Characterization of PGRP-LB and immune deficiency in the white-backed planthopper Sogatella furcifera (Hemiptera: Delphacidae)

Peptidoglycan recognition proteins (PGRPs) participate in insect defense against bacterial pathogens by recognizing bacterial cell wall peptidoglycans (PGNs). Here, we identified the PGRP-LB gene in the white-backed planthopper Sogatella furcifera (SfPGRP-LB). SfPGRP-LB is a secreted protein with a typical PGN-binding domain and five conserved amino acid (aa) residues required for amidase activity. Expression analysis showed that the SfPGRP-LB transcript levels were significantly higher in the midgut than in other tissues. Silencing SfPGRP-LB with dsRNA significantly downregulated the expression of Toll pathway genes Toll and Dorsal and Imd pathway genes Imd and Relish after Escherichia coli challenge. However, only Toll and Dorsal expressions were downregulated after Staphylococcus aureus challenge. E. coli and S. aureus challenges rapidly and strongly upregulated SfPGRP-LB expression. Recombinantly expressed SfPGRP-LB (rSfPGRP-LB) had strong affinities for E. coli Dap-type PGN and S. aureus Lys-type PGN and agglutinated the bacteria. However, rSfPGRP-LB inhibited S. aureus but not E. coli growth. Furthermore, rSfPGRP-LB had amidase activity, degraded Lys-type PGN, and destroyed S. aureus cell walls but had no such effects on E. coli Dap-type PGN. Thus, SfPGRP-LB recognizes and binds various bacterial PGNs but only has amidase activity against Lys-type PGN.


Introduction
The immune system enables insects to adapt to their ambient environment, contend with biotic stress, and resist various microbial pathogens. Our current understanding of insect immunity is based mainly on analyses of species with complete metamorphosis such as Drosophila melanogaster (Diptera: Drosophilidae) (Dziarski and Gupta 2018;Gottar et al. 2002;Hillyer 2016), Aedes aegypti (Diptera: Culicidae) (Koh et al. 2018;Ramirez et al. 2019;Wang et al. 2018), and Bombyx mori (Lepidoptera: Bombyxidae) (Chen et al. 2018a; Chen and Lu 2018;Li et al. 2019). However, subsequent genome sequencing has clarified immune system function in other insect species as well (Bao et al. 2013;Chen et al. 2018b;Laughton et al. 2011). Studies on completely metamorphic insects revealed that the first step in their immune response is pathogen detection via different pattern recognition receptors. Peptidoglycan recognition proteins (PGRPs) are the major insect pathogen pattern recognition receptors and confer protection against bacterial challenge.
PGRPs occur in most insect immune recognition systems. They detect and are induced by the peptidoglycans (PGNs) that are abundant in certain bacterial cell walls. The proportion of PGNs in Gram-positive bacterial cell walls is ~ 50-90% of the cell dry weight (DW). In contrast, PGNs comprise only ~ 10% of the cell DW in Gram-negative bacterial cell walls. Most PGRPs have a conserved domain that recognizes and binds bacterial cell wall PGNs. According to the size of the transcript and structure of the domains, insect PGRP can be divided into short extracellular PGRP (shortform PGRPs, PGRP-S) and longform PGRP (longform PGRPs, PGRP L) across or within the membrane (Royet et al. 2011). Insect PGRPs distinguish bacteria by the differences in the third amino acid (aa) of their short-peptide PGNs. PGRP-SA in D. melanogaster can recognize Lys-type PGN, which possesses a lysine residue in the third position of its short peptide. PGRP-SB, PGRP-SC, PGRP-LB, PGRP-LC, and PGRP-LE in D. melanogaster preferentially interact with diaminopimelate residues (Dap type). Most Gram-negative and Gram-positive bacteria have these PGNs (Guan et al. 2004;Leulier et al. 2003;Reiser et al. 2004).
The innate immune response of insects is mainly mediated by two different nuclear factor-κB (NF-κB) signaling pathways, Toll and Imd (Lemaitre et al. 1995). In Drosophila, the main genes involved in the Toll pathway (Tolls, Spatzle, Dorsal and Dif) and Imd pathway (Imd, Relish) can be activated by Gram-positive bacteria, Gram-negative bacteria and fungi through the identification and combination of pathogens and different PGRPs and GNBPs. The expressions of Toll and Imd pathway gene are activated, and the expressions of related antibacterial peptide protein are finally induced to achieve the purpose of antibacterial (Belvin and Anderson 1996;Myllymaki et al. 2014;Ramet 2012;Valanne et al. 2011). For example, PGRP-LE activates the phenoloxidase pro-PPO system and Imd pathway (Takehana et al. 2002). PGRP-SA, PGRP-SC1, and PGRP-SD may induce the Toll pathway (Bischoff et al. 2004;Garver et al. 2006;Michel et al. 2001). PGRP-LA, PGRP-LC, PGRP-LB, and PGRP-LF promote the Imd pathway in response to Gram-negative bacterial challenge (Gendrin et al. 2013;Iatsenko et al. 2016). D. melanogaster harbors one secreted and two cytosolic PGRP-LB isoforms; the former hydrolyzes PGN, whereas the latter regulates regional activation (Charroux et al. 2018). In B. mori, PGRP-LC1, PGRP-S1, and PGRP-S3 expressions are upregulated in response to Gram-positive and Gram-negative bacteria and fungi while PGRP-S2 expression is upregulated by bacteria only (Yang et al. 2015). In B. mori, PGRP-LB and PGRP-L5 expressions are upregulated by fungi; whereas, PGRP-LB and PGRP-L6 participate in the Imd signaling pathway (Tanaka and Sagisaka 2016;Zhan et al. 2018). PGRP-S4 initiates the B. mori immune response by activating the pro-PPO system. PGRP-S5 activates the phenoloxidase pro-PPO system and the Imd signaling pathway (Chen et al. 2016;Yang et al. 2017). In A. aegypti, PGRP-S1, PGRP-SC2, and PGRP-LB expressions are upregulated by both Gram-negative and Gram-positive bacteria. In contrast, PGRP-LA, PGRP-LC, and PGRP-LD are not induced by either bacteria type (Wang and Beerntsen 2015). In Tribolium castaneum (Coleoptera: Paracaridae), PGRP-LA expression is upregulated by Grampositive and Gram-negative bacteria and PGRP-LC and PGRP-LE are induced mainly by Gram-negative bacteria via the Imd signaling pathway. PGRP-S (short type) did not participate in the T. castaneum immune response (Koyama et al. 2015). In the incompletely metamorphic insect N. lugens, PGRP-LB is induced by Gram-positive and Gramnegative bacteria; whereas, PGRP-LC is only activated by Gram-positive bacteria (Bao et al. 2013).
The white-backed planthopper S. furcifera is an important insect pest on rice (Oryza sativa L.) and is incompletely metamorphic. However, little is known about its immune recognition, pattern-recognition receptor (PRR) signaling pathways, or innate immune response. Here, we used PGRP-LB sequencing data obtained by transcriptomics to investigate the structural and immune function characteristics of SfPGRP-LB. The results of this study will help us better understand the innate immune system of S. furcifera.

Sequence cloning, phylogenetic tree construction, and structure prediction
The ORF encoding SfPGRP-LB was amplified by PCR with PGRP-LB-full-F and PGRP-LB-full-R (Table 1). The PCR conditions were 95 °C for 5 min followed by 30 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 10 min. The amplified product was purified using a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA). Purified DNA was ligated into a pGM-T vector (TIANGEN Biotech, Beijing, China) and sequenced completely from both directions (Sangon Biotech, Shanghai, China).
The phylogenetic tree was plotted by the neighborjoining (

RNA isolation and cDNA synthesis
Total RNA was extracted from S. furcifera with an RNA extraction kit (Axygen Scientific Inc., Union City, CA, USA). First-strand cDNA was synthesized with the Pri-meScript™ IV first-strand cDNA synthesis mix (Takara Biomedical Technology Co. Ltd., Dalian, China). One microgram first-strand cDNA served as a template for PCR and qRT-PCR.

Tissue distribution analysis
To investigate the tissue distribution of SfPGRP-LB mRNA, total RNA was extracted from the head, midgut, thorax, epidermis, fat body, spermary, and ovary using fifth instars and adults. The organs were rinsed several times in 1 × phosphate-buffered saline (PBS, 1.47 mM KH 2 PO 4 , 8.1 mM NaH 2 PO 4 , 137 mM NaCl, and 2.68 mM KCl; pH 7.4) and mixed with those from 30 to 80 adults. Organs were used in high-quality RNA extraction and cDNA synthesis. The qRT-PCR-specific primers PGRP-LBRTF/PGRP-LBRTR (Table 1) with 103-bp amplicons were designed according to full-length SfPGRP-LB cDNA.

Bacterial challenge
Gram-negative E. coli strain (K12) and Gram-positive S. aureus strain (ATCC6538) were used to infect S. furcifera. E. coli and S. aureus were cultured in LB medium at 37 °C with shaking at 200 rpm until OD 600 = 0.6. The bacteria were sedimented at 37 °C by centrifugation at 5000×g for 10 min, washed, resuspended in 1 × PBS to a density of 5 × 10 8 CFU mL −1 , and heat-killed by boiling for 30 min. For the immune challenge experiment, fourth-instar S. furcifera larvae (day 1) were randomly assigned to the E. coli, S. aureus, and PBS treatment groups. Each larva was anesthetized by 10 s exposure to 5 mPa CO 2 and positioned supine in the grooves of an agarose gel placement plate. Then, 0.5 μL of devitalized bacterial suspension or PBS solution was injected with a FemtoJet microinjection system (Eppendorf, Hamburg, Germany) into the abdominal segment junction between the second and third appendages (Chen et al. 2018b). The treated larvae were raised in an artificial incubation chamber and fed fresh rice seedlings. Samples were collected at 0, 6, 12, and 24 and 48 h postinjection and there were three replicates per time point. RNA extraction from midgut and fat body, cDNA synthesis, and qRT-PCR were conducted and specific qRT-PCR primers (Table 1) were prepared as previously described.

RNA interference
SfPGRP-LB and GFP (GenBank ID: KU306402.1) cDNA fragments were amplified by PCR using primers containing the T7 promoter (5′-TAA TAC GAC TCA CTA TAG GG-3; Table 1). The products were purified and used as templates to synthesize dsRNA in a T7 high-yield RNA transcription kit (Vazyme Biotech Co. Ltd., Nanjing, China). The reactions were conducted as follows: 95 °C for 5 min followed by 30 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. The dsRNA products were dissolved in ultrapure water and their concentration and purity were determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by 1.5% agarose gel electrophoresis, respectively. Fourth-instar nymphs were either microinjected with dsSfPGRP-LB (treatment) or dsGFP (control). Then, 200 ng of dsRNA from SfPGRP-LB or GFP was injected into the segment between the second and third appendages with a FemtoJet microinjection system (Eppendorf, Hamburg, Germany) (Chen et al. 2018b). After microinjection, each nymph group was fed TN1 rice under the previously described conditions.

Effects of SFPGRP-LB on Toll and Imd pathways in vivo
To determine whether SfPGRP-LB modulates the expression of genes involved in the Toll and Imd pathways after bacterial challenge, the Toll, Imd, Dorsal, and Relish transcription levels were measured and compared among the dsSfPGRP-LB group injected with E. coli (dsSfPGRP-LB + E. coli), dsSfPGRP-LB group injected with S. aureus (dsSfPGRP-LB + S. aureus), dsGFP group injected with E. coli (dsGFP + E. coli), and dsGFP group injected with S. aureus (dsGFP + S. aureus). The specific qRT-PCR primers are shown in Table 1.

Recombinant vector construction
The ORF encoding SfPGRP-LB was amplified by PCR with PGRP-LB-protein-F/PGRP-LB-protein-R primers ( Table 1). The PCR conditions were 95 °C for 5 min followed by 30 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR product was digested with the restriction enzymes BamHI and XhoI (Takara Biomedical Technology Co. Ltd., Dalian, China) and ligated to the BamHI/XhoI site of the pET28a (+) vector (Novagen, Darmstadt, Germany). The recombinant vectors transformed BL21-competent cells for protein expression.

Recombinant protein purification
BL21 colonies were incubated in fresh LB medium at 37 °C with shaking at 200 rpm until OD 600 = 0.6. Then 0.5 mM isopropyl-β-dithiogalactoside was added to induce PGRP-LB expression with poly-His tags at the C-terminus. After incubation at 200 rpm for 4 h, the cells were collected and resuspended in Buffer B (8 M urea, 50 mM Tris-HCl, and 300 mM NaCl; pH 8.0). After sonication on ice for 10 min (5 s on, 10 s off), the supernatant was collected by centrifugation at 12,000×g and 4 °C for 10 min, mixed with nickel nitrilotriacetate (Ni-NTA; Sangon Biotech, Shanghai, China) and gently shaken at 4 °C overnight. The bound protein was eluted in PBS-T buffer with 20, 50, and 100 mM imidazole.

Western blotting assay
Purified recombinant rSfPGRP-LB was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Western blotting was conducted using mouse primary and goat anti-rabbit immunoglobulin G (IgG)conjugated horseradish peroxidase secondary antibodies (Sangon Biotech, Shanghai, China). Western blot signals were developed in an enhanced chemiluminescence (ECL) detection kit (Bio-Rad Laboratories, Hercules, CA, USA) and photographed with a Molecular Imager® ChemiDoc™ XRS + system (Bio-Rad Laboratories, Hercules, CA, USA). β-actin polyclonal rabbit serum was used to ensure equal protein loading.

Amidase activity assay
Amidase activity was measured as described in a previous report (Yang et al. 2019) with some modifications. Briefly, Dap-type PGN from E.coli and Lys-type PGN from S. aureus were purchased from Sigma-Aldrich (Merck KgAA, Darmstadt, Germany). Twenty micrograms of bovine serum albumin (BSA; Sangon Biotech, Shanghai, China), 20 µg of rSfPGRP-LB, or 20 μg of lysozyme (Sangon Biotech, Shanghai, China) was resuspended in 100 μL of PBS, respectively, and mixed with 100 μL of reaction buffer (2 mM ZnSO 4 , 40 mM MgCl 2 , and 100 mM Tris HCl; pH 7.9). Then, the suspensions were incubated at 37 °C for 60, 120, or 180 min, respectively. Afterward, 40 μL of this mixture was added to 100 μL of 1 M NaOH and the suspension was incubated at 37 °C for 30 min. Then, 10 μL of 0.5 M H 2 SO 4 and 1 mL of 98% (v/v) H 2 SO 4 were added in succession and the mixtures were boiled for 5 min. After cooling, 10 μL of 4% (w/v) CuSO 4 ·5H 2 O and 20 μL of 95% (v/v) ethanol containing 1.5% (v/v) p-hydroxydiphenyl were added and the mixture was incubated at 30 °C for 30 min. Then, 200 μL of this mixture was taken and its OD 560 was measured in a CMax Plus multiskan (Molecular Devices LLC, San Jose, CA, USA).

Scanning electron microscopy
Escherichia coli and S. aureus were incubated at 37 °C with shaking at 200 rpm until OD 600 = 0.4-0.6. For each treatment group, 2 mL of bacterial suspension was centrifuged at 5000×g for 5 min and the cells were collected and washed with 1 × PBS. The E. coli or S. aureus was resuspended in 500 μL of 1 × PBS and mixed with 150 μL of 1 × PBS, 150 μL of rSfPGRP-LB (0.45 μg/μL), or 150 μL of ampicillin (0.45 μg/μL). All treatment groups were incubated at 25 °C for 12 h and washed with 1 × PBS. Then, 200 μL of prechilled 2.5% (v/v) glutaraldehyde was added to the cells and the suspensions were incubated at 4 °C overnight. The bacteria were rinsed with 1 × PBS and centrifuged at 4 °C for 20 min and the supernatant was discarded. The bacteria were dehydrated twice with an ethanol gradient (30% (v/v), 50% (v/v), 70% (v/v), 80% (v/v), 95% (v/v), and 100% (v/v)) and 100% isoamyl acetate. The cells were resuspended, incubated at 4 °C for 20 min, and centrifuged at 5000×g at 4 °C for 20 min to collect the pellet. The dehydrated samples were immersed in isoamyl acetate and subjected to zero-point drying. The dried samples were coated and examined and photographed under a scanning electron microscope (SEM) (Hitachi, Tokyo, Japan).

Inhibition zone assay
SfPGRP-LB antibacterial activity was determined using an inhibition zone assay. E. coli and S. aureus were incubated in LB broth at 37 °C until OD 600 = 0.6. Each 100-μL bacterial suspension was spread onto LB agar plates. Filter disks 5 mm in diameter were soaked in 0.45 μg/μL rSfPGRP-LB and set on the media. The plates were incubated at 37 °C until bacterial growth was visible. Ampicillin (0.45 μg/μL) and BSA (0.45 μg/μL) were the positive and negative controls, respectively.

Statistical analysis
The mRNA expression levels were determined by one-way ANOVA and Student's t test in SPSS v. 19.0 (IBM Corp., Armonk, NY, USA). Differences between treatment means were considered statistically significant at p < 0.05.

SfPGRP-LB sequence and evolutionary analyses
We identified and cloned full length cDNA of SfPGRP-LB by searching the S. furcifera transcriptome database (BioSample accession: SAMN12612920) with BLASTX at e-value = 10 -5 and using PCR, respectively. The nucleotide sequence of SfPGRP-LB obtained by transcriptome sequencing was exactly the same as the result of PCR fulllength amplification (S1.seq). SfPGRP-LB is long and resembles D. melanogaster PGRP-LB. SfPGRP-LB cDNA has a 606-bp ORF encoding a putative 201-aa protein (Gen-Bank ID: MW323547.1). The predicted MW of SfPGRP-LB is ~ 22.60 kDa and its theoretical pI = 6.39. SfPGRP-LB has five Zn 2+ -dependent amidase active sites (H-Y-H-T-C) (Fig. 1A). This protein might have amidase activity in the presence of Zn 2+ . SfPGRP-LB had no transmembrane region but did have a signal peptide at the protein N-terminus (aa: 1-20) (Fig. 1A).
To clarify the evolutionary position of SfPGRP-LB, we constructed a phylogenetic tree using the sequence of SfPGRP-LB and other insect PGRP proteins available. Twenty-one PGRP aa sequences from seven insect species were used in the sequence alignment and evolutionary analysis. A phylogenetic tree was constructed by the NJ method and showed that all PGRP proteins identified here were in groups PGRP-LA to PGRP-LF and SfPGRP-LB was in PGRP-LB. Moreover, SfPGRP-LB has the highest homology with N. lugens PGRP-LB (sequence similarity, 80%), which belongs to the same planthopper family (Fig. 1B).

SfPGRP-LB tissue expression analyses
SfPGRP-LB mRNA expression was detected in the head, thorax, epidermis, fat body, midgut, spermary, and ovary. It was highly expressed in the male and female midgut and to a lesser degree in the female fat body but its expression levels were low in the head, thorax, spermary, and ovary (Fig. 2).

SfPGRP-LB expression analysis in response to bacterial challenge
Bacteria-induced SfPGRP-LB expression profiles were analyzed to determine whether SfPGRP-LB has immune-related function in Gram-positive and Gram-negative bacteria. Whether in the midgut or in the fat body, the mRNA expression of SfPGRP-LB increased under the challenge of different pathogens. Especially in the midgut, after 6 h of E. coli and S. aureus induction, the expression level of SfPGRP-LB increased significantly, and continued to increase at 12-48 h. In the fat body, the expression of SfPGRP-LB did not increase until 12 h after the bacterial challenge, and the rate of increase was lower than that in the midgut (Fig. 3).

Relationship between SfPGRP-LB and NF-κB pathway
To clarify NF-κB pathway regulation by SfPGRP-LB, we measured the Toll, Imd, Dorsal, and Relish transcript levels in the dsSfPGRP-LB + E. coli and dsSfPGRP-LB + S. aureus treatment groups. The transcript level of SfPGRP-LB substantially declined by 87-98% at 12-72 h after dsSfPGRP-LB injection compared with that after dsGFP injection group (Fig. 4A). Additionally, the Toll, Imd, Dorsal, and Relish mRNA levels were significantly downregulated in the dsSf-PGRP-LB + E. coli injection group at 24 and 72 h compared with those in the dsGFP + E. coli injection group (p < 0.05) (Fig. 4B). However, only the Toll and Dorsal mRNA levels were downregulated in the dsSfPGRP-LB + S. aureus injection group at 24 and 72 h compared with those in the dsGFP + S. aureus injection group (p < 0.05) (Fig. 4C).

Protein purification
To identify the immune-related functions of SfPGRP-LB, recombinant protein was expressed in E. coli. Purified recombinant protein was detected by Coomassie brilliant blue (CBB) staining and western blotting. The rSfPGRP-LB target was detected with anti-HisTag antibody and appeared as a single ~ 23 kDa band (Fig. S2).

rSfPGRP-LB agglutination analysis
The rSfPGRP-LB agglutination test results are shown in Fig. 6. After 3 h of incubation, the E. coli and S. aureus in the control groups ( Fig. 6A and B) were diffuse and did not agglutinate. In contrast, E. coli (Fig. 6C) and S. aureus the GW and R residues determining peptidoglycan-binding specificity are marked by three red dots above the alignment. B Phylogenetic tree was constructed by NJ (bootstrap test; 1000 replicates) from 21 insect PGRP proteins. Numbers of branch lengths are shown beside branches (Fig. 6D) treated with rSfPGRP-LB were bound to Lys-and Dap-type PGN. Thus, SfPGRP-LB agglutinates various bacteria.

Effects of rSfPGRP-LB on bacterial morphology
After 12 h of incubation with rSfPGRP-LB, S. aureus cells were destroyed and their cell content had leaked. Nevertheless, there was no obvious damage to E. coli cells subjected to rSfPGRP-LB. No pore formation or leakage was detected in BSA-treated E. coli or S. aureus. Ampicillin-treated E. coli and S. aureus presented with cell damage (Fig. 7). Hence, SfPGRP-LB severely damaged S. aureus membrane integrity.

SfPGRP-LB antibacterial activity
SfPGRP-LB has key residues that enable it to cleave PGNs and inhibit bacterial growth. We performed inhibition zone and CCK-8 assays to evaluate bacterial growth in the presence of rSfPGRP-LB. After rSfPGRP-LB application, inhibition zones expanded on each bacterial plate. rSfPGRP-LB had antibacterial efficacy against S. aureus (Fig. 8A) but not E. coli. Ampicillin had strong antibacterial efficacy against E. coli and S. aureus.
In the innate immune response, insect PGRP is a PRR bindingbacterial peptidoglycan and activating the Toll and Imd signaling pathways (Gottar et al. 2002;Ramet Fig. 2 SfPGRP-LB expression patterns. SfPGRP-LB expression in midgut, thorax, epidermis, head, fat body, ovary, and spermary. β-actin was internal control. Black bars represent standard deviation (SD) Fig. 3 Analysis on the expression level of SfPGRP-LB in fat body a and midgut b after E. coli or S. aureus challenge, respectively. PBS was negative control. Asterisks above bars indicate statistically significant differences between E. coli and S. aureus injection groups and control group (p < 0.05). Black bars represent standard deviation (SD) et al. 2002). It also hydrolyzes amide bonds in bacterial peptidoglycan, has bactericidal activity (Cheng et al. 1994), acts as an opsonin, and promotes phagocytosis (Garver et al. 2006;Royet et al. 2011). The PGRP gene family is relatively conserved among insects. Certain PGRPs have a ~ 160-aa domain structurally homologous to bacteriophage T7 lysozyme/Zn-dependent N-acetylmuramoyl-L-alanine amidase (Liu et al. 2001;Mellroth et al. 2003;Werner et al. 2000). The sequence analysis (Fig. 1) revealed that SfPGRP-LB has all the T7 lysozyme amidase activity sites required for Zn 2+ binding. As SfPGRP-LB protein was thought to have amidase activity dsSfPGRP-LB and dsGFP groups. Asterisks above bars indicate statistically significant differences between E. coli or S. aureus injection groups and control group (p < 0.05). Black bars represent standard deviation (SD) against bacterial peptidoglycan, we analyzed tissue-specific and bacteria-induced SfPGRP-LB expression profiles. SfPGRP-LB expression was highly upregulated in the midgut (Fig. 2). Thus, it operates mainly in the intestinal tract which may be a challenge route in S. furcifera. The gut is one of the most important interfaces between insects and the internal and external environment. However, owing to its special function, the gut is often infected by external pathogenic bacteria (Huang et al. 2015;Zheng et al. 2017). In Rhynchophorus ferrugineus, PGRP-LB is of great significance for the development of new management strategies to destroy intestinal pathogenic bacteria (Dawadi et al. 2018). In our study, the mRNA expression of SfPGRP-LB was highly upregulated in the midgut (Fig. 2). In addition, E. coli and S. aureus injections significantly upregulated SfPGRP-LB expression and the rate of increase in fat body was lower than that in the midgut (Fig. 3); thus, we supposed SfPGRP-LB may prevent bacteria from penetrating midgut cells. A previous study showed that PGRP induction confers resistance to microbial pathogen invasion. After E. coli or S. aureus injection, PGRP-B and PGRP-C mRNA levels are upregulated in Helicoverpa armigera (Lepidoptera: Noctuidae) (Yang et al. 2013). In addition, PGRP-S5 transcription significantly increases when B. mori is infected with Pseudomonas aeruginosa and S. aureus (Chen et al. 2014). E. coli and Bacillus subtilis challenges significantly upregulate PGRP-LB expression in N. lugens (Bao et al. 2013). These results suggest that SfPGRP-LB plays vital roles in natural immune responses to pathogen invasion.
PGRPs recognize and bind pathogens and initiate downstream immune responses such as Toll and Imd pathway activation and antimicrobial peptide biosynthesis (Garver et al. 2006;Michel et al. 2001). The Toll pathway is activated mainly by Lys-type PGN, whereas, Dap-type PGN activates the Imd pathway (Leulier et al. 2003). Drosophila PGRP-SCla recognizes invading E. coli and Micrococcus luteus and activates the Toll pathway (Garver et al. 2006). Drosophila PGRP-LC and PGRP-LE recognize Dap-type PGN on Gram-negative bacterial cell wall and activate the Imd pathway (Choe et al. 2002;Gottar et al. 2002;Iatsenko et al. 2016;Kurata 2010). When dsSfGFRP-LB was injected into S. furcifera and induced by E. coli, Toll and Dorsal (Toll pathway) and Imd and Relish (Imd pathway) expressions were significantly upregulated compared with that in the control. When dsSfGFRP-LB was injected into S. furcifera and induced by S. aureus, Toll and Dorsal (Toll pathway) expressions were declined relative to the control, whereas Imd and Relish (Imd pathway) expression did not significantly change (Fig. 4). Therefore, SfGFRP-LB may participate in E. coli resistance via the Toll and Imd pathways. In contrast, SfGFRP-LB resistance to S. aureus is mediated by the Toll pathway.
A previous study demonstrated that PGRP proteins cleaving bacterial peptidoglycan might have antibacterial activity. PGRP-LC of Bombus lantschouensis (Hymenoptera: Apoidea) directly binds Dap-type PGN and responds to E. coli challenge (Liu et al. 2020). The same is true for PGRP-L protein in humans, mice, and Amphioxus (Li et al. 2012;Wang et al. 2003;Yang et al. 2010). PGRP proteins are bactericidal factors in immunization. However, not all proteins with amidase activity are bactericidal. Drosophila PGRP-SC has a conserved lactamase protein structure with no direct antibacterial activity (Mellroth and Steiner 2006). To verify whether SfPGRP-LB has amidase activity, we used Dap-type and Lys-type PGN as research objects. The rSfPGRP-LB protein hydrolyzed only Lys-type PGN (Fig. 5). In Drosophila, PGRP-LB, PGRP -SC1(a/b), and PGRP-SC2 are known or predicted to have amidase activity, and they also modulate activation of the Imd pathway (Bischoff et al. 2006;Mellroth et al. 2003;Paredes et al. 2011;Royet et al. 2011;Zaidman-Remy et al. 2006. In our study, PGRP-LB had amidase activity and resisted pathogen challenge through the NF-κB pathway. In Drosophila, there are 13 members of the PGRP family, and the division of labor is more detailed. In S. furcifera, there are only two PGN degradation detected by changes in OD 560 . Asterisks above bars indicate statistically significant differences between lysozyme or rSfPGRP-LB groups and BSA group (p < 0.05). Black bars represent standard deviation (SD) family members of PGRP (PGRP-LB and PGRP-LC); thus, the function may be more comprehensive.
Certain PGRPs recognize and bind PGNs in bacterial cell walls and promote aggregation. B. mori PGRP-S5 binds PGNs and promotes E. coli and P. aeruginosa aggregation (Chen et al. 2014). In the presence of Zn 2+ , H. armigera PGRP-B and PGRP-C induce E. coli and S. aureus aggregation (Yang et al. 2013). In the present study, rSfPGRP-LB bound E. coli and S. aureus in vitro and promoted E. coli and S. aureus agglutination (Fig. 6). The E. coli and S. aureus activity tests showed that rSfPGRP-LB significantly inhibited the growth of S. aureus but not that of E. coli  . 8). Moreover, rPGRP-LB damaged the cell walls of S. aureus but not those of E. coli (Fig. 7). For these reasons, we speculated that the antibacterial/bactericidal mechanisms of rSfPGRP-LB differ between E. coli and S. aureus.
In conclusion, our results suggest that SfPGRP-LB regulates the balance between the invasion of pathogenic bacteria and reproduction of pathogenic bacteria in the gut. In this process, SfPGRP-LB may have multiple functions. First, it prevents the overproduction of Gram-positive bacteria Lystype PGN by degrading it and realizes the innate immune response of S. furcifera to intestinal Gram-positive bacteria. Second, it acts as a PRR to activate the NF-κB signaling pathway to resist the growth of pathogenic bacteria in the gut.
Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 31860505) and the Joint Project from Zunyi science and Technology Bureau and Zunyi Normal University Natural Science Foundation (No.ZSKH HZ (264)).
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Fig. 8 Antibacterial activity analysis. A rSfPGRP-LB activity against E. coli and S. aureus determined by inhibition zones. E-1 and S-1: 0.45 mg/mL BSA were negative controls. E-2 and S-2: 0.45 mg/mL rSfPGRP-LB, and E-3 and S-3: 0.45 mg/ mL ampicillin were positive controls. B rSfPGRP-LB antibacterial efficacy measured by CCK-8 assay in liquid culture. CK: bacteria-free liquid culture. 0.45 mg/mL BSA: negative control. 0.45 mg/mL ampicillin: positive control. Asterisks above bars indicate statistically significant differences relative to ampicillin (p < 0.05). Black bars represent standard deviation (SD)