Identification and functional characterization of a peptidoglycan recognition protein from the cotton bollworm, Helicoverpa armigera.

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IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF A PEPTIDOGLYCAN RECOGNITION PROTEIN FROM THE COTTON BOLLWORM, Helicoverpa armigera Li Li, Yu-Ping Li, Cai-Xia Song, Min Xiao, Jia-Lin Wang, and Xu-Sheng Liu Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, China

Peptidoglycan recognition proteins (PGRPs) specifically bind to peptidoglycans, and play crucial roles as pattern recognition receptors (PRRs) in mediating innate immune responses. In this study, we identified and characterized a PGRP (HaPGRP-D) from the cotton bollworm, Helicoverpa armigera. Sequence analysis indicated that HaPGRP-D is an amidase-type PGRP. Expression of HaPGRP-D was upregulated in the hemocytes of H. armigera larvae after injecting Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, or chromatography beads. To test the biological activity of HaPGRP-D, purified recombinant protein was prepared. Subsequent analysis showed that rHaPGRP-D (i) could bind and agglutinate Gram-negative E. coli and Gram-positive S. aureus in a zinc-dependent manner, (ii) functioned as an amidase to degrade peptidoglycans in the presence of Zn2+ , (iii) strongly inhibited the growth of E. coli and S. aureus in the presence of Zn2+ , (iv) could bind to the surface of hemocytes, (v) increased the phagocytosis of E. coli cells by hemocytes in vitro, and (vi) promoted hemocyte encapsulation on chromatography beads in vitro. These results suggest that HaPGRP-D plays important roles as PRR, amidase, and C opsonin in H. armigera humoral and cellular immune responses. 2014 Wiley Periodicals, Inc. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 31000982, 31101672. Correspondence to: Xu-Sheng Liu, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, China. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 86, No. 4, 240–258 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21174

PGRP-D in Helicoverpa armigera

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Keywords: peptidoglycan recognition protein; Helicoverpa armigera; encapsulation; phagocytosis

INTRODUCTION The innate immune system is the only effective defense mechanism against invading microbes in invertebrates. Recognition and distinction of self from nonself is the first and the most critical step in insect innate immunity and is mediated by pattern recognition receptors (PRRs; Medzhitov and Janeway, 2002; Leulier et al., 2003; Royet, 2004). PRRs recognize conserved pathogen-associated molecular patterns (PAMPs) present on microbial pathogen surfaces such as bacterial lipopolysaccharide (LPS), lipoteichoic acid, peptidoglycan (PGN), fungal β-1,3-glucan, and double-stranded RNA from viruses (Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2002; Pal and Wu, 2009). Once a pathogen is recognized by PRRs, immune responses are stimulated by activating humoral responses, including activation of prophenoloxidase (PPO) cascade and synthesis of antimicrobial peptides and cellular responses, including hemocyte-mediated immune responses such as phagocytosis, encapsulation, and nodulation (Strand and Pech, 1995; Schmidt et al., 2001; Kurata, 2004; Lemaitre and Hoffmann, 2007; Williams, 2007). Peptidoglycan recognition proteins (PGRPs) are a type of PRRs that recognize PGN, which is an essential and unique cell wall component of virtually all bacteria but is not present in eukaryotic cells (Kurata, 2014). PGRP was initially isolated from the silkworm, Bombyx mori (Yoshida et al., 1996). Its binding to PGN from Micrococcus luteus triggered a serine proteinase cascade that led to PPO activation and melanin formation. Subsequently, PGRP homologues have been identified in several species, particularly in insects and mammals (Kang et al., 1998; Dziarski and Gupta, 2006). In Drosophila melanogaster, up to 19 different PGRPs have been identified and are classified into short (S) and long (L) forms (Lemaitre and Hoffmann, 2007; Kurata, 2014). In addition, PGRPs have been characterized in several insect species, including Trichoplusia ni (Kang et al., 1998), B. mori (Ochiai and Ashida, 1999; Tanaka et al., 2008), Anopheles gambiae (Christophides et al., 2002), Manduca sexta (Yu et al., 2002), Holotrichia diomphalia (Lee et al., 2004), Samia cynthia ricini (Hashimoto et al., 2007; Onoe et al., 2007), Tenebrio molitor (Park et al., 2007; Yu et al., 2010), Bombus ignites (You et al., 2010), Ostrinia nubilalis (Khajuria et al., 2011), Helicoverpa armigera (Yang et al., 2013), and Ostrinia furnacalis (Sun et al., 2014). Insect PGRPs play important and diverse roles in activating innate immune reactions such as recognizing and binding bacterial PGN to activate Toll pathway (Michel et al., 2001; Garver et al., 2006; Park et al., 2007; Yu et al., 2010), immune deficiency pathway (Choe et al., 2002), and PPO activation system (Yoshida et al., 1996; Lee et al., 2004; Park et al., 2007; Sumathipala and Jiang, 2010; Sun et al., 2014); hydrolyzing bacterial PGN to exhibit bactericidal activity (Kim et al., 2003; Mellroth et al., 2003); and induction of phagocytosis (Rämet et al., 2002; Garver et al., 2006). Recent transcriptome analysis of H. armigera hemocytes identified a PGRP gene (HaPGRP-D). In this study, we investigated the physiological function of HaPGRP-D in immune responses of H. armigera.

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MATERIALS AND METHODS Insects Rearing H. armigera larvae were reared on an artificial diet (Li et al., 2009) at 28 ± 1°C with a 14 h light/10 h dark photoperiod.

Identification and Sequence Analysis of HaPGRP-D Gene The immune transcriptome of H. armigera hemocytes was sequenced as described (Yang et al., 2013). A cDNA encoding protein homologous to PGRP was identified and named HaPGRP-D. Physical and chemical parameters of the deduced protein were analyzed using ExPASy (http://web.expasy.org/protparam/). Signal sequence prediction was performed using Signal P (http://www.cbs.dtu.dk/services/SignalP/), and domain prediction was performed using SMART (http://smart.embl-heidelberg.de/). ClustalX (http://www.clustal.org) was used to make multiple alignments.

Tissue Expression Profiles Reverse-transcription polymerase chain reaction (RT-PCR) was used to compare the transcript abundance of HaPGRP-D in different tissues of H. armigera. Total RNA was isolated from epidermis, midgut, fat body, and hemocytes of the sixth instar larvae by using Total RNA Purification System (Omega, Norcross, GA) combined with On-Column DNase (Qiagen, Hilden, Germany) digestion to remove any genomic DNA contamination. The first strand cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA). PCR was performed using the following primer sets: HaPGRP-D forward 5 -AGTATAGACCGTGCCTCATG-3 and reverse 5 -TAATTAAACCGGCAAAGTACC-3 ; rpS3 forward 5 -GTGCGCGTCACTCCGACTC-3 and reverse 5 -TCATGAGGCCGTCCACGAAC-3 . The rpS3 gene from H. armigera (HarpS3) was used as the control. The annealing temperature and number of cycles for HaPGRP-D and Ha-rpS3 were 53.3°C and 35 cycles and 55°C and 28 cycles, respectively. The PCR products (3 μl each) were electrophoresed in 1% agarose gel. The DNA was stained using ethidium bromide and imaged.

Injection of Bacteria and Chromatography Beads Escherichia coli and Staphylococcus aureus were harvested from cultures by centrifugation at 6,000 rpm for 3 min, washed three times with PBS (0.138 M NaCl, 0.0027 M KCl, 0.0073 M Na2 HPO4 , 0.00147 M KH2 PO4 , pH 7.4), and resuspended in PBS. After gentle mixing, the number of bacteria was calculated using a hemocytometer. Sephadex DEAE A-25 chromatography beads (Pharmacia, Uppsala, Sweden) were washed four times with PBS and finally resuspended in PBS. H. armigera larvae (sixth instar, 1-day old) were anesthetized on ice and approximately 1 × 105 colony-forming units (CFU) bacteria or 30 chromatography beads resuspended in 5 μl of PBS were injected into each larval hemocoel by using a microsyringe. Control larvae were injected with 5 μl of PBS buffer. Hemocyte samples were collected at selected time points (2, 6, 12, and 24 h after injection) for RNA extraction. Archives of Insect Biochemistry and Physiology


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Quantitative Real-Time PCR Quantitative real-time PCR (qRT-PCR) was used to measure the changes in transcript levels of HaPGRP-D in hemocytes after the injection of bacteria or beads. Extraction of total RNA and synthesis of the first strand cDNA were performed as previously described. The qRT-PCR was performed using the following primer sets: HaPGRP-D forward 5 GCAGCTGGGGTAGAACTTG-3 and reverse 5 -AGAGTAGTGGTCCCACGTT-3 ; rpS3 forward 5 -CGGCGTGGAGGTGCGCGTC-3 and reverse 5 -CGATGGCGCACAGACCGCG-3 . The qRT-PCR was performed using a MiniOpticon System (Bio-Rad, Hercules, CA) with TransStart Eco Green qPCR SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Each 20 μl reaction contained 10 μl of 2× TransEco qRCR Mix, 2 μl of the cDNA template, and 4 μl of each primer (1 μM). The conditions for the qRT-PCR were as follows: initial denaturing at 95°C for 2 min, followed by 40 cycles of denaturing at 95°C for 5 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s. The relative expression level of HaPGRP-D was calculated using the 2−CT method (Schmittgen and Livak, 2008). The obtained data from three independent repeats were statistically analyzed using Student’s t-test, and significant difference was accepted when P < 0.05. Recombinant Expression and Purification of HaPGRP-D The sequence encoding mature peptide of HaPGRP-D was amplified using a pair of gene specific primers (forward 5 -CGCGGATCCATGCCGAGTGCTTTTAGTA-3 and reverse 5 CCCAAGCTTTTAAACCGGCAAAGTACCG-3 ; italic indicates BamHI and HindIII sites, respectively). After cutting with BamHI and HindIII, the DNA fragments were cloned into expression vector pET-32a (Novagen, Madison, WI) and transformed into E. coli BL21 (DE3). The pET-32a vector lacking the insert fragment was used as a negative control, which expressed a thioredoxin (Trx) with 6× His-tag in the prokaryotic express system. Positive transformants were incubated in LB medium at 37°C with shaking at 200 rpm. When the optical density (OD) at 600 nm reached 0.6, IPTG was added at a final concentration of 0.3 mM, and the medium was shaken for another 4 h. Bacterial cells were harvested by centrifugation, resuspended with PBS, and sonicated on ice. Soluble protein fractions of bacterial cells were applied on High-Affinity Ni–NTA Resin (GenScript, Nanjing, China) to purify recombinant proteins according to the manufacturer’s instruction. The proteins were finally dissolved in PBS, and their concentrations were quantified by the Bradford method. The obtained proteins were stored at −80°C before use. Binding of rHaPGRP-D to Bacteria E. coli and S. aureus were selected to test the binding ability of rHaPGRP-D. Bacteria in midlogarithmic phase were pelleted by centrifugation at 6,000 rpm for 3 min, washed three times with Tris-buffered saline (TBS; 50 mM Tris–HCl, 150 mM NaCl, pH 7.5), and resuspended in TBS at a concentration of 2 × 108 cells/ml. A total of 500 μl of bacterial suspensions were mixed with 200 μl of rHaPGRP-D (900 μg/ml) in the presence or absence of 10 mM ZnCl2 . The mixture was incubated at room temperature for 1 h with gentle rotation. Microorganisms were pelleted, washed four times with TBS, and eluted with 7% SDS. As negative and positive controls, TBS and rTrx, respectively, were incubated with bacterial cells and subjected to the same treatments. The eluted samples were applied to Western blotting analysis. The samples were first treated with loading buffer, Archives of Insect Biochemistry and Physiology

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electrophoresed on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked with blocking buffer (3% BSA in TBS) for 1.5 h at room temperature and were incubated with mouse anti-His monoclonal antibody (diluted 1:2500 in blocking buffer) overnight at 4°C and with horseradish peroxidase conjugated goat anti-mouse immunoglobulin G (IgG) for 2 h at room temperature. 4-Chloro-1-naphthol was used for color development.

Bacterial Agglutination Assay Fluorescein isothiocyanate (FITC) labeling of bacteria (E. coli and S. aureus) was performed using the method described by Mavrouli et al. (2005). In brief, heat-killed bacteria (109 cells/ml) were incubated in carbonate buffer (0.1 M Na2 CO3 , 0.1 M NaHCO3 , pH 9.6) containing FITC (0.5 mg/ml) for 1 h in the dark. FITC-conjugated bacteria was rinsed three times with PBS, resuspended in PBS at a concentration of 1 × 108 cells/ml, and stored at −20°C before use. Bacterial agglutination assay was performed according to the method described by Yang et al. (2013). Briefly, 10 μl of FITC-labeled bacterial suspensions were added to 20 μl of rHaPGRP-D (600 μg/ml) in the presence or absence of 10 mM ZnCl2 . The mixtures were incubated at room temperature for approximately 1 h, and the cells were then observed and photographed under a fluorescence microscope. rTrx was used as the control protein.

Isolation of Insoluble PGNs Insoluble PGNs from S. aureus were prepared using a modified protocol (Rosenthal and Dziarski, 1994). Briefly, S. aureus was cultured overnight in LB medium at 37°C. Bacterial cells were collected and resuspended in PBS and then boiled for 20 min. The cells were washed with PBS, water, and acetone and then dried at 37°C. Dried cells were resuspended in 10% trichloroacetic acid and boiled for 30 min to break the cells. After cooling, cell walls not containing teichoic acid were pelleted by centrifugation at 12,000 rpm for 15 min and were washed three times with water. Trypsin (3 mg/ml) was added, and the suspensions were incubated for 3 h at 37°C. Insoluble trypsin was removed by centrifugation at 3,000 rpm for 15 min, and the supernatant was centrifuged at 12,000 rpm for 15 min. The pellet (insoluble PGNs) was washed three times with water and three times with ether, dried, and stored at −20°C before use.

Amidase Activity Analysis The relative enzymatic activity of rHaPGRP-D against insoluble PGNs from S. aureus was assayed according to a modified protocol (Mellroth et al., 2003). Briefly, insoluble PGNs (5 mg/ml) were incubated with rHaPGRP-D (1 mg/ml) in Hepes buffer (20 mM Hepes, 150 mM NaCl, pH 7.2) with or without 10 mM ZnCl2 . The OD at 540 nm was recorded every 5 min during a 180-min period. rTrx was used as the control protein. The decreased OD value per min in rHaPGRP-D + Zn2+ group was standardized to 100%. The relative activities of other groups were calculated by comparing with rHaPGRP-D + Zn2+ group. The assay was performed thrice independently. Archives of Insect Biochemistry and Physiology


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Antibacterial Activity Assay The antibacterial activities of rHaPGRP-D against E. coli and S. aureus were evaluated. Bacteria were cultured in LB medium at 37°C until the OD at 600 nm reached 0.5. Five microliters of bacteria were pelleted by centrifugation, washed three times with PBS, and resuspended in PBS. Bacteria were incubated with rHaPGRP-D (final concentration 500 μg/ml in the presence or absence of 10 mM ZnCl2 ) for 4 h at room temperature. Next, the samples were serially diluted and spread on LB agar plates. The plates were incubated at 37°C overnight, and the number of colonies was counted. PBS and rTrx were used as negative and positive controls, respectively. The CFU per centimeter square of LB plate in the PBS group was standardized to 100%. The relative viabilities (%) of bacteria in the treated groups were calculated by comparing with negative control.

In Vitro Phagocytosis Assay In vitro phagocytosis assay was performed according to a modified protocol (Wootton et al., 2003). Briefly, 10 μl of hemolymph was collected from the sixth instar larvae, mixed immediately with 40 μl of ice-chilled anticoagulant (62 mM NaCl, 100 mM glucose, 20 mM EDTA, 26 mM citric acid, 30 mM sodium citrate, pH 4.6), and centrifuged at 4,000 rpm for 6 min to separate hemocytes from the plasma. The hemocytes were resuspended in 200 μl of TBS buffer. rHaPGRP-D was added at a final concentration of 400 μg/ml. After incubating for 30 min at room temperature, 5 μl of TBS containing E. coli (OD600 = 0.4) was added and incubated for another 1 h at room temperature. Aliquots of the mixture (50 μl) were mounted on microscope slides and kept in a moist chamber for 1 h. The slides were washed gently with TBS, air-dried, fixed for 10 min in 4% formaldehyde in PBS, and washed with TBS. Subsequently, the slides were stained with Giemsa and washed with TBS. The phagocytic activity of hemocytes attached to the slide was examined using a light microscope. In all, 200 hemocytes were counted on each slide. Phagocytic rate (PR) and phagocytic index (PI) representing the phagocytic activities were expressed as follows: PR = (phagocytic hemocytes)/(total hemocytes) × 100%; PI = average number of bacteria in phagocytic hemocytes. For each treatment, assay was performed in three different slides for statistical analysis. rTrx was used as the control protein.

In Vitro Encapsulation Assay An in vitro encapsulation assay was performed using a modified protocol (Ling and Yu, 2006). Briefly, Sephadex DEAE A-25 chromatography beads were stained in 0.1% Congo Red solution for 2 h. Beads were then dried under UV light, resuspended in TBS, and incubated with rHaPGRP-D or rTrx (as control protein) overnight in a 1.5 ml Eppendorf tube at room temperature. The recombinant protein-coated beads were washed six times with TBS and were finally resuspended in TBS at 100–120 beads per microliter. The hemocyte samples were prepared as previously described. A total of 1 μl of protein-coated beads were added to 200 μl of hemocyte suspensions, and the mixture was incubated overnight with slow rotation at room temperature. After incubation, the hemocytes were removed, and the beads were washed six times with TBS. The beads were resuspended in TBS and observed under a microscope. Archives of Insect Biochemistry and Physiology

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Binding of rHaPGRP-D to Hemocytes The hemocyte samples were prepared as previously described. Next, 20 μl of hemocyte suspensions were dropped onto a slide. The hemocytes were allowed to settle down for 20 min and then fixed with 4% paraformaldehyde in TBS for 20 min. After washing three times with TBS, the hemocytes were incubated with 0.2% Triton X-100 for 15 min. After washing another three times with TBS, the hemocytes were blocked with 3% BSA in TBS for 1 h. Subsequently, 150 μl of rHaPGRP-D (160 μg/ml) was added and incubated with hemocytes for 1 h. After washing, the hemocytes were incubated with mouse anti-His monoclonal antibody (diluted 1:1000 in 1% BSA) for 1.5 h, washed three times with TBS, and incubated with FITC-labeled goat anti-mouse IgG (diluted 1:200 in 1% BSA) for 1.5 h. Finally, 4 -6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to stain the nuclei. rTrx was used as the control protein. A Nikon fluorescence microscope 2000 was used to detect fluorescence.

RESULTS Identification and Sequence Analysis of HaPGRP-D A PGRP cDNA was identified from the transcriptome of H. armigera hemocytes and named HaPGRP-D (GenBank accession no. KF985962). The HaPGRP-D cDNA contained an open reading frame (ORF) of 699 bp. The predicted protein encoded by HaPGRP-D was 232aa long and included a signal peptide (1–18 aa), a PGRP domain (38–181 aa), and an overlapping Ami 2 domain (50–187 aa). The mature HaPGRP-D had a predicted molecular weight (MW) of 26.081 kDa and a theoretical isoelectric point (pI) of 5.82. Multiple sequence alignment of the deduced protein sequences of the lepidopteran PGRP genes indicated that HaPGRP-D amino acid sequence had significant similarities with the PGRP sequences from other lepidopterans (Fig. 1). Comparative analysis indicated that five amino acids that were required for T7 lysozyme Zn2+ binding and amidase activity were also conserved in HaPGRP-D (Fig. 1, asterisks), suggesting that HaPGRP-D is an amidase-type PGRP. Tissue Distribution of HaPGRP-D mRNAs Expression of HaPGRP-D mRNAs was examined using RT-PCR in four tissues (hemocytes, fat body, midgut, and epidermis) of H. armigera larvae (Fig. 2). HaPGRP-D was expressed at high levels in the midgut and low levels in the hemocytes. No expression was detected in the epidermis and fat body. Expression Profiles of HaPGRP-D Induced by Bacterial or Chromatography Beads Challenge qRT-PCR was used to examine the expression levels of HaPGRP-D mRNAs in the hemocytes of H. armigera larvae at various time points after the injection of E. coli, S. aureus, or Sephadex DEAE A-25 chromatography beads (Fig. 3). Our results showed that HaPGRP-D mRNA expression was upregulated at all the four time points after the E. coli challenge. After S. aureus challenge, the expression of HaPGRP-D mRNA increased in the first 6 h, returned to original level at 12 h, and increased again at 24 h. After chromatography beads challenge, the expression of HaPGRP-D mRNA increased until 6 h after the injection and Archives of Insect Biochemistry and Physiology


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Figure 1. Multiple alignments of the amino acid sequences of PGRPs from lepidopteron. Identical and similar amino acid residues are shaded in black and gray, respectively. Predicted signal peptides are underlined. The asterisks indicate the amino acids required for T7 lysozyme Zn2+ binding and amidase activity. Sequences from the following insects were used in this analysis: Ha-B (H. armigera-B, AFP23116); Ha-C (H. armigera-C, AFP23117); Ha-D (H. armigera-D, KF985962); On-A (O. nubilalis-A, ADU33184); On-B (O. nubilalis-B, ADU33185); On-C (O. nubilalis-C, ADU33186); On-D (O. nubilalis-D, ADU33187); Scr-A (S. cynthia ricini-A, BAF03522); Scr-B (S. cynthia ricini-B, BAF03520); Scr-C (S. cynthia ricini-C, BAF03521); Scr-D (S. cynthia ricini-D, BAF74637); Bm (Bombyx mori, NP_001036836); Ms-1A (M. sexta-1A, AAO21509); Tn (Trichoplusia ni, AAC31820); Of-S (Ostrinia furnacalis-S, EU289210).

Figure 2. Tissue-specific expression of HaPGRP-D in various tissues of H. armigera larvae. rpS3 serves as a positive control. Hc, hemocytes; Fb, fat body; Mg, midgut; Ep, epidermis.


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Figure 3. Expression profiles of HaPGRP-D induced by microbial and beads challenge. Error bars show means ± SD (n = 3). Asterisks indicate significant differences (Student’s t-test, *P < 0.05, **P < 0.01).

Figure 4. Expression and purification of recombinant HaPGRP-D. Lane 1, crude protein extract of bacteria without induction. Lane 2, crude protein extract of bacteria induced for 4 h. Lane 3, soluble protein fraction of bacterial cells induced for 4 h. Lane 4, insoluble protein fraction of bacterial cells induced for 4 h. Lane 5, purified rHaPGRP-D. Lane 6, protein standard.

dropped to the original level at 12 h after the injection. These results suggested that HaPGRP-D gene expression was inducible upon bacterial or beads challenge. Expression and Purification of rHaPGRP-D To express recombinant mature HaPGRP-D, a cDNA fragment encoding residues 19–232 of HaPGRP-D was obtained by PCR and was cloned into the bacterial expression vector pET-32a. Recombinant plasmids were transformed into competent E. coli BL21 (DE3) cells, and protein expression was induced by IPTG (Fig. 4). Recombinant HaPGRP-D was purified using the High-Affinity Ni–NTA Resin. Meanwhile, the transformants with parent vector expressed a unique product representing rTrx (Yang et al., 2013). rTrx was also purified to use as the control protein in the subsequent assays. Archives of Insect Biochemistry and Physiology


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Figure 5. Binding activity of rHaPGRP-D to E. coli (A) and S. aureus (B) in the presence or absence of Zn2+ . (A) Lane 1, wash solution of E. coli alone in TBS as a negative control; lanes 2–5, wash solutions of E. coli incubated with rHaPGRP-D; lane 6, 7% SDS elution of E. coli; lane 7, purified rHaPGRP-D as protein standard. (B) Lane 1, wash solution of S. aureus alone in TBS as a negative control; lanes 2–6, wash solutions of S. aureus incubated with rHaPGRP-D; lane 7, 7% SDS elution of S. aureus; lane 8, purified rHaPGRP-D as protein standard.

Binding Activity of rHaPGRP-D to Bacteria E. coli and S. aureus were used to test the microbial binding activity of rHaPGRP-D. SDSPAGE and Western blotting analysis were performed to evaluate the binding ability of rHaPGRP-D to live microbial cells. In the absence of Zn2+ , rHaPGRP-D could weakly bind to E. coli and S. aureus while in the presence of Zn2+ , rHaPGRP-D could strongly and significantly bind to these bacteria (Fig. 5). As expected, control protein rTrx could not bind to any of the tested bacteria in the presence or absence of Zn2+ (data not shown). These results indicated that HaPGRP-D could directly bind to bacteria in a zinc-dependent manner. Bacterial Agglutination Assay The agglutination activities of rHaPGRP-D toward E. coli and S. aureus were assessed (Fig. 6). In the absence of Zn2+ , rHaPGRP-D could weakly agglutinate E. coli and S. aureus. However, significant agglutination was observed in the presence of Zn2+ . As expected, rTrx could not agglutinate any of the tested bacteria in the presence or absence of Zn2+ . These results indicated that agglutination activities of HaPGRP-D toward bacteria were Zn2+ -dependent. Amidase Activity Analysis Sequence analysis indicated that HaPGRP-D is an amidase-type PGRP. To confirm this, insoluble PGNs were purified from S. aureus and used to measure the amidase activity of rHaPGRP-D. The OD decreased dramatically when rHaPGRP-D was incubated with PGNs in the presence of 10 mM ZnCl2 (Fig. 7), indicating its high-degrading activity toward PGNs in the presence of Zn2+ . Much lower degrading activity toward PGNs was detected in the non-Zn2+ group. Very little activity was observed in the rTrx group (data for rTrx in the absence of Zn2+ are not shown). These results proved that HaPGRP-D is a Zn2+ -dependent amidase. Archives of Insect Biochemistry and Physiology

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Figure 6. Agglutination toward E. coli and S. aureus induced by rHaPGRP-D in the presence or absence of Zn2+ . rTrx was used as the control protein.

Figure 7. Enzymatic degradation of PGNs. (A) rHaPGRP-D was incubated with PGNs from S. aureus in the presence or absence of Zn2+ , and the OD at 540 nm was recorded every 5 min during a 180-min period. The enzyme activity was recorded as a decrease in optical density. rTrx was used as the control protein. (B) The relative enzyme activity was quantitatively analyzed and shown in graph. The assay was performed independently for three times. Asterisks indicate significant differences (Student’s t-test, **P < 0.01).

Antibacterial Activity Assay To assess whether the amidase activity of HaPGRP-D was correlated with bacterial killing, the antibacterial activity of rHaPGRP-D was determined against E. coli and S. aureus (Fig. 8). In the absence of Zn2+ , rHaPGRP-D showed an apparent growth inhibition of E. coli (51% killing); however, in the presence of Zn2+ , rHaPGRP-D showed an increased antibacterial activity (67% killing). In the absence of Zn2+ , rHaPGRP-D showed no antibacterial effect against S. aureus; however, in the presence of Zn2+ , rHaPGRP-D showed significant antibacterial activity against S. aureus (65% killing). rTrx showed no antibacterial activity in the presence or absence of Zn2+ (data for rTrx in the absence of Zn2+ are not shown). Archives of Insect Biochemistry and Physiology


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Figure 8. Antibacterial activity of rHaPGRP-D against E. coli (A) and S. aureus (B) in the presence or absence of Zn2+ . rTrx was used as the control protein. Asterisks indicate significant differences (Student’s t-test, compared with rTrx+Zn2+ , **P < 0.01).

Figure 9. rHaGRP-D promoted hemocyte phagocytosis in vitro. (A) Phagocytic rate. (B) Phagocytic index. rTrx was used as the control protein. For each treatment, assay was performed in three different slides for statistic analysis. Asterisks indicate significant differences (Student’s t-test, **P < 0.01).

HaPGRP-D Promotes Hemocyte Phagocytosis An in vitro phagocytosis assay was performed to test whether HaPGRP-D was involved in the phagocytosis response. Compared with rTrx, rHaPGRP-D significantly enhanced the phagocytic activity of H. armigera hemocytes against E. coli both with respect to PR and PI (Fig. 9). These results indicated that HaPGRP-D may be involved in the phagocytosis process of H. armigera. HaPGRP-D Promotes Hemocyte Encapsulation An in vitro encapsulation assay was performed to test whether HaPGRP-D was involved in encapsulation response. Up to 71% beads coated with rHaPGRP-D were encapsulated by hemocytes (Fig. 10), which was significantly higher than that in control group where only 13% beads were encapsulated. These results suggested that HaPGRP-D may be involved in the encapsulation response of H. armigera. Archives of Insect Biochemistry and Physiology

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Figure 10. rHaGRP-D promoted hemocyte encapsulation in vitro. (A) Beads coated with rHaPGRP-D were encapsulated by hemocytes, while beads coated with rTrx (as the control protein) could hardly be encapsulated. (B) The encapsulation rates of the beads. Asterisks indicate significant differences (Student’s t-test, **P < 0.01).

Binding of HaPGRP-D to Hemocytes Because HaPGRP-D enhanced hemocyte-mediated phagocytosis and encapsulation responses, it was predicted that HaPGRP-D in the hemolymph might bind to hemocytes. To confirm this hypothesis, rHaPGRP-D or Trx (as control protein) was incubated with hemocytes from H. armigera larvae. The binding of recombinant proteins to hemocytes was detected using anti-His monoclonal antibody because the recombinant proteins contained a 6× His-tag. Fluorescence detected in hemocytes indicated that rHaPGRP-D was bound to granulocytes and plasmatocytes (Fig. 11), which may account for enhanced phagocytosis and encapsulation by HaPGRP-D.

DISCUSSION PGRPs play a significant role in innate immunity and they are involved in various responses against pathogen infection (Dziarski, 2004; Dziarski and Gupta, 2006; Lemaitre and Hoffmann, 2007). Recently, we identified two PGRPs (HaPGRP-B and -C) from H. armigera and investigated their roles in H. armigera immunity (Yang et al., 2013). In the present study, we identified another PGRP gene (HaPGRP-D) from H. armigera. HaPGRPD was predicted to have a putative signal peptide in the deduced amino acid sequence (Fig. 1); however, no predicted transmembrane domain was found in the mature protein sequence, indicating that this PGRP should belong to the short PGRP family (Dziarski and Gupta, 2006). Comparative analysis indicated that five amino acids that were required for T7 lysozyme Zn2+ binding and amidase activity (Cheng et al., 1994; Mellroth et al., 2003) are also conserved in HaPGRP-D (Fig. 1). This is a common feature of all amidase-type Archives of Insect Biochemistry and Physiology


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Figure 11. rHaPGRP-D directly bound to hemocytes. Hemocytes collected from H. armigera larvae were incubated with rHaPGRP-D or rTrx (as the control protein). Binding of recombinant proteins to hemocyte surface was detected using mouse anti-His monoclonal antibody and FITC (green) labeled goat anti-mouse IgG. DAPI (blue) was used to stain the nuclei. Gr, granulocyte; Pl, plasmatocyte. Scale bar = 10 μm.

PGRPs (Mellroth et al., 2003). These results suggest that HaPGRP-D is an amidase-type PGRP. Some amidase-type PGRPs such as Drosophila PGRP-SC1, PGRP-LB, and PGRP-SB1 and mouse and human PGRP-L have N-acetylmuramoyl-L-alanine amidase activity to degrade PGNs by hydrolyzing the amide bond that links peptide units to muramic acid residues of the glycan strands (Gelius et al., 2003; Mellroth et al., 2003; Kim et al., 2003; Wang et al., 2003; Mellroth and Steiner, 2006). In this study, amidase activity assay showed that rHaPGRP-D possessed a high-degrading activity toward PGNs (Fig. 7), confirming that HaPGRP-D also had amidase activity. In addition to amidase activity, some amidase-type PGRPs have antibacterial activity, such as Drosophila PGRP-SB1 (Mellroth and Steiner, 2006); the scallop Chlamys farreri and the amphioxus Branchiostoma japonicum PGRP-S (Yang et al., 2010; Yao et al., 2012); the teleost Sciaenops ocellatus, mouse, and human PGRP-L (Gelius et al., 2003; Wang et al., 2003; Li et al., 2012); and H. armigera PGRP-B and PGRP-C (Yang et al., 2013). These PGRPs may play role in innate immunity by acting directly as antibacterial factors (Mellroth and Steiner, 2006). In the present study, rHaPGRP-D strongly inhibited the growth of E. coli and S. aureus in the presence of Zn2+ (Fig. 8), suggesting that HaPGRP-D had antibacterial activity against bacteria. Whether HaPGRP-D has in vivo antibacterial activity during the immune response of H. armigera requires further study. Immune recognition, which distinguishes self from the potentially harmful nonself, is the premier and crucial step in innate immunity. The predominant PAMP recognition and binding abilities of PGRP make it an indispensable molecule in the first defense line against pathogen infection (Kurata, 2014). In the present study, the expression levels of HaPGRP-D mRNAs were significantly upregulated after H. armigera larvae were stimulated by E. coli or S. aureus (Fig. 3), indicating HaPGRP-D was involved in the immune response induced by these microbes. Binding and agglutination activity assays showed that rHaPGRP-D could bind and agglutinate E. coli and S. aureus (Figs. 5 and 6), indicating that HaPGRP-D could recognize bacteria and served as a PRR. There are two types of PGNs in nature: Lys-type PGNs from most Gram-positive bacteria and Dap-type PGNs from all Gram-negative bacteria and some Gram-positive Archives of Insect Biochemistry and Physiology

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bacteria (Dziarski, 2004). Some amidase-type PGRPs can only recognize and degrade one type of PGNs or have antibacterial activity against either Gram-negative or Grampositive bacteria. For example, Drosophila PGRP-LB specifically degrades PGNs from Gramnegative bacteria (Zaidman-Remy et al., 2006). B. ignite PGRP shows antibacterial activity against some Gram-positive bacteria (You et al., 2010). S. ocellatus PGRP is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria (Li et al., 2012). However, some amidase-type PGRPs have broad-spectrum activities against both Gramnegative and Gram-positive bacteria. For example, C. farreri PGRP shows agglutination and antibacterial activities against both Gram-negative E. coli and Gram-positive S. aureus (Yang et al., 2010). Our previous studies have shown that H. armigera PGRP-B and PGRP-C also show agglutination and antibacterial activities against both E. coli and S. aureus (Yang et al., 2013). In this study, HaPGRP-D showed broad-spectrum activities against bacteria. Challenges with both E. coli and S. aureus upregulated the expressions of HaPGRP-D mRNAs (Fig. 3); moreover, rHaPGRP-D showed binding, agglutination, and antibacterial activities against both E. coli and S. aureus (Figs. 5, 6, and 8). Presence of conserved Zn2+ -binding sites is the common feature of all amidase-type PGRPs (Mellroth et al., 2003), and the actions of many amidase-type PGRPs are zincdependent (Mellroth and Steiner, 2006; Li et al., 2012; Yao et al., 2012; Yang et al., 2013). The binding and agglutination activities of HaPGRP-D were zinc-dependent (Figs. 5 and 6). It is interesting to note that the antibacterial activity of HaPGRP-D toward S. aureus was zinc-dependent while those toward E. coli were not zinc-dependent because rHaPGRP-D strongly inhibited the growth of E. coli in the absence of Zn2+ (Fig. 8). Similar phenomena were also observed in other studies. For example, antibacterial activities of B. ignite PGRP were not zinc-dependent (You et al., 2010). Moreover, the antibacterial activities of H. armigera PGRP-B toward E. coli were not zinc-dependent (Yang et al., 2013). In addition to playing important roles in humoral immune responses, PGRPs function in cellular immune responses (Lemaitre and Hoffmann, 2007; Kurata, 2014). In Drosophila, PGRP-SC1a in the hemolymph may bind to bacteria and enhance phagocytosis of S. aureus as an opsonin through phagocytic receptors such as Eater (Garver et al., 2006). In vitro phagocytosis assay showed that incubation of the seastar Asterias rubens phagocytes with recombinant PGRP-S2a increased the phagocytosis of Micrococcus luteus cells, suggesting that this PGRP was also involved in phagocytosis (Coteur et al., 2007). In this study, we tested whether HaPGRP-D is involved in phagocytosis. Our results showed that rHaPGRPD significantly enhanced the phagocytic activity of H. armigera hemocytes against E. coli (Fig. 9), suggesting that HaPGRP-D may be involved in the phagocytosis response of H. armigera. The expression levels of HaPGRP-D mRNAs were significantly upregulated after H. armigera larvae were stimulated by chromatography beads, indicating that HaPGRP-D may be involved in the encapsulation response of H. armigera. In vitro encapsulation assay was conducted to test whether HaPGRP-D was involved in the encapsulation response of H. armigera. The results showed that incubation of H. armigera hemocytes with rHaPGRP-D enhanced the encapsulation of chromatography beads by hemocytes (Fig. 10), indicating that HaPGRP-D may be involved in the encapsulation response of H. armigera. To the best of our knowledge, this is the first study to demonstrate that PGRP can regulate encapsulation response in insects. Hemocytes play important roles in innate immune responses. The main defense responses mediated by hemocytes are phagocytosis, nodulation, and encapsulation (Lavine and Strand, 2002). Four main circulating hemocyte types called plasmatocytes, granulocytes, oenocytoids, and spherulocytes have been identified in all the lepidopteran species Archives of Insect Biochemistry and Physiology


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studied to date (Ribeiro and Brehélin, 2006). Normally, the hemocytes involved in encapsulation and phagocytosis responses are granulocytes and plasmatocytes (Schmidt et al., 2001; Lavine and Strand, 2002; Ribeiro and Brehélin, 2006). This study showed that HaPGRP-D participated in phagocytosis and encapsulation of H. armigera. To determine the possible mechanism by which HaPGRP-D regulated phagocytosis and encapsulation responses, we conducted an immunocytochemistry assay to test whether HaPGRP-D could bind to hemocytes of H. armigera. The results showed that HaPGRP-D could bind to the surface of plasmatocytes and granulocytes (Fig. 11), the two classes of hemocytes that mediate phagocytosis and encapsulation responses. Moreover, HaPGRP-D could bind to microbes (Fig. 5). In vitro encapsulation assay showed that HaPGRP-D could also bind to chromatography beads (Fig. 10). Taken together, our results suggest a possible involvement of HaPGRP-D in phagocytosis and encapsulation responses of H. armigera. After invasion by foreign objects, HaPGRP-D served as an opsonin to bind to both invaders and hemocytes, thus triggering phagocytosis or encapsulation response against the invaders. In conclusion, the HaPGRP-D gene was identified from H. armigera. Its expression in the hemocytes of H. armigera larvae is inducible upon bacterial or beads challenge. HaPGRP-D can bind and agglutinate E. coli and S. aureus and bind to the surface of hemocytes. HaPGRP-D possesses amidase and antibacterial activities. Moreover, HaPGRPD participates in phagocytosis and encapsulation responses of H. armigera.

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Identification and characterization of three chemosensory receptor families in the cotton bollworm Helicoverpa armigera.

Organophosphate and pyrethroid hydrolase activities of mutant Esterases from the cotton bollworm Helicoverpa armigera.

Molecular Characterization and Function Analysis of the Vitellogenin Receptor from the Cotton Bollworm, Helicoverpa armigera (Hübner) (Lepidoptera, Noctuidae).

Molecular cloning and mRNA expression of a ryanodine receptor gene in the cotton bollworm, Helicoverpa armigera.

NMR structure and function of Helicoverpa armigera sterol carrier protein-2, an important insecticidal target from the cotton bollworm.

High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections.

Acute, sublethal, and combination effects of azadirachtin and Bacillus thuringiensis on the cotton bollworm, Helicoverpa armigera.

Expression analysis of GSK-3β in diapause pupal brains in the cotton bollworm, Helicoverpa armigera.

Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera.

Histopathology of cotton bollworm midgut infected with Helicoverpa armigera cytoplasmic polyhedrosis virus.

Mitochondrial P-glycoprotein ATPase contributes to insecticide resistance in the cotton bollworm, Helicoverpa armigera (Noctuidae: Lepidoptera).

Efficacy of venom from tentacle of jellyfish Stomolophus meleagris (Nemopilema nomurai) against the cotton bollworm Helicoverpa armigera.

A comparison of the life-history traits between diapause and direct development individuals in the cotton bollworm, Helicoverpa armigera.

Distribution and Metabolism of Bt-Cry1Ac Toxin in Tissues and Organs of the Cotton Bollworm, Helicoverpa armigera.

Antibiotics influence the toxicity of the delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa armigera.

Identification of the 2-tridecanone responsive region in the promoter of cytochrome P450 CYP6B6 of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

Isomer-specific comparisons of the hydrolysis of synthetic pyrethroids and their fluorogenic analogues by esterases from the cotton bollworm Helicoverpa armigera.

Insecticidal and antifeedant activities of clerodane diterpenoids isolated from the Indian bhant tree, Clerodendron infortunatum, against the cotton bollworm, Helicoverpa armigera.

Influence of Dual-Bt Protein Corn on Bollworm, Helicoverpa zea (Boddie), Survivorship on Bollgard II Cotton.

Global metabolomic analyses of the hemolymph and brain during the initiation, maintenance, and termination of pupal diapause in the cotton bollworm, Helicoverpa armigera.

Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

Co-silence of the coatomer β and v-ATPase A genes by siRNA feeding reduces larval survival rate and weight gain of cotton bollworm, Helicoverpa armigera.

Cas9 system.

Fitness costs of reproductive capacity and ovarian development in a Bt-resistant strain of the cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).