Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs.

Xenorhabdus hominickii, an entomopathogenic bacterium, inhibits eicosanoid biosynthesis of target insects to suppress their immune responses by inhibiting phospholipase A2 (PLA2) through binding to a damage-associated molecular pattern (DAMP) molecule called dorsal switch protein 1 (DSP1) from Spodoptera exigua, a lepidopteran insect. However, the signalling pathway between DSP1 and PLA2 remains unknown. The objective of this study was to determine whether DSP1 could activate Toll immune signalling pathway to activate PLA2 activation and whether X. hominickii metabolites could inhibit DSP1 to shutdown eicosanoid biosynthesis. Toll-Spätzle (Spz) signalling pathway includes two Spz (SeSpz1 and SeSpz2) and 10 Toll receptors (SeToll1-10) in S. exigua. Loss-of-function approach using RNA interference showed that SeSpz1 and SeToll9 played crucial roles in connecting DSP1 mediation to activate PLA2. Furthermore, a deletion mutant against SeToll9 using CRISPR/Cas9 abolished DSP1 mediation and induced significant immunosuppression. Organic extracts of X. hominickii culture broth could bind to DSP1 at a low micromolar range. Subsequent sequential fractionations along with binding assays led to the identification of seven potent compounds including 3-ethoxy-4-methoxyphenol (EMP). EMP could bind to DSP1 and prevent its translocation to plasma in response to bacterial challenge and suppress the up-regulation of PLA2 activity. These results suggest that X. hominickii inhibits DSP1 and prevents its DAMP role in activating Toll immune signalling pathway including PLA2 activation, leading to significant immunosuppression of target insects.

Introduction

Xenorhabdus hominickii is an entomopathogenic bacterium that is mutualistic to <span class="Species">Steinernema monticolum, a nematode [1]. In general, infective juveniles (IJs) of host nematodes carry pathogenic bacteria in specific receptacles of intestine [2,3]. After entering target insect’s hemocoel through anus, spiracle, and mouth, IJs will release symbiotic bacteria to induce insect immunosuppression [4].

Upon pathogen infection, insects can recognize the nonself with their specific molecular patterns such as <span class="Chemical">lipopolysaccharide, peptidoglycan, or β-1,3-glucan using pattern recognition receptors [5]. The nonself signal is then propagated to nearby immune effectors via various immune mediators such as cytokine, biogenic monoamine, nitric oxide (NO), and eicosanoids [6]. Subsequently, acute cellular immune responses are triggered by hemocytes. Later, humoral immune responses can remove pathogens with melanin formation through the catalysis activity of plasma phenoloxidase or the activity of antimicrobial peptides (AMPs) [4].

For successful pathogenicity, X. hominickii needs to produce and release secondary metabolites such as <span class="Chemical">phenylethylamides to inhibit phospholipase A2 (PLA2) and shutdown eicosanoid biosynthesis [7,8]. PLA2 catalyzes hydrolysis of phospholipids at sn-2 ester bond to release arachidonic acid. This is a committed step for eicosanoid biosynthesis in insects as well as in other organisms [9]. Immune challenge can activate PLA2 to produce eicosanoids in insects [10]. Thus, inhibiting te activity of PLA2 by secondary metabolites of X. hominickii will lead to immunosuppression, which is favorable for these bacteria to express potent pathogenicity.

Dorsal switch protein 1 (DSP1) plays a crucial role in activating PLA2 in <span class="Species">Spodoptera exigua, a lepidopteran insect, in response to immune challenge [11]. DSP1 is a homolog of vertebrate high mobility group protein 1 (HMGB1) that is ubiquitously expressed and localized in the nucleus to bind to DNA for transcriptional regulation and chromatin organization [12]. It is released passively from dead cells or actively from activated immune cells, enterocytes, hepatocytes, and other cells [13]. Released HMGB1 can act as a damage-associated molecular pattern (DAMP) molecule and activate innate immune responses [14]. In insects, DSP1 in the nucleus can act as a corepressor of Dorsal protein in Drosophila melanogaster [15]. In Aedes aegypti (a mosquito), DSP1 can facilitate chromatin remodelling for Toll-associated transcriptional factor to bind to promoter in response to immune challenge [16]. In S. exigua, DSP1 is released to plasma upon bacterial challenge. It can activate PLA2 to mediate various immune responses [11]. Toll immune signalling is likely to serve as a functional link between DSP1 and PLA2 activation because Pelle kinase, an adaptor molecule of Toll receptor, is required for PLA2 activation in S. exigua upon bacterial challenge [17]. Furthermore, HMGB1 in mammals is known to mediate immune signals via Toll-like receptors [18].

In insects, Toll receptor is activated by binding to its ligand, Spätzle, which is activated by sequential activation of <span class="Chemical">serine proteases after pathogen recognition [19]. This led us to pose a hypothesis that DSP1 acts as an initial immune inducer by activating the Toll-Spätzle signal pathway to activate PLA2. This hypothesis suggested that X. hominickii could suppress DSP1 to inhibit PLA2 activation using its secondary metabolites. For screening secondary metabolites, we used a purified recombinant DSP1 protein to test its binding to a specific metabolite. Chemical identification of DSP1-binding compound supported the hypothesis of DSP1 mediation of PLA2 activity via the Toll-Spätzle signal pathway.

Results

Se-DSP1 is released to plasma in exosome cargo

An immunofluorescence assay showed that Se-DSP1 was localized in the nuclei of fat bodies collected from naïve larvae (Fig 1A). When larvae were challenged with a Gram-postive bacterium, <span class="Species">Enterococcus mundtii, an entomopathogen, Se-DSP1 was detected in the nuclei at less intensity (t = 5.67; df = 4; P = 0.0048) than in those of naïve larvae, in which relative fluorescence intensity per cell was 92.8 ± 6.94 in naïve larvae and 56.7 ± 8.58 in immune-challenged larvae. An immunoblotting analysis showed that some Se-DSP1 were detected in the plasma of immune-challenged larvae, but not in the plasma of naïve larvae (Fig 1B). When exosomes were extracted from the plasma of immune-challenged larvae, Se-DSP1 was detected in these exosomes which reacted with CD9, an exosome-specific protein (Fig 1C). Plasma after removing exosomes still reacted with an antibody specific to Se-DSP1, indicating the presence of unbound free form of Se-DSP1 in the plasma.

Fig 1

Secretion of Se-DSP1 from nuclei to plasma upon infection to Gram-positive bacterium, E. mundtii (‘Em’, 4 × 105 cells/larva), in S. exigua.

(A) An immunofluorescence assay of Se-DSP1 in fat body at 6 h after bacterial injection. F-actin and nucleus were stained with phalloidin and DAPI, respectively. Se-DSP1 was detected with its polyclonal antibody. (B) Western blotting analysis of Se-DSP1 in the plasma of naïve or Em-challenged larvae. Plasma samples were collected at 6 h after bacterial challenge. Each lane was loaded with 5 μL of plasma. (C) Exosome analysis for secreted Se-DSP1 in the plasma using western blotting against CD9 (an exosome-specific protein) and Se-DSP1. (Left panel) Exosomes isolated from plasma of naïve or Em-challenged larvae. (Right panel) Plasma samples of Em-challenged larvae before and after exosome extraction. Each lane was loaded with 20 μg proteins. Coomassie-staining bands against a larval storage protein (‘LSP’) indicated the same amount of protein loading for plasma samples. A cytoskeletal protein, α-tubulin, was detected by western blotting to indicate the same amount of protein loading in exosome analysis.

(A) An immunofluorescence assay of Se-DSP1 in fat body at 6 h after bacterial injection. F-actin and nucleus were stained with <span class="Chemical">phalloidin and DAPI, respectively. Se-DSP1 was detected with its polyclonal antibody. (B) Western blotting analysis of Se-DSP1 in the plasma of naïve or Em-challenged larvae. Plasma samples were collected at 6 h after bacterial challenge. Each lane was loaded with 5 μL of plasma. (C) Exosome analysis for secreted Se-DSP1 in the plasma using western blotting against CD9 (an exosome-specific protein) and Se-DSP1. (Left panel) Exosomes isolated from plasma of naïve or Em-challenged larvae. (Right panel) Plasma samples of Em-challenged larvae before and after exosome extraction. Each lane was loaded with 20 μg proteins. Coomassie-staining bands against a larval storage protein (‘LSP’) indicated the same amount of protein loading for plasma samples. A cytoskeletal protein, α-tubulin, was detected by western blotting to indicate the same amount of protein loading in exosome analysis.

Se-DSP1 alone mediates immune responses induced by a bacterial challenge

The release of Se-DSP1 to plasma in reemical">sponse to challenge by Gram-positive bacteria suggested that Se-<span class="Gene">DSP1 alone without bacterial infection might mediate immune responses usually induced by such bacterial challenge. Before testing this hypothesis, we showed that the bacterial pathogen induced immune responses of S. exigua in the present study (Fig 2). Phenoloxidase (PO) activity was significantly (p < 0.05) induced by bacterial challenge (Fig 2A). PO activity was also significantly induced after injecting a recombinant Se-DSP1 (rSe-DSP1), but not by injecting a heat-inactivated rSe-DSP1. However, RNAi against Se-DSP1 expression suppressed the induction of PO activity after bacterial challenge, suggesting a role of Se-DSP1 in mediating PO activation. Eicosanoids are known to mediate immune responses [4]. To monitor eicosanoid biosynthesis, activity of PLA2, the enzyme for the synthesis of eicosanoid, was measured after bacterial or rSe-DSP1 injection (Fig 2B). Activities of both sPLA2 and cPLA2 were significantly (p < 0.05) induced by either bacterial or rSe-DSP1 injection. However, heat-inactivated rSe-DSP1 did not induce PLA2 activities. Expression levels of AMP genes were also significantly upregulated after bacterial or rSe-DSP1 injection (Fig 2C). When 11 AMP genes were assessed, bacterial challenge significantly (p < 0.05) induced 10 AMP genes except attacin-2. Especially, expression levels of apolipophorin-III, cecropin, defensin, gallerimycin, gloverin, and lysozyme were increased more than five-fold increases after bacterial challenge. rSe-DSP1 injection induced all AMP genes assessed, with expression levels of gallerimycin, gloverin, and lysozyme being up-regulated by more than five-fold. Some of these AMP genes were induced even by heat-inactivated rSe-DSP1. However, their up-regulated levels were much less than five-fold and regarded as a stress response to a physical damage by injection. There was a high correlation (r = 0.659; p < 0.027) between the up-regulation of AMP genes between after bacterial challenge and that after rSe-DSP1 injection. Expression levels of three AMP genes (Gal, Glv, and Lyz) were highly induced by either bacterial challenge or rSe-DSP1 injection.

Fig 2

Immune mediation of Se-DSP1 in S. exigua.

rSe-DSP1 (‘DSP1’) was injected into L5 larvae at a dose of 0.8 μg/larva. Inactivation of DSP1 used heat treatment at 95°C for 10 min. Immune challenge used an injection of E. mundtii (‘Em’) to L5 larvae at a dose of 4 × 105 cells/larva. At 8 h PI, hemolymph and fat body were collected. Hemolymph was used for analyzing activities of phenoloxidase (‘PO’) and sPLA2. Fat body was used for analyzing cPLA2 activity. For antimicrobial peptide (‘AMP’) analysis, fat body from treated larvae was collected at 12 h PI. (A) Up-regulation of PO activity by Se-DSP1. RNAi used injection of gene-specific dsRNA against Se-DSP1 (dsDSP1) at a dose of 1 μg/larva. At 24 h PI, Em was used for treatment. Control dsRNA (dsCON) used dsRNA specific to a viral gene, CpBV302. (B) Up-regulation of PLA2 activity by Se-DSP1. (C) Up-regulation of AMP gene expression. Expression levels of AMP were presented as fold changes in comparison with those in naïve larvae. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

rSe-<span class="Gene">DSP1 (‘DSP1’) was injected into L5 larvae at a dose of 0.8 μg/larva. Inactivation of DSP1 used heat treatment at 95°C for 10 min. Immune challenge used an injection of E. mundtii (‘Em’) to L5 larvae at a dose of 4 × 105 cells/larva. At 8 h PI, hemolymph and fat body were collected. Hemolymph was used for analyzing activities of phenoloxidase (‘PO’) and sPLA2. Fat body was used for analyzing cPLA2 activity. For antimicrobial peptide (‘AMP’) analysis, fat body from treated larvae was collected at 12 h PI. (A) Up-regulation of PO activity by Se-DSP1. RNAi used injection of gene-specific dsRNA against Se-DSP1 (dsDSP1) at a dose of 1 μg/larva. At 24 h PI, Em was used for treatment. Control dsRNA (dsCON) used dsRNA specific to a viral gene, CpBV302. (B) Up-regulation of PLA2 activity by Se-DSP1. (C) Up-regulation of AMP gene expression. Expression levels of AMP were presented as fold changes in comparison with those in naïve larvae. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

Toll-Spz signalling pathway is functionally linked with DSP1

Toll immune signalling pathway is mainly triggered by Gram-positive bacteria [20]. <span class="Gene">Toll immune signalling is operating in S. exigua [21]. Thus, we analyzed Toll signalling in response to Se-DSP1. Ten Toll genes (SeToll1 ~ SeToll10) were obtained from S. exigua genome (GenBank accession number: WNNL 01000015.1). Of them, at least eight SeToll genes were localized on the same chromosome (Fig 3A). All SeTolls were predicted to be transmembrane proteins. Most of them possessed Toll/interleukin-1 receptor homology domain except for SeToll7 (Fig 3B). Phylogeny tree suggested that SeToll9 and SeToll7 were closely related to each other, different from other SeToll genes. All predicted SeToll genes were expressed in S. exigua (S1 Fig). Among these ten Toll genes, SeToll3 was predominantly expressed in the fat body (Fig 3C).

Fig 3

Toll receptors (Se-Tolls) of S. exigua.

(A) Gene map of 10 Se-Tolls predicted in this study on chromosome(s), showing three linkage groups (LGs). (B) Their domain and phylogeny analyses. Predicted domains included SP (signal peptide), LRR (leucine-rich-repeat), LRR-CT (leucine-rich-repeat C terminal), LRR-NT (leucine-rich-repeat N terminal), Q-rich region (glutamine rich region), transmembrane region, and TIR (Toll-interleukin receptor). Neighbor-joining method was applied for constructing a phylogenetic tree. Bootstrap values on nodes are obtained from 1,000 repetitions. (C) Expression profile of 10 Toll genes (‘T1-T10’) in naive fat body tissues of S. exigua. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

(A) Gene map of 10 Se-Tolls predicted in this study on chromosome(s), showing three linkage groups (LGs). (B) Their domain and phylogeny analyses. Predicted domains included SP (signal peptide), LRR (<span class="Chemical">leucine-rich-repeat), LRR-CT (leucine-rich-repeat C terminal), LRR-NT (leucine-rich-repeat N terminal), Q-rich region (glutamine rich region), transmembrane region, and TIR (Toll-interleukin receptor). Neighbor-joining method was applied for constructing a phylogenetic tree. Bootstrap values on nodes are obtained from 1,000 repetitions. (C) Expression profile of 10 Toll genes (‘T1-T10’) in naive fat body tissues of S. exigua. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

Injection of individual dsRNA specific to each of 10 SeToll genes suppressed its target gene at 24 or 48 h after treatment compared to control dsRNA injection (Figs 4A and S2). However, some Se<span class="Gene">Toll genes recovered their expression levels at 72 h after injection. Under these RNAi conditions, induction of PO activity in response to rSe-DSP1 injection was significantly suppressed compared to that after control RNAi treatment for three dsRNA treatments (against SeToll5, SeToll6, or SeToll9 expression) (Fig 4B). PLA2 activities in response to rSe-DSP1 injection were not induced by RNAi treatment against SeToll1, SeToll2, SeToll5, SeToll6, SeToll7, or SeToll9 for sPLA2, or against SeToll2, SeToll3, SeToll4, SeToll5, SeToll6, SeToll7, or SeToll9 for cPLA2 (Fig 4C). Induction of AMP gene expressions in response to rSe-DSP1 was also inhibited by RNAi specific to SeToll genes (Table 1). Especially, RNAi treatments against SeToll2 and SeToll9 expression significantly (p < 0.05) suppressed the induction of three AMPs (Gal, Glv, and Lyz), with RNAi against SeToll9 being the most significant (P < 0.05) in suppressing the induction of these three AMP genes (Fig 4D). From these analyses, only RNAi treatment against SeToll9 expression impaired all the immune responses mediated by Se-DSP1. These results suggest that SeToll9 plays a crucial role in mediating Se-DSP1 to induce immune responses in S. exigua.

Fig 4

Functional assay of 10 Se-Tolls (‘T1-T10’) for immune mediation of Se-DSP1 in S. exigua by individual knocking-down of gene expression using RNAi.

(A) RNAi efficiencies of 10 Se-Toll genes by injecting gene-specific dsRNAs (‘dsToll1-dsToll10’, 1 μg/larva) to L5 larvae. A viral gene, CpBV302, was used to prepare control dsRNA (‘dsCON’). Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. (B-D) Changes in immune responses after individual RNAi treatments. At 24 h PI dsRNA, rSe-DSP1 (‘DSP1’) was injected to L5 larvae at a dose of 0.8 μg/larva. At 8 h PI, hemolymph and fat body were collected. Hemolymph was used for analyzing activities of phenoloxidase (‘PO’) and sPLA2. Fat body was used for analyzing cPLA2 activity. For analysis of expression levels antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes, fat bodies were collected from treated larvae at 12 h PI. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

Table 1

Effect of individual RNAi treatments against 10 Toll receptors of S. exigua on expression of antimicrobial peptide (AMP) genes.

Gene-specific dsRNA (‘dsToll’, 1 μg per larva) was injected into L5 larvae. After 24 h PI of dsRNA, rSe-DSP1 (0.8 μg per larva) was injected. After 12 h post injection of Se-DSP1, tissue samples (hemocyte (‘HC’), fat body (‘FB’), and midgut (‘GT’)) were collected and used for RT-qPCR.

dsToll	Tissue	AMP genes1
		Apol	Att1	Att2	Cec	Def	Gal	Glv	Hem	Lyz	Tra1	Tra2
Toll1	HC	-	-	-	-	-	-	-	-	-	-	-
	FB	-	-	-	-	-	-	+	-	-	-	-
	GT	-	-	+	-	-	-	-	-	-	-	-
Toll2	HC	+	-	+	-	+	+	+	+	-	+	+
	FB	-	+	+	-	-	+	+	-	+	-	-
	GT	+	-	+	+	+	-	-	+	-	+	+
Toll3	HC	-	+	+	-	+	-	-	-	-	-	+
	FB	-	-	-	-	-	-	-	+	-	-	+
	GT	+	-	-	+	-	-	-	+	-	+	+
Toll4	HC	-	-	+	-	-	-	+	-	+	-	-
	FB	-	-	+	+	+	+	+	-	-	-	+
	GT	+	+	+	+	+	+	+	+	+	+	+
Toll5	HC	+	+	+	+	+	+	+	-	+	+	+
	FB	-	+	+	+	-	-	-	-	-	-	-
	GT	+	+	+	+	+	+	-	+	+	+	+
Toll6	HC	+	-	+	+	+	+	+	-	+	+	+
	FB	-	+	+	+	-	-	+	-	+	-	+
	GT	+	+	+	+	+	+	+	+	+	+	+
Toll7	HC	-	+	+	-	-	+	+	+	+	-	-
	FB	+	-	+	+	-	+	-	+	-	+	-
	GT	+	+	+	+	-	+	-	+	+	+	+
Toll8	HC	-	-	-	-	+	-	-	-	-	-	-
	FB	+	-	-	+	-	+	-	-	-	+	-
	GT	-	-	-	-	+	-	-	+	-	-	-
Toll9	HC	+	+	+	+	+	-	+	-	+	-	+
	FB	+	+	+	+	+	+	+	+	+	+	+
	GT	+	+	+	+	+	+	+	+	+	+	+
Toll10	HC	+	-	-	-	-	+	+	+	-	+	-
	FB	-	-	+	+	-	+	+	-	-	-	+
	GT	+	+	+	-	+	+	-	+	-	+	+

1 ‘+’ represents significant (P < 0.05) decrease of AMP expression after Toll RNAi compared to control while ‘-’ represents no change from control.

Gene-specific dsRNA (‘dsToll’, 1 μg per larva) was injected into L5 larvae. After 24 h PI of dsRNA, <span class="Gene">rSe-DSP1 (0.8 μg per larva) was injected. After 12 h post injection of Se-DSP1, tissue samples (hemocyte (‘HC’), fat body (‘FB’), and midgut (‘GT’)) were collected and used for RT-qPCR.

1 ‘+’ represents significant (P < 0.05) decrease of AMP expression after <span class="Gene">Toll RNAi compared to control while ‘-’ represents no change from control.

Spz is a ligand for <span class="Gene">Toll receptor [22]. Two Spz genes were predicted from the genome of S. exigua. They shared a Toll-binding domain (Fig 5). Activation of proSpz to Spz requires proteolytic cleavage which releases the C-terminal clip domain (Fig 5A). These two Spz appeared to be distinct (Fig 5B). In response to bacterial challenge, Se-Spz1 was not induced in hemocytes, although its expression level was significantly increased in fat bodies (Fig 5C). In addition, rSe-DSP1 did not induce Se-Spz1 in fat bodies while it significantly induced the two Spz genes in hemocytes. These suggest that both Se-Spz expressions might be not directly induced by the bacterial challenge or Se-DSP1 injection.

Fig 5

Two Spätzles (Se-Spz) of S. exigua.

(A) Functional domains of two proSpätzles (‘Se-ProSpz1’ and ‘Se-Spz2’), including a SP (signal peptide), a D/ERR (aspartic/glutamic acid rich region), a TRR (threonine rich region), and a Toll binding domain. Scissors indicate cleavage sites during post-translational modification from Se-ProSpz1/2 to active Se-Spz1/2. Disulfide bonds are indicated by linking cysteine residues. (B) Phylogeny analysis of Se-ProSpz1/2 and those of other insects. The analysis was performed using MEGA6 program with a Neighbor-joining method. Bootstrapping values were obtained with 1,000 repetitions to support branching and clustering. Amino acid sequences were retrieved from GenBank with accession numbers shown in S2 Table. (C) Expression profile of Se-Spz1/2 in hemocyte (‘HC’) and fat body (‘FB’) of L5 larvae of S. exigua. rSe-DSP1 (‘DSP1’) was injected into L5 larvae at a dose of 0.8 μg/larva. Immune challenge was performed by injecting E. mundtii (‘Em’) to L5 larvae at a dose of 4 × 105 cells/larva. At 8 h PI, hemolymph and fat body were collected for RT-qPCR analysis. Control insects were injected with PBS. Expression levels of Se-Spz1/2 were presented as fold changes in comparison with those in control larvae. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

(A) Functional domains of two proSpätzles (‘Se-ProSpz1’ and ‘<span class="Chemical">Se-Spz2’), including a SP (signal peptide), a D/ERR (aspartic/glutamic acid rich region), a TRR (threonine rich region), and a Toll binding domain. Scissors indicate cleavage sites during post-translational modification from Se-ProSpz1/2 to active Se-Spz1/2. Disulfide bonds are indicated by linking cysteine residues. (B) Phylogeny analysis of Se-ProSpz1/2 and those of other insects. The analysis was performed using MEGA6 program with a Neighbor-joining method. Bootstrapping values were obtained with 1,000 repetitions to support branching and clustering. Amino acid sequences were retrieved from GenBank with accession numbers shown in S2 Table. (C) Expression profile of Se-Spz1/2 in hemocyte (‘HC’) and fat body (‘FB’) of L5 larvae of S. exigua. rSe-DSP1 (‘DSP1’) was injected into L5 larvae at a dose of 0.8 μg/larva. Immune challenge was performed by injecting E. mundtii (‘Em’) to L5 larvae at a dose of 4 × 105 cells/larva. At 8 h PI, hemolymph and fat body were collected for RT-qPCR analysis. Control insects were injected with PBS. Expression levels of Se-Spz1/2 were presented as fold changes in comparison with those in control larvae. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

RNAi treatment against Se-Spz1 or Se-Spz2 suppressed their expression levels for at least 72 h PI (Figs 6A and S2). Under RNAi conditions, induction of PO activity in response to bacterial or rSe-DSP1 injection was not suppressed (Fig 6B). However, the induction of PLA2 activity was significantly (p < 0.05) suppressed by RNAi treatment against either Se-Spz1 or Se-Spz2 (Fig 6C). Regarding AMP gene induction after injection of Se-DSP1, RNAi against Se-Spz1 suppressed the induction of expression of three AMP genes (Gal, Glv, and Lyz) while RNAi against Se-Spz2 suppressed the expression of Glv and Lyz, but not Gal (Fig 6D). These results suggest that Toll-Spz immune signalling pathway is mediated by Se-DSP1 in response to challenge by Gram-positive bacteria.

Fig 6

Functional assay of two Spätzles (‘Se-Spz1 and Se-Spz2’) for immune mediation of Se-DSP1 in S. exigua by individual knocking-down of gene expression using RNAi.

(A) RNAi efficiencies of two Se-Spz genes by injecting gene-specific dsRNAs (‘dsSpz1 and dsSpz2’, 1 μg/larva) to L5 larvae. A viral gene, CpBV302, was used to prepare control dsRNA (‘dsCON’). Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. (B-D) Changes in immune responses after individual RNAi treatment. At 24 h PI dsRNA, rSe-DSP1 (‘DSP1’) was injected to L5 larvae at a dose of 0.8 μg/larva. For bacterial immune challenge, Enterococcus mundtii (Em, 4 × 105 cells/larva) was injected into larvae. Naïve larvae were injected with PBS. At 8 h PI, hemolymph and fat body were collected. Hemolymph was used for analyzing activities of phenoloxidase (‘PO’) and sPLA2. Fat body was used for analyzing cPLA2 activity. For analyzing expression levels of antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes, fat bodies were collected from treated larvae at 12 h PI. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

(A) RNAi efficiencies of two Se-Spz genes by injecting gene-specific dsRNAs (‘ds<span class="Gene">Spz1 and dsSpz2’, 1 μg/larva) to L5 larvae. A viral gene, CpBV302, was used to prepare control dsRNA (‘dsCON’). Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. (B-D) Changes in immune responses after individual RNAi treatment. At 24 h PI dsRNA, rSe-DSP1 (‘DSP1’) was injected to L5 larvae at a dose of 0.8 μg/larva. For bacterial immune challenge, Enterococcus mundtii (Em, 4 × 105 cells/larva) was injected into larvae. Naïve larvae were injected with PBS. At 8 h PI, hemolymph and fat body were collected. Hemolymph was used for analyzing activities of phenoloxidase (‘PO’) and sPLA2. Fat body was used for analyzing cPLA2 activity. For analyzing expression levels of antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes, fat bodies were collected from treated larvae at 12 h PI. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

SeToll9 deletion mutants of S. exigua lose responsiveness to Se-DSP1 injection

We performed CRISPR/Cas9-mediated mutagenesis of Se-<span class="Gene">DSP1 (Fig 7). Se-DSP1 has an exon without intron. Based on this, we designed two sgRNA sites (covering 362 bp fragment, Fig 7A). A mixture of sgRNAs and Cas9 was injected into newly-deposited eggs. After eggs were injected with normal food dye, the hatching rate decreased from normally > 90% to 54.9%. Injecting the CRISPR/Cas9 construct reduced hatching rate to 11.3%. We then generated 12 mutants and confirmed by sequencing gDNA. Deletion sizes of these mutants ranged from 15 to 182 bp. Insertions between two sgRNA sites were also observed. Knocking out Se-DSP1 impaired the responsiveness of S. exigua to DSP1 injection. Injection of DSP1 to these mutants did not induce PLA2 activities in these mutants, although such injection induced PLA2 activities in the wild type (Fig 7B). Injection of DSP1 did not up-regulate the expression of AMP genes in these mutants either compared to the wild type larvae (Fig 7C).

Fig 7

Deletion mutants of SeToll9 using CRISPR/Cas9 and their insensitivity to Se-DSP1 to express immune responses in S. exigua.

(A) Construction of SeToll9-deletion mutants (ΔToll9) with two single-stranded guide RNAs (sgRNAs), with protospacer adjacent motif (PAM) denoted in red color. Twelve different types of ΔToll9 (‘M1-M12’) are confirmed by sequence analysis of 808 bp around both sgRNA-specific deletion sites by comparing with the corresponding sequence of wild type (‘Wild’). Deletion or insertion sizes by CRISPR/Cas9 are denoted by ‘+’ or ‘-’ in parentheses. (B) Insensitivity of mutants to Se-DSP1 in activation of PLA2. rSe-DSP1 (‘DSP1’) was injected into L5 larvae at a dose of 0.8 μg/larva. Control (‘CON’) larvae were injected with PBS. At 8 h PI, hemolymph and fat bodies were collected. Hemolymph was used for sPLA2 activity analysis. Fat body was used for cPLA2 activity analysis. (C) Insensitivity of mutants to Se-DSP1 in activating expression of antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes. Fat bodies were collected from treated larvae at 12 h PI of Se-DSP1. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

Inhibition of Se-DSP1 immune signalling enhances Bt pathogenicity

Toll-<span class="Chemical">Spz immune signalling pathway mediated by Se-DSP1 may play a crucial role in defending Gram-positive bacteria. To test this hypothesis, B. thuringiensis (Bt), a well-known Gram-positive insect pathogen, was applied to S. exigua larvae after individual RNAi treatment against each signaling component (Fig 8). At 24 h after indicidual dsRNA injections against ten Se-Toll genes, 500 ppm of Bt was applied to larvae by feeding with diet (Fig 8A). Compared to control RNAi treatment, four RNAi treatments against SeToll5, SeToll6, SeToll7, and SeToll9 significantly (p < 0.05) enhanced Bt pathogenicity. Treatment with RNAi specific to SeToll9 expression was the most effective one in enhancing the insecticidal activity of Bt. Similarly, two Se-Spz genes were tested. Results showed that both Se-Spz genes should be expressed to defend against Bt pathogenicity (Fig 8B).

Fig 8

Comparative analysis of susceptibility of S. exigua larvae to an entomopathogenic bacterium, Bacillus thuringiensis (‘Bt’), after individual RNAi treatment targeting each of 10 SeTolls (‘T1-T10’) (A) and two Spätzle (‘SeSpz1 and SeSpz2’) (B) genes.

dsRNA-specific to individual genes were injected to L5 larvae (1 μg/larva). Control larvae were injected with dsRNA (‘CON’) specific to a viral gene CpBV302. At 24 h PI, 500 ppm of Bt was orally fed to larvae with a leaf-dipping method. Mortality was recorded at 3 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).

X. hominickii secretes secondary metabolites that inhibit DSP1 translocation

To look for effective inhibitor(s) of Se-DSP1, bacterial culture broth of <span class="Species">X. hominickii, an insect pathogen known to induce immunosuppression [7], was fractionated (S4 Fig) and the resulting fractions were assessed for their binding affinities for rSe-DSP1 (Fig 9A). Four organic extracts were assessed. Of them, the butanol extract (‘BX’) was the most effective one in binding to Se-DSP1 with the lowest Kd value (Fig 9B). BX was further fractionated into 15 subfractions (Fig 9C). Three subfractions (F2, F4, and F6) showed the lowest Kd values. Thus, they were subjected to further fractionation. From F2 subfraction, nine subfractions were obtained and three subfractions (F2-2, F2-5, and F2-9) were selected after performing rSe-DSP1 binding assays. From F4 fractions, 11 subfractions were obtained and two subfractions (F4-3 and F4-8) were selected after rSe-DSP1 binding assays. From F6 fractions, nine subfractions were obtained and three subfractions (F6-3, F6-4, and F6-8) were selected after rSe-DSP1 binding assays. These eight subfractions selected were then subjected to GC-MS (S5 Fig).

Fig 9

Screening bacterial metabolites of X. hominickii (Xh) for their binding affinities for Se-DSP1.

(A) Thermal shift assay for screening binding affinities using protein denaturation curve occurring with increasing ambient temperature. Shifting of the maximal value at higher temperature presumes that Se-DSP1 is completely denatured and maximally bound to a fluorescence dye. Dissociation constant (Kd) is estimated based on the relation between the maximal dissociation temperature and test compound concentration. (B) Binding assays for four organic extracts of Xh culture broth after 48 h of grown in TSB (S2 Fig). Extracts used included hexane (HEX), ethyl acetate (EAX), chloroform (CX), and butanol (BX) extracts. (C) Binding assay of 15 fractions (‘F1-F15’) of BX. (D) Binding assays for nine subfractions isolated from F2 fraction of BX (‘BX-F2’). (E) Binding assays for 11 subfractions isolated from F4 fraction of BX (‘BX-F4’) (F) Binding assays for nine subfractions isolated from F6 of BX (‘BX-F6’). Each measurement was replicated three times (three independent samples for each replicate). Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).

Seven metabolites were predicted from active subfractions using GC-MS (Fig 10A). When these compounds were tested to bind to Se-DSP1, they showed binding affinities at a low micromolar range (Fig 10B). Especially, <span class="Chemical">3-ethoxy-4-methoxyphenol (EMP) exhibited the highest binding affinity. Although its chemical structure was similar to salicylic acid (SA), EMP had higher binding affinity to rSe-DSP1 than SA known to bind to rSe-DSP1 and inhibit immune responses of S. exigua [11].

Fig 10

Identification of compounds binding to Se-DSP1 from bacterial culture broth of X. hominickii.

(A) Prediction of Se-DSP1-binding compounds from purified butanol extract (‘Xh-BX’) of X. hominickii culture broth using GC-MS analysis. For example, ‘F2-2’ stands for subfraction #2 from ‘BX-F2’ fraction in Fig 9. See GC-MS chromatograms of compounds in S3 Fig (B) Binding affinity (Kd) estimations of seven bacterial metabolites and salicylic acid to rSe-DSP1 assessed by thermal shift assay. Each treatment was replicated three times with individual samples. Different letters following standard deviation (‘SD’) indicate significant difference among means at Type I error = 0.05 (LSD test).

(A) Prediction of Se-DSP1-binding compounds from purified <span class="Chemical">butanol extract (‘Xh-BX’) of X. hominickii culture broth using GC-MS analysis. For example, ‘F2-2’ stands for subfraction #2 from ‘BX-F2’ fraction in Fig 9. See GC-MS chromatograms of compounds in S3 Fig (B) Binding affinity (Kd) estimations of seven bacterial metabolites and salicylic acid to rSe-DSP1 assessed by thermal shift assay. Each treatment was replicated three times with individual samples. Different letters following standard deviation (‘SD’) indicate significant difference among means at Type I error = 0.05 (LSD test).

EMP induces immunosuppression and enhances Bt pathogenicity

The high binding affinity of EMP to <span class="Gene">rSe-DSP1 was further analyzed for its effect on immune responses mediated by Se-DSP1. EMP treatment appeared to inhibit rSe-DSP1 release from damage fat bodies (Fig 11A) and hemocytes (Fig 11B). Se-DSP1 was localized in nuclei of hemocytes and fat bodies of naïve larvae. Upon bacterial challenge, Se-DSP1 was released into plasma (Fig 11C), However, EMP treatment prevented the release of Se-DSP1 after bacterial challenge.

Fig 11

Inhibitory activity of 3-ethoxy-4-methoxyphenol (‘EMP’) on Se-DSP1 secretion to plasma in response of S. exigua to immune challenge.

L5 larvae were injected with E. mundtii (‘Em’, 4 × 105 cells/larva) and EMP (1 μg/larva). At 6 h PI, hemolymph and fat body were collected. Immunofluorescence assays of Se-DSP1 in hemocytes (A) and fat body (B) are shown. F-actin and nucleus were stained with phalloidin and DAPI, respectively. Se-DSP1 was detected with its polyclonal antibody. (C) Western blotting analysis of Se-DSP1 in the plasma of naïve or treated larvae. Each lane was loaded with 5 μL of plasma. Coomassie-staining bands against a larval storage protein (‘LSP’) indicated that the same amount of proteins in plasma samples was loaded.

L5 larvae were injected with E. mundtii (‘Em’, 4 × 105 cells/larva) and <span class="Chemical">EMP (1 μg/larva). At 6 h PI, hemolymph and fat body were collected. Immunofluorescence assays of Se-DSP1 in hemocytes (A) and fat body (B) are shown. F-actin and nucleus were stained with phalloidin and DAPI, respectively. Se-DSP1 was detected with its polyclonal antibody. (C) Western blotting analysis of Se-DSP1 in the plasma of naïve or treated larvae. Each lane was loaded with 5 μL of plasma. Coomassie-staining bands against a larval storage protein (‘LSP’) indicated that the same amount of proteins in plasma samples was loaded.

The suppression of Se-DSP1 release by treatment with <span class="Chemical">EMP also significantly suppressed the induction of PO activity after Se-DSP1 injection (Fig 12A). This suppression of Se-DSP1 activity was also observed after SA treatment. EMP and SA both suppressed PLA2 activity induction after Se-DSP1 treatment, with EMP being more potent than SA (Fig 12B). The induction of expression of three AMP genes was significantly suppressed by treatment with EMP or SA (Fig 12C). Sch immunosuppressive activity of EMP significantly enhanced Bt insecticidal activity (Fig 12D).

Fig 12

Inhibitory effect of 3-ethoxy-4-methoxyphenol (‘EMP’) or salicylic acid (‘SA’) on immune responses mediated by Se-DSP1 in S. exigua.

L5 larvae were injected with rSe-DSP1 (‘DSP1’, 0.8 μg/larva) and EMP or SA (1 μg/larva). At 8 h PI, hemolymph and fat body were collected. Hemolymph was used for analyzing activities of phenoloxidase (‘PO’) and sPLA2. Fat body was used for analysis of cPLA2 activity. For antimicrobial peptide (‘AMP’) analysis, fat bodies were collected from treated larvae at 12 h PI. (A) Inhibition of up-regulated PO activity by EMP or SA. (B) Inhibition of up-regulated PLA2 activities by EMP or SA. (C) Inhibition of up-regulation of antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes by EMP or SA. Expression levels of AMP were presented as fold changes in comparison with those in naïve larvae. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of targeted genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test). (D) Enhanced susceptibility of S. exigua larvae to Bacillus thuringiensis (‘Bt’), an entomopathogenic bacterium, after treatment with RNAi specific to Se-DSP1 or EMP. RNAi was performed by injecting gene-specific dsRNA against Se-DSP1 (dsDSP1) at a dose of 1 μg/larva. At 24 h PI, Bt was used for treatnet. Control dsRNA (dsCON) used dsRNA specific to a viral gene, CpBV302. EMP treatment was performed by mixing with Bt suspension. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

L5 larvae were injected with rSe-<span class="Gene">DSP1 (‘DSP1’, 0.8 μg/larva) and EMP or SA (1 μg/larva). At 8 h PI, hemolymph and fat body were collected. Hemolymph was used for analyzing activities of phenoloxidase (‘PO’) and sPLA2. Fat body was used for analysis of cPLA2 activity. For antimicrobial peptide (‘AMP’) analysis, fat bodies were collected from treated larvae at 12 h PI. (A) Inhibition of up-regulated PO activity by EMP or SA. (B) Inhibition of up-regulated PLA2 activities by EMP or SA. (C) Inhibition of up-regulation of antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes by EMP or SA. Expression levels of AMP were presented as fold changes in comparison with those in naïve larvae. Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of targeted genes. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test). (D) Enhanced susceptibility of S. exigua larvae to Bacillus thuringiensis (‘Bt’), an entomopathogenic bacterium, after treatment with RNAi specific to Se-DSP1 or EMP. RNAi was performed by injecting gene-specific dsRNA against Se-DSP1 (dsDSP1) at a dose of 1 μg/larva. At 24 h PI, Bt was used for treatnet. Control dsRNA (dsCON) used dsRNA specific to a viral gene, CpBV302. EMP treatment was performed by mixing with Bt suspension. Each treatment was replicated three times. Different letters above standard deviation bars denote significant difference among means at Type I error = 0.05 (LSD test).

Discussion

Upon infection or physical damage, tissues and specific cells can release damage signals via D<span class="Chemical">AMP [14]. Se-DSP1 was the first DAMP molecule reported in insects [11]. The present study further investigated the release of Se-DSP1 and its immune signalling via Toll-Spz pathway. X. hominickii, a well-known insect pathogen, can induce insect immunosuppression by inhibiting eicosanoid biosynthesis [23]. This study tested a hypothesis that X. hominickii inhibits the DAMP to induce host immunosuppression.

Se-DSP1 in the nucleus was secreted to the plasma in a form of exosome in <span class="Disease">infected larvae of S. exigua. Se-DSP1 is a homologous protein of a vertebrate HMGB1 [11]. HMGB1 is a ubiquitously expressed and highly conserved nuclear protein that plays important roles in chromatin organization and transcriptional regulation [12]. This nuclear HMGB1 is released to the plasma under stress either passively from dead cells or actively by secretion from activated immune cells, enterocytes, hepatocytes, and possibly several other types of cells [13]. Released HMGB1 can act as a DAMP and activate the innate immune system by interacting with pattern recognition receptors [14]. It has been demonstrated that several receptors are involved in HMGB1-mediated functions, including RAGE (receptor for advanced glycation end products), TLR2 (Toll-like receptor 2), TLR4, and TLR9 [18,24]. Therefore, Se-DSP1 has been regarded as a DAMP molecule and a key player in activating immune responses in insects [11]. However, how Se-DSP1 is released from the nucleus to the extracellular plasma remains unclear. In mammals, HMGB1 is released from the nucleus using a caspase pathway and delivered to the plasma via exosomes. This has been well demonstrated in hepatocytes in response to lipopolysaccharide via Toll-like receptor 4/caspase-11-dependent cleavage [25]. The present study observed that Se-DSP1 was located in the exosome collected from larval plasma of S. exigua. Exosomes are membrane-bound extracellular vesicles produced in the endosomal compartment of most eukaryotic cells. They have been detected in biological fluids including blood, urine, and cerebrospinal fluid, with an diameter of at about 30 ~ 150 nm [26]. Exosomes can transfer molecules from one cell to another via membrane vesicle trafficking, thereby playing a crucial role in communicating immune signalling between cells [27]. In insects, exosome-like vesicles have been found in hypopharyngeal gland secretomal products (honey, royal jelly, and bee pollen) of honeybee, Apis mellifera. They are known to possess antibacterial and pro-regenerative effects [28]. In Drosophila, exosomes play a direct immunological role in defending viral infection by delivering short interfering double stranded RNA derived from infected hemocytes [29]. In our current study, isolated exosomes containing Se-DSP1 reacted with antisera raised against CD9, a tetraspanin protein known to be rich in exosomes [30]. These results suggest that Se-DSP1 is released from the nucleus upon bacterial infection and secreted to the plasma in a form of exosome.

The Se-DSP1 secreted in plasma used the <span class="Gene">Toll/Spz signalling pathway to mediate immune responses. PO activity was significantly increased after bacterial challenge. However, RNAi against Se-DSP1 expression prevented PO activation against bacterial challenge. In contrast, Se-DSP1 alone without bacterial challenge significantly increased the activity of PO. In response to Gram-positive bacterial challenge, S. exigua larvae express Toll immune signalling [17]. In insects, pathogen recognition accompanies the activation of a group of serine proteases with multiple modular regulatory domains [31] and proteases with amino-terminal clip domains [32]. Terminal clip proteases in the PO-activation pathway can activate PO which catalyzes the formation of reactive compounds and melanin to kill and sequester pathogens [33]. This suggests that Se-DSP1 may interact with the serine protease cascade to up-regulate PO activity. The activation of serine protease(s) by Se-DSP1 also cleaves pro-Spz to active Spz, which in turn binds to the Toll receptor to induce synthesis of AMPs [34]. The present study revealed that two Spz genes annotated were expressed in S. exigua. It is known that D. melanogaster genome encodes six Spz homologs [35]. All these six orthologs have been identified in genomes of mosquitoes Anopheles gambiae [36] and Aedes aegypti [37]. However, only two Spz homologs are present in the genome of honeybee Apis mellifera [38] and the genome of red flour beetle Tribolium castaneum [39]. Spz can activate immune responses via Toll signal pathway in insects. RNA interference experiments have demonstrated that A. aegypti Spz1 has a function in antifungal immunity [37]. Injection of the active form of B. mori Spz1 protein can induce antimicrobial peptide expression [40]. RNAi treatments against two Spz genes of S. exigua significantly interfered with immunity modulation of Se-DSP1 in the present study.

A total of 10 Toll receptor homologs in <span class="Species">S. exigua were predicted. Although all Toll genes were expressed and associated with AMP expression, SeToll9 was found to have the strongest association with the Se-DSP1 signalling pathway based on individual RNAi treatment with Se-DSP1 injection. Toll immune signalling pathway is activated when Spz binds to Toll receptor [20]. Binding of Spz to Toll receptor promotes Toll multimerization which stimulates downstream signalling through adaptor protein MyD88 and two kinases, Tube and Pelle [41]. This results in phosphorylation and degradation of IκB, Cactus, which releases NF-κB transcriptional factors Dif and Dorsal to translocate to the nucleus and induce AMP gene expression [42]. In B. mori, 14 Toll genes are identified, of which six genes are predicted to be associated with immunity based on a phylogenetic analysis [43]. So far, only BmToll9-1 has been confirmed to have function in innate immunity [44,45]. Shafeeq et al. [17] have shown that Pelle can activate PLA2 and lead to eicosanoid biosynthesis. Although PLA2 activity is required in various physiological processes, it is essential to mediate various immune responses in insects [4,6]. Eicosanoids can mediate various immune responses, including PO activation by releasing inactive proPO to the plasma [4]. These results indicate that Se-DSP1 can activates immune response via the Toll/Spz immune signalling pathway. This was further supported by findings that RNAi treatment against SeToll or Spz expression made larvae become susceptible to Bt pathogens.

The role of Se-DSP1 in mediating various immune reemical">sponses of <span class="Species">S. exigua suggests that microbial pathogens can inhibit Se-DSP1 to induce host immunosuppression for their survival and growth. To test this hypothesis, this study screened secondary metabolites of X. hominickii after organic extraction. A previous study has shown that organic extract is more potent in inhibiting immune responses of S. exigua than an aqueous extract of secondary metabolities of another pathogen, Xenorhabdus nematophila [46]. Potent metabolites include phthalimide, indole, and salicylic acid (SA) derivatives. Especially, EMP, a SA derivative, was the most potent in binding to Se-DSP1. Treatment with EMP alone induced significant immunosuppression. Interestingly, Se-DSP1 was not secreted to the plasma in EMP-treated larvae. This suggests that EMP can bind to Se-DSP1 and prevent its translocation from the nucleus. Pathogenic bacteria including X. hominickii possess a gene cluster (polyketide ketone synthase: PKS) associated with the biosynthesis of virulent factors [47]. EMP is likely to be a product of PKS [48].

In summary, X. hominickii, an insect pathogen, can synthesize and secrete secondary metabolites including <span class="Chemical">EMP (Fig 13). EMP then enters immune-associated cells to prevent secretion of Se-DSP1 for mediating immune responses via the Toll/Spz signalling pathway.

Fig 13

A working hypothesis of Se-DSP1 for mediating immune responses via Toll-Spätzle (‘Spz’) signalling pathway and its inhibition by a bacterial metabolite, 3-ethoxy-4-methoxyphenol (‘EMP’), in S. exigua.

Upon challenge with bacteria including an entomopathogenic bacterium, X. hominickii, Se-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. Activated Spz can bind to SeToll receptor to activate PLA2 and the expression of antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes. Activated PLA2 can catalyze eicosanoid biosynthesis to mediate cellular immune responses to defend bacterial infection along with AMPs. To overcome tmmune responses mediated by Se-DSP1, X. hominickii produces and secretes secondary metabolites including EMP to inhibit the secretion of Se-DSP1 and prevent its immune-mediating activity.

Upon challenge with bacteria including an entomopathogenic bacterium, X. hominickii, Se-<span class="Gene">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. Activated Spz can bind to SeToll receptor to activate PLA2 and the expression of antimicrobial peptide (gallerimycin (‘Gal’), gloverin (‘Glv’), and lysozyme (‘Lyz’)) genes. Activated PLA2 can catalyze eicosanoid biosynthesis to mediate cellular immune responses to defend bacterial infection along with AMPs. To overcome tmmune responses mediated by Se-DSP1, X. hominickii produces and secretes secondary metabolites including EMP to inhibit the secretion of Se-DSP1 and prevent its immune-mediating activity.

Materials and methods

Insect rearing and bacteria culture

Larvae of S. exigua were collected from <span class="Species">Welsh onion (Allium fistulsum L.) fields in Andong, Korea and reared on an artificial diet [49]. Larvae underwent five larval molts (L1-L5). Adults were fed with 10% sucrose solution. Rearing conditions were 25 ± 2°C with relative humidity at 60 ± 5% and 16 h of day light length. X. hominickii was isolated from S. monticolum and grown in tryptic soy broth (TSB: Difco, Sparks, MD, USA) at 28°C for 48 h [1]. Enterococcus mundtii (Em), a Gram-positive bacterium, was also cultured overnight in TSB medium at 28°C with shaking at 180 rpm. For immune challenge, E. mundtii was centrifuged at 10,000 × g for 5 min. The cell pellet was dissolved in sterile distilled water and heat-killed at 90°C for 10 min. Heat-killed E. mundtii was then injected into L5 larvae (4 x 105 cells/larva) with a microsyringe (Hamilton, Reno, NV, USA) after counting with a hemocytometer (Neubauer improved bright line, Superior Marienfeld, Lauda-Konigshofen, Germany) under a phase contrast microscope (BX41, Olympus, Tokyo, Japan).

Chemicals

Phthalimide (PM), <span class="Chemical">3-Ethoxy-4-methoxyphenol (EMP), o-cyanobenzoic acid (CBA), dibutylamine (DBA), bis-2 (ethylhexyl) phthalate (BEP), indole (IND), hexahydro-3-(2-methylpropyl)-pyrrolopyrazine-1,4-dione (HMPP), and salicylic acid (SA) were purchased from Sigma-Aldrich Korea (Seoul, Korea) and dissolved in dimethylsulfoxide (DMSO). Phosphate-buffered saline (PBS, pH 7.4) was prepared with 100 mM phosphoric acid and 0.7% sodium chloride. Anticoagulant buffer (ACB, pH 4.5) was prepared to contain 186 mM NaCl, 17 mM Na2EDTA, and 41 mM citric acid. A transfection reagent (Metafectene Pro) was purchased from Biontex (Plannegg, Germany).

Bioinformatics and sequence analysis

Toll sequences of <span class="Species">Bombyx mori [50] were used as queries to obtain S. exigua Tolls (Se-Tolls) from the transcriptome of S. exigua with the following GenBank accession numbers: GGRZ01034653.1 for Se-Toll1, GGRZ01152585.1 for Se-Toll2, GGRZ01091454.1 for Se-Toll3, GGRZ01062293.1 for Se-Toll4, GGRZ01239670.1 for Se-Toll5, GGRZ01119041.1 for Se-Toll6, GARL01056056.1 for Se-Toll7, GGRZ01148168.1 for Se-Toll8, GGRZ01158569.1 for Se-Toll9, and GGRZ01242837.1 for Se-Toll10. Using the same method, S. exigua Spätzles (Se-Spz) genes were obtained from GenBank (accession numbers of GGRZ01098176.1 and GGRZ01162530.1 for Se-Spz1 and Se-Spz2, respectively). Phylogenetic relationship and domain prediction analyses were performed using MEGA6 and Clustal W programs from EMBL-EBI (www.ebi.ac.uk). Bootstrapping values were obtained with 1,000 repetitions to support branches. Protein domains were predicted using SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.xfam.org).

RNA extraction and RT-PCR or RT-qPCR

Total RNA extraction, cDNA preparation, and RT-qPCR followed the procedure described by Mollah et al. [48]. Briefly, the synthesized single-stranded cDNA was used as template for RT-PCR with 35 rounds of a temperature cycle of 94°C for 30 sec, different annealing temperatures for 30 sec, and 72°C for 30 sec after an initial heat treatment at 94°C for 2 min with gene-specific primers (S1 Table). RT-qPCR was conducted using a Step One Real-Time PCR System (Applied Biosystem, Marsiling, Singapore) wit the following conditions: 95°C for 10 min for initial heat followed by 40 cycles of 95°C for 15 sec, different annealing temperature for 30 Sec, and 72°C for 30 sec, and 1 cycle of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec for dissociation using gene-specific primers (S1 Table). A ribosomal gene, RL32, was used as reference gene. Quantitative analysis was done with a comparative CT method as reported by Livak and Schmittgen [51] to estimate mRNA expression levels. Each experiment was replicated three times.

RNA interference (RNAi) for Se-Toll and Se-Spz expression

Template cDNA was amplified using Se-Toll or Se-Spz primers (S1 Table) containing T7 RNA polymerase promoter sequence (5´-TAATACGACTCACTATAGGGAGA-3´) at 5´ ends. These amplified PCR products were used for double-stranded RNA (dsRNA) synthesis using a MEGAscript RNAi kit (Ambion, Austin, TX, USA) based on the manufacturer’s instructions. Control dsRNA was synthesized from a viral gene, CpBV302 [52]. Purified dsRNA was injected to the hemocoel of S. exigua larvae (1 μg/larva) after mixing with the same volume of transfection reagent and incubating at 25°C for 30 min for liposome formation. From this mixture, 1 μg of dsRNA in 2 μL volume was micro-injected into S. exigua larval hemocoel. RNAi efficacy for reducing Se-Toll or Se-Spz expression was determined by RT-qPCR at 0, 12, 24, 48, and 72 h post-injection (PI). At 24 h PI, treated larvae were used for immune challenge experiments.

Extraction of bacterial secondary metabolites and thin layer chromatography (TLC)

X. hominickii was cultured in 1 L of <span class="Chemical">TSB at 28°C for 48 h. After centrifuging cultured broth at 10,000 × g for 20 min at 4°C, the resulting supernatant was used for organic extraction as described by Mollah et al. [48]. Briefly, the supernatant was mixed with the same volume of hexane. After 30 min of incubation 4°C, the hexane extract (HEX) was separated from the aqueous fraction. The same procedure was sequentially used to obtain chloroform (CX), ethylacetate (EAX), and butanol (BX) extracts. Resulting organic extracts containing bacterial metabolites were dried with a rotary evaporator (Eyela N-1110, Rikakikai, Tokyo, Japan). After weighing, extracts were resuspended in methanol. TLC was performed for resulting extracts to obtain metabolites on a silica gel plate (20×20 cm; Merck, Darmstadt, Germany). Different compositions of chloroform and methanol (v/v) were used as eluents. The developed silica gel plate was incubated with a mixture (19:1, g/g) of sea sand (Merck) and iodine (Duksan, Ansan, Korea). Spots were visualized and marked in a fluorescence analysis cabinet (Spectroline, CM-10, Westbury, NY, USA).

Fractionation of bacterial secondary metabolites using column chromatography

Potent organic extract in the binding assay (see below) was subjected to column chromatography filled with silica gel 60 (0.063–0.200 mm; Merck) using a <span class="Chemical">chloroform/methanol eluent with increasing amount of methanol from 100:0 to 0:100 (v/v) (S2 Fig). Each resulting fraction was dried and dissolved in methanol for binding assay with a recombinant Se-DSP1. Active fractions were further separated by a preparatory thin layer chromatography (Merck) with chloroform: methanol: acetic acid (7.5:2:0.5, v/v). Resulting potent subfractions were subjected to GC-MS analysis for compound identification.

Chemical determination using gas chromatography-mass spectrometry (GC-MS)

GC-MS analysis of potent bacterial fractions followed the method described by Mollah et al. [7]. Briefly, an MS (5977A Network, Agilent Technologies; Santa Clara, CA, USA) was coupled with GC (7890B, Agilent Technologies) equipped with a non-polar column (HP5 MS column, Agilent Technologies). The carrier gas was helium at a flow rate of 1 mL/min. The injector temperature was set at 200°C. Each potent active fraction from binding assays was dissolved in methanol. The result suspension (1 μL) was injected at a split mode with a split ratio of 10:1. The oven temperature was initiated at 100°C for 3 min and then raised to 300°C at a rate of 5°C/min. The final temperature (300°C) was continued for 10 min. The total run-time was 53 min. Mass spectra were recorded in EI mode at 70 eV with a scanning range of 33–550 m/z. Samples were identified by comparing their mass spectra with those in the database (NIST 11, Version 2.0, NIST, Gaithersburg, MD, USA).

Preparation of recombinant Se-DSP1 (rSe-DSP1) protein

Recombinant Escherichia coli [11] expressing Se-<span class="Gene">DSP1 was grown at 37°C for 4 h with shaking at 200 rpm. Soluble recombinant protein was extracted from E. coli pellet using a sonication after two freezing-thawing cycles with liquid nitrogen. After centrifugation at 14,000 × g for 15 min at 4°C, the resulting supernatant was used for protein purification. A native condition method was used to purify 6xHis-tagged soluble proteins. The supernatant (10 mL) obtained from the previous step was mixed with 1 mL of Ni-NTA agarose resin (Qiagen, Hilden, Germany) in 15 mL Econo-Pac Chromatopgaphy column (1.5 ×12 cm, BioRad, Hercules, CA, USA) and agitated for 60 min at 4°C on a rocker platform. The resin containing rSe-DSP1 was washed four times with 10 mL of native wash buffer (50 mM potassium phosphate, pH 8.0, 500 mM NaCl, and 20 mM imidazole). Finally, proteins remained in the resin of the column were eluted with 10 mL of native elution buffer (50 mM NaH2PO4, 500 mM NaCl, 250 mM imidazole, pH 8.0). Presence or absence of purified protein was detected using 10% SDS-PAGE and confirmed by western blotting. Dialysis was done using a semipermeable tube (3 kDa cut-off size, Sigma-Aldrich Korea) against PBS by changing fresh buffer three times at 6 h intervals. Finally, the protein solution was concentrated using a TFD5503 Bench-Top freeze dryer (Ilshine, Seoul, Korea) overnight and dissolved in 1 mL of PBS. Protein solution was then kept at -20°C.

Binding of X. hominickii metabolites with rSe-DSP1 using thermal shift assay

rSe-<span class="Gene">DSP1 binding to X. hominickii metabolites was evaluated using a thermal shift assay [53] with a Protein Thermal Shift dye kit (Applied Biosystem, Foster City, CA, USA) according to the manufacturer’s instruction. Binding assay was conducted as described by Mollah et al. [11]. Briefly, a reaction mixture consisted of 5 μL of protein thermal shift buffer, 2.5 μL of protein thermal shift dye, 10 μL of rSe-DSP1 (500 ng), and 2.5 μL of test metabolite at different final concentrations (0, 2, 4, 6 and 8 μM). A melting curve experiment type was set up using a Step One real time PCR system (Applied Biosystems, Foster City, CA, USA). Thermal profile was obtained at 25°C for 2 min and 99°C for 2 min. Melting temperatures resulting from the experiment were plotted with SigmaPlot 10.0 (Systat Software, San Jose, CA, USA). Dissociation constant (Kd) was calculated using ligand binding equation category.

Immunofluorescence assay

For immune challenge, 4 × 105 cells of E. mundtii were injected into L5 larva. For inhibitor assay, 1 μg of <span class="Chemical">EMP was injected into L5 larva. At 6 h PI, the hemolymph (∼250 μL) was collected into 750 μL of ACB and incubated on ice for 30 min. After centrifugation at 800 × g for 2 min, 800 μL of supernatant was discarded and 250 μL of TC100 insect tissue culture medium (Welgene, Gyeongsan, Korea) was added. Fat body was collected from the insect hemocoel into ACB after removing whole gut. The same procedure was used to isolate hemocytes. Tissue suspension (10 μL) was loaded onto a glass coverslip and incubated in a wet chamber for 30 min in a dark condition. Cells were then fixed with 4% formaldehyde for 10 min at room temperature (RT). After washing thrice with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 2 min at RT. Cells were washed thrice in PBS and blocked with 3% BSA in PBS for 10 min. After washing once with PBS, cells were incubated with Alexa Fluor 555 phalloidin and a primary antibody (Abclone, Seoul, Korea) raised against Se-DSP1 for 1 h 20 min at RT. After washing thrice, cells were incubated with a secondary antibody conjugated with FITC (Sigma-Aldrich Korea) for 1 h. Following three washings with PBS, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL) (Thermo Scientific, Rockford, IL, USA) in PBS for nucleus staining. Finally, after washing thrice with PBS, cells were adhered to slide glass and observed under a fluorescence microscope (DM2500, Leica, Wetzlar, Germany) at 400 × magnification.

Exosome isolation and western blotting

Exosome was extracted using an ExoQuick (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instruction. Briefly, 500 μL of hemolymph was collected from 20 L5 larvae and centrifuged at 3,000 × g for 15 min. The supernatant (250 μL) was mixed with 63 μL of ExoQuick solution and incubated at 4°C for 30 min. Then the mixture was centrifuged at 1,500 × g for 30 min at 4°C. A white exosome pellet was resuspended in <span class="Chemical">PBS. For western blot analysis, extracted exosome proteins (20 μg per sample) were separated on 10% SDS-PAGE. These separated samples in the gel were transferred onto 0.2 μm pore nitrocellulose membranes (BioRad) for 45 min at 100 V in chilled transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, pH 8.5). Membranes were briefly rinsed with Tris-buffered saline containing Tween-20 (TBST) (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) and then blocked with 3% bovine serum albumin (BSA) in TBST at RT for 1 h. Membranes were then incubated with an anti-CD9 Rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) specific to exosome as a primary antibody diluted 1,000 times with TBST containing 3% BSA at 4°C for 2 h. Membranes were then washed three times with TBST (5 min per washing) and then incubated with an anti-Rrbbit IgG-alkaline phosphatase secondary antibody (Sigma-Aldrich Korea) at a dilution of 1:2,000 in TBST containing 3% BSA for 1 h at RT. Blots were rinsed three times with TBST. To detect alkaline phosphatase activity, nitrocellulose membrane blots were incubated with a substrate (BICP/NBT, Sigma-Aldrich Korea).

PLA2 and PO enzyme activity assay

For measuring PLA2 enzyme activity of the treated larvae sample, PLA2 Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) was used as described by Vatanparast et al. [54]. Activity of phenoloxidase (PO) from plasma was determined using the method described by Sadekuzzaman et al. [23]. Briefly, <span class="Chemical">L-3,4-dihydroxyphenylalanine (DOPA) was used as substrate to check the enzyme activity of PO in the plasma collected from treated larvae. A reaction volume of 200 μL consisted of 180 μL of 10 mM DOPA in PBS and 20 μL of the plasma. Absorbance was taken at 490 nm using a VICTOR multi label Plate reader (PerkinElmer, Waltham, MA, USA). PO activity was expressed as ΔABS/min/μL of plasma. Each treatment was replicated three times (each time with three independent samples).

Construction of a deletion mutant using CRISPR/Cas9

The structure of Se-Toll9 genomic DNA was ana<span class="Chemical">lyzed after sequencing the cognate gDNA. After confirming no intron, the exon sequence was submitted to an online tool (http://www.chopchop.com) to determine the optimal target site. Based on a target containing a protospacer adjacent motif (PAM), two 20 bp (5′-AGATCGGAGTTTCCGTATCGAGG-3′ and 5′-ATAGATTAAGTATAACCATACGG-3′) targeting sites were selected for sgRNA1 and sgRNA2, respectively. sgRNA was generated using Guide-it sgRNA In Vitro Transcription kit (Takara Korea Biomedical, Seoul, Korea) according to the manufacturer’s manual. Briefly, PCR was performed using target oriented forward primer and company-provided reverse primer. PCR conditions included a pretreatment at 98°C for 5 s and subsequent amplification with 33 cycles of 98°C for 10 s and 68°C for 10 s. The resulting PCR product was used for in vitro transcription using T7 RNA polymerase to produce sgRNA, which was purified using a spin column provided by the kit. The final amount was quantified with a spectrophotometer (NanoDrop, Thermo Fisher Scientific Korea, Seoul, Korea). Females were kept in the dark to lay eggs on a kitchen paper towel for 1 h. Eggs were dried in a desiccator for 10 min at RT. Dried eggs were then fixed on a cover slip with a double-sided tape. Before injection, glass capillaries (10 μL quartz, World Precision Instrument, Sarasota, FL, USA) with sharp points (< 20 μm diameter) were prepared with a Narishige magnetic glass microelectrode horizontal puller model PN30 (Tritech Research, Los Angeles, CA, USA). Eggs were injected through a micropyle using a Sutter CO2 Pico pump injector (PV830, World Precision Instrument) under a stereomicroscope (SZX-ILLK200, Olympus). The injection volume per egg was 10 nL of a mixture containing Cas9 (500 ng/μL) and sgRNA (50 ng/μL). All injections were finished within 30 min after egg collection, including drying time. Injection-treated eggs were incubated at RT for 4 h before transferring to a growing chamber (25°C). They were then observed for 4 days until hatching. Genomic DNA extraction, PCR, and sequencing were carried out to detect mutant insects using gene-specific primers (S1 Table) producing a 808 bp product. Genomic DNA was extracted using 10% Chelex (Biorad, Hercules, CA, USA) from hemolymph of L5 larvae (~10 μL/larva). PCR conditions had a pretreatment at 94°C for 2 min, an amplification step with 35 cycles of 94°C for 1 min, 56.8°C for 1 min, and 72°C for 1 min, and a final extension step at 72°C for 10 min. PCR products were then cloned into pCR2.1 vector (Thermo Fisher Scientific Korea) and bidirectionally sequenced.

Bioassay of RNAi-treated S. exigua against Bacillus thuringiensis (Bt)

To assess Bt virulence to RNAi-treated S. exigua, L5 larvae were injected with 1 μg of dsRNA. At 24 h after dsRNA injection, feeding assay was applied with a leaf-dipping method. Briefly, a piece of <span class="Species">cabbage leaf (3 × 3 cm) was soaked in 500 ppm of Bt suspension for 5 min. Treated leaves were then provided to dsRNA treated larvae for 24 h. Treated larvae were then incubated for another 2 days under rearing conditions to observe mortality. Each replication consisted of 10 larvae. Each treatment was replicated three times. A commercial product of B. thuringiensis var. kurstaki (serotype IIIa & IIIb, Hanearl Science Corporation, Taebaek, Korea) was used for mortality test at concentration of 500 ppm.

Statistical analysis

All data for continuous variables were subjected to one-way analysis of variance (ANOVA) using PROC GLM in SAS program [55]. Mortality data were subjected to <span class="Disease">arcsine transformation and used for ANOVA. Means were compared with the least significant difference (LSD) test at Type I error = 0.05. Median lethal dose (LD50) was subjected to Probit analysis using EPA Probit Analysis Program, ver. 1.5 (Environmental Protection Agency, USA).

List of primers used in this study.

(DOCX)

Click here for additional data file.

GenBank accession numbers used for phylogenetic analysis.

(DOCX)

Click here for additional data file.

Expression profile of 10 Toll genes (‘T1-T10’) of S. exigua.

(A) Egg developmental stage. (B) Larval hemocytes. (C) Larval midgut. A ribosomal RNA, RL32, was used as endogenous control. Expression level was calculated as fold change from the lowest expression value. Each treatment was independently replicated three times.

(DOCX)

Click here for additional data file.

RNAi efficiencies of 10 Se-Toll genes by injecting gene-specific dsRNAs (‘dsToll1-dsToll10’, 1 μg/larva) to L5 larvae.

A viral gene, CpBV302, was used to prepare control dsRNA (‘dsCON’). Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was independently replicated three times.

(DOCX)

Click here for additional data file.

RNAi efficiencies of two Se-Spz genes by injecting gene-specific dsRNAs (‘dsSpz1 and dsSpz2’, 1 μg/larva) to L5 larvae.

A viral gene, CpBV302, was used to prepare control dsRNA (‘dsCON’). Expression level of a ribosomal gene, RL32, was used as reference to normalize expression levels of target genes. Each treatment was independently replicated three times.

(DOCX)

Click here for additional data file.

A diagram illustrating fractionation steps of culture broth of X. hominickii.

Organic extracts were obtained using hexane (‘<span class="Chemical">HEX’), ethyl acetate (‘EAX’), chloroform (‘CX’), and butanol (‘BX’). BX was fractionated using a chromatography column filled with silica gel where a gradient chloroform/methanol mixture with increasing amount of methanol from 100:0 to 0:100 (v/v) was used. Active butanol fractions were separated using a preparatory thin layer chromatography (‘TLC’).

(DOCX)

Click here for additional data file.

GC-MS analysis of active fractions and prediction of compounds from X. hominickii culture broth extracted by butanol.

EMP, 3-ethoxy-4-methoxy phenol; HMPP, <span class="Chemical">hexahydro-3-(2-methylpropyl)-pyrrolo[1,2-a]pyrazine-1,4-dione; BEP, bis (2-ethylhexyl) phthalate; IND, indole; DBA, dibutylamine; PM, phthalimide; CBA, o-cyanobenzoic acid.

(DOCX)

Click here for additional data file.

14 Feb 2021

Dear Dr. Kim,

Thank you very much for submitting your manuscript "Immune mediation of HMG-like <span class="Gene">DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Francis Michael Jiggins

Associate Editor

PLOS Pathogens

Karla Satchell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript characterizes the mechanisms by which X. hominikii suppresses the immune reemical">sponse in the insect <span class="Species">S. exigua. It identified the genes involved and uses both ectopic expression and dsRNA knockdown and/or CRISPR mutants to define the roles of DSP1, Toll receptors, and Spatzle (a Toll ligand) in the immune response in S. exigua. It goes on to define the secondary metabolite from X. hominikii that suppresses this immune response. These findings provide significant insight into how x. hominikii causes immunosuppression. The manuscript is well-written, and the data presented are generally clear and convincing. However, there are a few areas that could be modified to improve the clarity.

Reviewer #2: This study investigated the immune signaling pathway of a damage-associated molecular pattern, DSP1. It reports that <span class="Gene">DSP1 activates Toll/Spz pathway for defending Gram-positive bacterial infection. Especially, Toll9 among 10 Toll receptors was a main signal component. This is further supported by a deletion mutant of Toll9, which lost immune activation. Furthermore this study identified an immune blocker, EMP, which exhibited a tight binding to DSP1 not to be released. Thus the entomopathogenic bacterium, X. hominickii induces a fatal pathogenicity by releasing EMP to suppress host immune defense. Overall, this reports a new immune signaling pathway in insects using a DAMP signal, DSP1 and a novel immunosuppressive agent, EMP. Thus, this study fits to the journal scope and standard of PLoS Pathogens. However, following issues need to be addressed.

1. This study showed the release of DSP1 from nucleus to plasma. The released <span class="Gene">DSP1 appears to be transported in plasma using exosome cargo. Here are questions. What does the intracellular signal stimulate DSP1 release from nucleus? Why does the DSP1 take exosome in the plasma?

2. In addition to Toll9, <span class="Gene">DSP1 may use Toll6, Toll7, or Toll8 to induce the AMP or PO activation from Fig. 4. Why did you determine Toll9 as DSP1 signal component?

3. I am not sure that DSP1 directly activated <span class="Chemical">Spz to trigger Toll sigaling pathway?

4. What is the relationship of Bt pathogenicity and immunosuppression? Is it required for the Bt to exhibit its insecticidal activity?

5. How can you explain the inhibitory activity of EMP on SP1 release from the nucleus?

Reviewer #3: Precis

Some insect pathogenic microbes overcome host immune responses to infection by secreting compounds that inhibit insect immune reactions to <span class="Disease">infection. Park and Kim (2000; doi: 10.1016/s0022-1910(00)00071-8) first reported that the bacterial lethality of the insect pathogen Xenorhabdus nematophilus was attenuated after injecting arachidonic acid (AA), a direct precursor to biosynthesis of prostaglandins and other eicosanoids, into the abdomens of infected lepidopterans, Spodoptera exigua. They put forth the idea the bacterium suppresses insect immunity by inhibiting biosynthesis of eicosanoids. Now, after 20 years and many publications on eicosanoid signaling in insect immunity, they turned attention to a related insect pathogen, X. hominickii. This pathogen also secretes metabolites that inhibit the first step in eicosanoid biosynthesis, phospholipase A2 (PLA2), which effectively suppresses eicosanoid signaling to suppress host immunity. They reported that a damage-associated molecular pattern called dorsal switch protein 1 (DSP1), which activates PLA2 early in infection in S. exigua. They used immunofluorescence to record increased hemocyte spreading and Se-DSP1 in fat body. They used western blots to show Se-DSP2 in hemolymph of infected, but not naïve larvae at 6 h post-infection. Their western blot analysis also indicated the Se-DSP1 was transported out the cells as via exosomes. The authors created a recombinant Se-DSP1 (rSe-DSP1) and used it to demonstrate that bacterial infection with another insect pathogen, E. mundtii, leads to increased PO activity, as does rDSP1 treatments, but not after denaturing the protein. Treating larvae with a dsRNA construct, dsDSP1 + E. mundtii blocked infection-triggered PO activity. They used enzyme activity assays to show rDSP1 treatments led to significant increases in secretory PLA2 and cellular PLA2 activities. Translating to the influence of rDSP1 on humoral immunity, the authors show that rDSP1 injections led to significant increases in mRNAs encoding a range of antimicrobial peptides and proteins, including apolipophoren-III, cecropin and lysozyme. They report ten Toll receptors and show accumulations of mRNAs encoding them. Expression of each of the ten genes was substantially reduced at 24 or 48 h after injecting dsRNA constructs specific to each gene. Co-injections with each dsRNA + rDSP1 led to substantial PO activity, sPLA2 activity and cPLA2 activity. Similar co-injections led to substantial expression of genes encoding three anti-microbial proteins. Digging deeper into humoral immunity, the authors report two genes encoding Spätzels, showing gene structures and a phylogenetic tree placing them withing Diptera-Lepidoptera. They show the genes are expressed in hemocytes and fat body. rDSP1 treatments led to increased gene expression in hemocytes, but not in fat body. Bacterial injections led to increased expression of both genes in fat body. They show dsRNA treatments led to decreased expression of both genes encoding Spätzes from 24 – 72 h post-injections. Bacterial injection and DSP1 injections led to increased PO activity, but not to increased PLA2 activity. Co-inections with dsSpätze1 + DSP1 did not lead to increased expression of three anti-microbial peptides, although dsSpätze2 +DSP1 did.

The authors continued drilling into humoral immune signaling by creating CRISPR/Cas9 deletion mutants in Se<span class="Gene">Toll9. Although rDSP1 injections into wild-type larvae led to increased sPLA2 and cPLA2 activities and to increased expression of the three anti-microbial proteins, similar injections into the deletion mutants did not. The changes in immune parameters just mentioned translated into increased mortality following co-injections with most dsRNAs designed to each of the ten toll receptors + the insect pathogen, Bacillus turingiensis Mortality did not increases following dsTOLL1, 8 or 10. Similarly, dsSpätze1 and -2 treatments also led to increased mortality. The authors identified X. hominickii metabolites that bind to rSeDSP1, and thus, block its translocation. They used GC-MS analysis to identify several metabolites with binding affinities in the low �M range, of which 3-eethoxy-4-methoxyphenol (EMP) had the highest binding affinity. In a direct test of DSP1 translocation, the authors presented a western blot showing the presence of Se-DSP1 in plasma from larvae injected with the pathogen E. mundtii, but not in larvae injected with the pathogen + EMP. EMP + rDSP1 treatments led to sharp reductions in PO, sPLA2 and cPLA2 activities and virtually eliminated accumulations of mRNAs encoding gallerimycin, gloverin and lyzozyme. Injections of pathogen B. thuringiensis led to increased mortality in control larvae. The bacterial injections into larvae treated with dsDSP1 led to higher mortality and still higher mortality followed in larvae treated with EMP.

The authors complete their story with a meaningful model of a hypothesized mechanism of DSP1 action. In their view, the bacterial product <span class="Chemical">EMP damages hemocytes and DSP1 is translocated from cells into hemolymph, where it activates one or more serine protease cascades that leads to active phenol oxidase and melanization reactions. It also activates two Spätzels, with interacts with toll receptors that lead to biosynthesis of eicosanoids that mediate cellular immune reactions and lead to expression of genes encoding lysozyme and other anti-microbial protains.

They present three supplementary figures. Fig. S1 reports accumulations a mRNAs encoding the 10 toll receptors. Fig. S2 shows their extraction scheme for analysis of the <span class="Species">X. hominickii metabolites and Fig. S3 shows the chemical structures of selected metabolites.

Critique

The authors present a well-reasoned and thorough series of experiments to test their hypothesis.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: n/a

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. Abstract: The abstract is incredibly long and it seems like much of this information belongs in the Introduction. Further, from the abstract, the reader expected eicosanoid biosynthesis to be studied in the m<span class="Chemical">anuscript and it was not.

2. Line 104 – explain to the reader what Pelle kinase is.

3. Paragraph starting at line 107 – it is not clear what the authors think the order of events is.

4. Figure 1 – The change in Se-DSP1 immunofluorescence isn’t convincing. It seems like it is relocalizes, perhaps to the nucleolus, but the intensity does not appear reduced. Some quantification is needed to clarify this.

5. The terminology in the results and in the figures is not always consistent. For example, Line 134-135, Se-<span class="Gene">DSP1 (rSe-DSP1) is not how it is written in the Figure 2. Another example is in relation to Figure 7

6. The authors need use PLA2 activity to indicate eicosanoid biosynthesis, but PLA2 has many other roles. The authors need to discuss this limitation. Further, in the results it is not discussed what samples are used for the analysis and why they are the correct samples.

7. It seems it is worth discussing why AMP genes are activated by heat inactivated <span class="Gene">rSe-DSP1

8. The description of the data in Figure 4 is difficult to follow. Sometimes the authors say which Toll receptors have no effects and for other data which <span class="Gene">Toll receptors play a role. It would be clearer to the reader to talk about the Toll receptors that are involved in the process. Also a discussion of the difference in the Toll receptor role in PLA activity vs AMP gene expression is warranted. It is unclear what the reader should conclude about who is involved.

9. Line 183 – the data in figure 5c would say that neither Spz gene is induced in the hemocytes in response to EM, and Spz1 may be inhibited. This is not what is stated in the results and it seems worth talking about. Lines 184-185 seems to contradict the prior sentence

10. In the section starting at 209, it would help the reader for the authors to explain their method of assessing pathogenicity.

11. Figure 1 legend talks about Tubulin western blots that are not shown in the figure

12. The RNAi knockdown data in Figure 4a and Figure6a are shown as connected lines. Such a graph implies the expression was assessed repeatedly in the same cells, when in reality it was done on different cells. The data should be presented as individual data points or bar graphs.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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Reviewer #2: No

Reviewer #3: Yes: David Stanley

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26 Feb 2021

Submitted filename: Response to reviewers.docx

Click here for additional data file.

9 Mar 2021

Dear Dr. Kim,

Thank you very much for submitting your manuscript "Immune mediation of HMG-like <span class="Gene">DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs" for consideration at PLOS Pathogens. Thank you for thoroughly revising the manuscript, it is a great paper. I am happy with all the revisions in the main text, but would like you to look again at the first comment about the abstract. At the moment the abstract is hard to read unless you have technical knowledge of this area. This is because it includes a large amount of technical detail and goes through many different experiments. In contrast, it does not make clear the wider significance and importance of the work. This contrasts with the author summary, which did this job very well. I would ask that the abstract is simplified and makes clearer the wider significance of your work. I apologise for returning the manuscript a second time, but I hope this will be a quick change to make that will help your work reach a wider audience.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the m<span class="Chemical">anuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Francis Michael Jiggins

Associate Editor

PLOS Pathogens

Karla Satchell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Thank you for thoroughly revising the manuscript which is a very interesting body of work. I am happy with all the revisions in the main text, but would like you to look again at the first comment about the abstract. At the moment the abstract is not suitable for PlOS Pathogens as it is very hard to read unless you have a technical knowledge of this area. This is because it includes a large amount of technical detail and goes through many different experiments. However, it does not make clear the wider significance and importance of the work. This contrasts with the author summary, which did this job very well. I would ask that the abstract is simplified and makes clearer the wider significance of your work.

Reviewer Comments (if any, and for reference):

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10 Mar 2021

Submitted filename: Response to reviewers-r2.docx

Click here for additional data file.

11 Mar 2021

Dear Dr. Kim,

We are pleased to inform you that your manuscript 'Immune mediation of HMG-like <span class="Gene">DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs' has been provisionally accepted for publication in PLOS Pathogens.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Francis Michael Jiggins

Associate Editor

PLOS Pathogens

Karla Satchell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

18 Mar 2021

Dear Dr. Kim,

We are delighted to inform you that your manuscript, "Immune mediation of HMG-like <span class="Gene">DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064