAE) or modular serine protease (MSP).

Fig 1. Molecular characterization of T. molitor Spätzle-Processing Enzyme (Tm-SPE).

(A) Phylogenetic analysis of Tm-SPE with other insect SPE genes using MEGA6 program with a Neighbor-joining method. Bootstrapping values were obtained with 1,000 repetitions to support branching and clustering. Amino acid sequences of SPE were retrieved from GenBank with accession numbers shown in S2 Table. (B) Domain analysis of Tm-SPE. Domains were predicted using Prosite (https://prosite.expasy.org/) and SMART protein (http://smart.embl-heidelberg.de/).

Fig 2. Expression profiles of Tm-SPE.

(A) Expression patterns of Tm-SPE at different developmental stages: egg, some larval instar (‘L1, L2, L6, L12’), pupa and adult. (B) Expression patterns in different tissues of L6 larvae: hemocyte (‘HC’), fat body (‘FB’), midgut (‘GT’) and epidermis (‘EPD’). (C) Inducible expression of Tm-SPE in whole body sample of L6 larvae at different time intervals after bacterial infection. For bacterial challenge, heat-killed E. mundtii (‘Em’, 1.8 × 10⁵ cells/larva) were injected into each larva. For control, larvae were injected with PBS. Expression was analyzed with real-time qPCR and up-regulated expression levels were calculated as fold change of the lowest expressed. A ribosomal gene, L27a, was used as internal control [24]. Each treatment was replicated three times with independent samples preparation. Different letters above the standard deviation bar indicate significant differences among means at Type I error = 0.05 (LSD test).

Fig 3. Role of SPE for the induction of immune responses by E. mundtii and rDSP1.

(A) RNAi efficiencies of Tm-SPE gene by injecting gene-specific dsRNA (‘dsSPE’, 1 μg/larva) to L6 larvae. A viral gene, CpBV302, was used as a control dsRNA (‘dsCON’). (B) E. mundtii and rDSP1 induced mediation of hemocyte nodulation, (C) PO activity and (D) Pictorial representation of change in plasma due of PO activity. L6 larvae of T. molitor were injected with E. mundtii (2.2 x 10⁵ cells/larvae) dissolved in PBS or E. mundtii (2.2 x 10⁵ cells/larvae) along with recombinant DSP1 (‘rDSP1, 0.6 μg/larva). At 8 h of E. mundtii or rDSP1 injection, nodules were counted in each larva by dissecting and plasma was collected for PO activity analysis. Naïve was injected with PBS. Knock-down of Tm-SPE expression was done by injecting dsRNA specific to Tm-SPE (1 μg/larva) into L6 larvae before 24 h of E. mundtii or rDSP1 injection. A viral gene, CpBV302, was used as a control dsRNA (‘dsCON’). Each treatment was replicated three times. Asterisks indicate significant differences between dsSPE and dsCON treated group at Type I error = 0.05 (LSD test). Different letters above standard deviation bars denotes significant difference among means at Type I error = 0.05 (LSD test).

Fig 4. Functional assay of spätzle processing enzyme (Tm-SPE) for PLA₂ activity mediation.

(A) Mediation of sPLA₂ activity induced by bacteria and rDSP1 via SPE. (B) Mediation of cPLA₂ activity by bacteria and rDSP1 via SPE. L6 larvae were injected with E. mundtii (2.2 x 10⁵ cells/larvae) or recombinant DSP1 (‘rDSP1’, 0.6 μg/larva). Naïve were injected with PBS. dsRNA specific to SPE or dsCON was injected 24 h before bacteria or rDSP1 injection. At 8 h after E. mundtii or rDSP1 injection, Fatbody and plasma was collected to determine cPLA₂ and sPLA₂ activity, respectively. dsRNA was made from a viral gene (CpBV302). Each treatment was replicated three times. Different letters above standard deviation bars denotes significant difference among means at Type I error = 0.05 (LSD test).

Fig 5. Expression pattern of AMP genes in T. molitor.

(A) Mediation of AMP genes expression via SPE upon E. mundtii challenge (B) Mediation of AMP genes expression via SPE upon rDSP1 challenge. For AMP genes expression upon bacterial challenge, E. mundtii were dissolved in PBS (‘Em’, 1.8 × 10⁵ cells/larva) and for rDSP1 challenge, purified rDSP1 (0.6 μg/larva) was injected into L6 larva. For analysing expression of Attachin1a (‘Att1a’), Attachin2 (‘Att2’), Cecropin2 (‘Cec2’), Coleoptericin1 (‘Col1’), Defensin1 (‘Def1’), Defensin2 (‘Def2’), Tenesin1 (‘Ten1’) and Tenesin3 (‘Ten3’) AMP genes, whole body samples were collected after 8 h of bacterial or rDSP1 challenge for RNA extraction. For knockdown of Tm-SPE gene, dsRNA specific to Tm-SPE was injected 24 h before E. mundtii or rDSP1 injection. For control, dsRNA made from a viral gene (CpBV302) was injected. Each treatment was replicated three times with independent tissue preparations. Different letters above the bar indicate significant differences among means at Type I error = 0.05 (LSD test).

Fig 6. Comparative virulence analysis of E. mundtii (‘Em’) and X. hominickii (‘Xh’) bacteria against T. molitor larvae.

(A) Individual RNAi treatment targeting SPE gene and (B) Immune induction by rDSP1 to increase survivability. For survivability test, Gram positive E. mundtii (3 x 10⁴ cells/larva) and Gram-negative X. hominickii (2.5 x 10⁴ cells/larva) were injected to L6 larvae of T. molitor already (24 h before) injected with dsSPE and dsCON (1 μg/larva). dsCON was made from a viral gene CpBV302. For immune mediation test of rDSP1, at 24 h post injection of dsRNA, E. mundtii (3 x10⁵ cells/larva) or E. mundtii along with rDSP1 (0.6 μg/larva) was injected to larvae with a Hamilton micro syringe. Mortality was recorded up to 7 days after treatment (‘DAT’). Each treatment was replicated three times and each replication used 10 larvae. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

Fig 7. A working hypothesis of Tm-DSP1 for mediating immune responses via Toll-Spatzle (‘Spz’) signalling pathway.

Upon challenge with gram positive bacteria including a non-entomopathogenic bacterium, E. mundtii, Tm-DSP1 is secreted to the plasma to activate serine protease (SP) cascade for activating phenoloxidase (‘PO’) and Spz. Activated PO can catalyze melanin formation to suppress the growth of pathogenic bacteria. Besides, activated Spz can bind to Tm-Toll receptor to activate PLA₂ and the expression of antimicrobial peptide [Cecropine2 (‘Cec2’), Defencine1 (‘Def1’), Defencine2 (‘Def2’), Tenecine1 (‘Ten1’) and Tenecine3 (‘Ten3’)) genes. Activated PLA₂ can catalyze eicosanoid biosynthesis to mediate cellular immune responses to defend bacterial infection along with AMPs.