Peptidoglycan Recognition Proteins Gene Research Articles

Identification and functional characterization of a long-type peptidoglycan recognition protein, PGRP-L in amphibian Xenopus laevis

Peptidoglycan recognition proteins (PGRPs) are a family of multifunctional proteins playing vital roles in PGN metabolism and antibacterial defense, and their functions have been well-characterized in mammals, bony fishes, and insects. However, the information about the functions of amphibian long-type PGRP is rather limited. Here, we identified and cloned a long-type PGRP gene (named Xl-PGRP-L) from African clawed frog, Xenopus laevis. Xl-PGRP-L gene was detected in all orangs/tissues examined, and was rapidly induced in intestine, liver, and lung following the stimulation of PGN. Sequence analysis showed that Xl-PGRP-L possesses four Zn2+-binding residues (His358, Tyr395, His470, and Cys478) required for amidase activity of catalytic PGRPs, and assays for amidase activity revealed that recombinant Xl-PGRP-L cloud degrade PGN in a Zn2+-dependent manner, indicating that Xl-PGRP-L is belonging to catalytic PGRPs. In addition, Xl-PGRP-L have antibacterial activity against Gram-negative bacteria Edwardsiella tarda and Gram-positive bacteria Streptococcus agalactiae. The present investigation represents the first characterization regarding the biological activities of amphibian long-type PGRPs, thus contributes to a better understanding of the functions of tetrapod PGRPs and the molecular mechanisms of amphibian antibacterial defense.

Peptidoglycan recognition protein MsPGRP in largemouth bass (Micropterus salmoides) mediates immune functions with broad nonself recognition ability

Peptidoglycan (PGN) recognition proteins (PGRPs) are important immune factors in innate immunity that function in recognising pathogens and activating the immune system. These ubiquitous proteins are conserved in invertebrates and vertebrates. In this study, a PGRP gene (MsPGRP) from largemouth bass (Micropterus salmoides) was identified and characterised, and its transcription distribution was explored. Recombinant protein (rMsPGRP) exhibited dose-dependent binding to PGN and glucan (GLU), but weak binding to lipopolysaccharide (LPS). MsPGRP exhibited agglutinating activity against several Gram-negative bacteria, Gram-positive bacteria and fungi, and it promoted phagocytosis activity of leukocytes against Micrococcus luteus and Aeromonas hydrophila. The protein also possessed amidase activity in the presence of Zn2+, degraded PGN, and disrupted the M. luteus cell wall. The results suggest that MsPGRP plays an important role in pathogen recognition, and acts as a opsonin during immune system responses and elimination of invading pathogens.

A short-type peptidoglycan recognition protein from Asian corn borer, Ostrinia furnacalis (Guenée) exhibits antibacterial activity, and regulates the expression of antimicrobial peptides

• OfPGRP8 exhibits the highest transcript level in fat body. • The mRNA level of OfPGRP8 significantly increases upon invading microbes. • OfPGRP8 exhibits intensive binding ability to peptidoglycans. • OfPGRP8 inhibits the growth of Staphylococcus aureus and Micrococcus luteus . • Knockdown of OfPGPR8 leads to a marked decrease in the expression of AMPs genes. Peptidoglycan recognition proteins (PGRPs) discriminate and bind peptidoglycans by acting as pattern recognition receptors (PRRs) in insects, and function pivotal roles in innate immune response. In the present study, we cloned a full-length PGRP gene designed as OfPGRP8 from the Asian corn borer, Ostrinia furnacalis (Guenée). Its mRNA exhibited the highest abundance in fat body, and its expression level upregulated dramatically after bacterial challenges. Purified recombinant OfPGRP8 exhibited intensive binding capacity to peptidoglycans from Staphylococcus aureus and Micrococcus luteus . Additionally, recombinant OfPGRP8 could inhibit the growth of S. aureus , M. luteus . However, recombinant OfPGRP8 could not cause agglutination of S. aureus , M. luteus or Escherichia coli . Furthermore, we also demonstrated that OfPGRP8 may be involved in modulating the signaling pathway of antimicrobial peptides (AMPs) synthesis. In sum, our results provided evidence that Of PGRP8 discriminates peptidoglycans from microbes and acts as a PRR to initiate downstream immune signaling pathways.

Molecular and Functional Characterization of a Short-Type Peptidoglycan Recognition Protein, Ct-PGRP-S1 in the Giant Triton Snail Charonia tritonis.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the potential function of PGRPs in the giant triton snail Charonia tritonis. In this study, a short-type PGRP gene (termed Ct-PGRP-S1) was identified in C. tritonis. Ct-PGRP-S1 was predicted to contain several structural features known in PGRPs, including a typical PGRP domain (Amidase_2) and Src homology-3 (SH3) domain. The Ct-PGRP-S1 gene was constitutively expressed in all tissues examined except in proboscis, with the highest expression level observed in the liver. As a typical PRR, Ct-PGRP-S1 has an ability to degrade peptidoglycan (PGN) and was proven to have non-Zn2+-dependent amidase activity and antibacterial activity against Vibrio alginolyticus and Staphylococcus aureus. It is the first report to reveal the peptidoglycan recognition protein in C. tritonis, and these results suggest that peptidoglycan recognition protein Ct-PGRP-S1 is an important effector of C. tritonis that modulates bacterial infection resistance of V. alginolyticus and S. aureus, and this study may provide crucial basic data for the understanding of an innate immunity system of C. tritonis.

Four peptidoglycan recognition proteins are indispensable for antibacterial immunity in the cigarette beetle Lasioderma serricorne (Fabricius)

The peptidoglycan recognition protein (PGRP), an important pattern recognition receptor of insects, is significant for reducing innate immunity and effective pest control. We cloned four PGRP genes (LsPGRP-LB, LsPGRP-LB1, LsPGRP-LE, and LsPGRP-SC2) from the cigarette beetle, Lasioderma serricorne (Fabricius), which encoded proteins of 216, 197, 317, and 190 amino acids, respectively. Three LsPGRPs were predominantly expressed in the larval and pupal stages, whereas LsPGRP-LE displayed high expression in adults. All the four LsPGRPs genes were highly expressed in the midgut and integument. Pathogen inoculation revealed that the four LsPGRPs actively responded to Escherichia coli and its peptidoglycan. The transcription levels of LsPGRP-LE and LsPGRP-SC2 increased significantly after Staphylococcus aureus stimulation. RNA interference-mediated knockdown of the four LsPGRPs led to increased larval mortality when challenged by E. coli, and the expression of four antimicrobial peptide genes (LsCole, LsAtt2, LsDef1 and LsDef2) had a significant decrease. Higher mortality and lower AMP expression were also observed in L. serricorne under S. aureus infection after silencing LsPGRP-LE and LsPGRP-SC2. Our results suggest that the four LsPGRP genes play important and distinct regulatory roles in the antibacterial defense response of L. serricorne.

Genome-wide identification and characteristic analysis of PGRP gene family in Tegillarca granosa reveals distinct immune response of the invasive pathogen

The peptidoglycan recognition proteins (PGRPs) are conserved innate immune molecular in invertebrates and vertebrates, which play important roles in immune system by recognize the peptidoglycans of bacterial cell walls. Although PGRPs have been extensively characterized in insects, a systematic analysis of PGRPs in bivalves is lacking. In the present study, the phylogenic relationships, gene structures and expression profiles of PGRPs in marine bivalves were analyzed. The results indicated that the most PGRPs of bivalves were predicted to degrade the peptidoglycans and prevent excessive immunostimulation of bacteria. In addition, the results of the present study showed that the protein diversity of PGRPs in most marine bivalves was mainly generated by the alternative splicing of genes, however the alternative splicing of PGRP gene family was absent in Tegillarca granosa. The differences of PGRPs might be related to the genetic and environmental differences of marine bivalves. Spatiotemporal expression profiling in T. granosa suggested that PGRPs play important roles in the immune response of invasive pathogens. The present study describes a comprehensive view of PGRPs in the blood clam T. granosa and provides a foundation for functional characterization of this gene family in innate immune of marine bivalves.

Peptidoglycan recognition protein-S1 (PGRP-S1) from Diaphania pyloalis (Walker) is involved in the agglutination and prophenoloxidase activation pathway.

Recognition of invading foreign exogenous pathogen is the first step to initiate the innate immune response of insects, which accomplished by the pattern recognition receptors (PRRs). Peptidoglycan recognition proteins (PGRPs) serve as an important type of PRRs, which activate immune response by detecting peptidoglycan of microbial cell wall. In this study, we have cloned the full-length cDNA of PGRP gene called PGRP-S1 from the Diaphania pyloalis (Walker). The open reading frame (ORF) of D. pyloalis PGRP-S1 encodes 211 amino acids which containing a secretion signal peptide and a canonical PGRP domain. Multisequence alignment revealed that PGRP-S1 possess the amino acid residues responsible for zinc binding and amidase activity. D. pyloalis PGRP-S1 exhibited the highest transcript level in fat body and followed in head. The mRNA concentration dramatically increased after an injection of Escherichia coli or Micrococcus luteus. Purified recombinant PGRP-S1 exhibit binding ability to peptidoglycans from Staphylococcus aureus or Bacillus subtilis and cause intensive agglutination of E. coli, M. luteus or S. aureus in the presence of zinc ions. Furthermore, phenoloxidase activity significantly increased when the plasma from larvae was incubated with recombinant PGPR-S1 and peptidoglycans from B. subtilis or M. luteus simultaneously. These results implied that PGRP-S1 was a member involving the prophenoloxidase activation pathway. Overall, our results indicated that D. pyloalis PGRP-S1 serve as a PRR to participate in the recognition of foreign pathogen and prophenoloxidase pathway stimulation.

A mini review on the short-type peptidoglycan recognition protein in Chinese soft-shelled turtle (Pelodiscus sinensis)

Peptidoglycan recognition proteins (PGRPs) function as the pattern recognition receptor involved in antibacterial innate immunity. Evidence have showed that the molecular structure and function of PGRPs was conserved in vertebrate. However, as the pivotal species in the evolution of vertebrates, reptiles are believed to be the first vertebrates that have escaped from the aquatic environment and are able to adapt to a variety of different terrestrial lives, few studies about the PGRPs in reptiles has been reported. The Chinese soft-shelled turtle, Pelodiscus sinensis, is an ancient, secondary aquatic reptile with high economic value and nutritional value in Asia, which occupies a unique position in the animal kingdom and has important research value. In the latest research, a PGRP gene which was classified into the member of short-type PGRP family was characterized in Pelodiscus sinensis. This paper presented the latest findings on the molecular structure, expression pattern and function feature of PGRP-S from Pelodiscus sinensis, aiming at revealing that PGRP in vertebrates is evolutionarily conserved.

Identification and functional characterization of a short-type peptidoglycan recognition protein, PGRP-S in the orange-spotted grouper, Epinephelus coioides

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) mediating the recognition of peptidoglycan. In the present study, a short-type PGRP gene named Eco-PGRP-S was identified in the orange-spotted grouper (Epinephelus coioides), a major commercial marine fish cultured in Asia. Eco-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in gill and skin. As a typical PRR, Eco-PGRP-S is inducible following the stimulation of peptidoglycan, and has an ability to bind peptidoglycan. Importantly, Eco-PGRP-S has been proven to have Zn2+-dependent amidase activity and direct antibacterial activity against Vibrio Harveri and Edwardsiella tarda. In addition, Eco-PGRP-S is mainly localized in cytoplasm and nucleus, and is able to significantly trigger the activation of NF-κB signalling. The present research thus contributes to a better understanding of the molecular mechanism of antibacterial defense and the containment of infectious bacterial diseases in grouper.

Rhodnius prolixus uses the peptidoglycan recognition receptor rpPGRP-LC/LA to detect Gram-negative bacteria and activate the IMD pathway.

Insects rely on an innate immune system to recognize and eliminate pathogens. Key components of this system are highly conserved across all invertebrates. To detect pathogens, insects use Pattern recognition receptors (PRRs) that bind to signature motifs on the surface of pathogens called Pathogen Associated Molecular Patterns (PAMPs). In general, insects use peptidoglycan recognition proteins (PGRPs) in the Immune Deficiency (IMD) pathway to detect Gram-negative bacteria, and other PGRPs and Gram-negative binding proteins (GNBPs) in the Toll pathway to detect Gram-positive bacteria and fungi, although there is crosstalk and cooperation between these and other pathways. Once pathogens are recognized, these pathways activate the production of potent antimicrobial peptides (AMPs). Most PRRs in insects have been reported from genome sequencing initiatives but few have been characterized functionally. The initial studies on insect PRRs were done using established dipteran model organisms such as Drosophila melanogaster, but there are differences in the numbers and functional role of PRRs in different insects. Here we describe the genomic repertoire of PGRPs in Rhodnius prolixus, a hemimetabolous hemipteran vector of the parasite Trypanosoma cruzi that causes Chagas disease in humans. Using a de novo transcriptome from the fat body of immune activated insects, we found 5 genes encoding PGRPs. Phylogenetic analysis groups R. prolixus PGRPs with D. melanogaster PGRP-LA, which is involved in the IMD pathway in the respiratory tract. A single R. prolixus PGRP gene encodes isoforms that contain an intracellular region or motif (cryptic RIP Homotypic Interaction Motif-cRHIM) that is involved in the IMD signaling pathway in D. melanogaster. We characterized and silenced this gene using RNAi and show that the PGRPs that contain cRHIMs are involved in the recognition of Gram-negative bacteria, and activation of the IMD pathway in the fat body of R. prolixus, similar to the PGRP-LC of D. melanogaster. This is the first functional characterization of a PGRP containing a cRHIM motif that serves to activate the IMD pathway in a hemimetabolous insect.

Molecular and functional identification of a short-type peptidoglycan recognition protein, PGRP-S, in the Chinese soft-shelled turtle Pelodiscus sinensis

Peptidoglycan recognition proteins (PGRPs), which are discovered in invertebrates and vertebrates, play an important role in antibacterial immunity. However, the function of PGRPs is largely uninvestigated in reptiles. In the present study, a short-type PGRP gene, designed as C-turtle-PGRP-S, was identified in the Chinese soft-shelled turtle, Pelodiscus sinensis. The C-turtle-PGRP-S contains a highly conserved PGRP domain and has close relationship with PGRP-S orthologues in other species according to sequence and phylogenetic analyses. C-turtle-PGRP-S gene was constitutively expressed in all detected tissues and was induced by Edwardsiella tarda. Additionally, recombinant C-turtle-PGRP-S showed PGN binding activity and antibacterial function against E. tarda. Therefore, it is suggested that the function of PGRP-S is likely to be conserved in reptile vertebrates, as observed in other vertebrates, shedding light on the evolutionary conservation of PGRPs.

Inhibition of Chlamydial Infection by CRISPR/Cas9-SAM Mediated Enhancement of Human Peptidoglycan Recognition Proteins Gene Expression in HeLa Cells.

The global problem of emerging resistance of microorganisms to antibiotics makes the search for new natural substances with antibacterial properties relevant. Such substances include peptidoglycan recognition proteins (PGLYRP), which are the components of the innate immunity of many organisms, including humans. These proteins have a unique mechanism of action that allows them to evade the resistance of bacteria to them, as well as to be active against both Gram-positive and Gram-negative bacteria. However, the use of antimicrobial recombinant proteins is not always advisable due to the complexity of local delivery of the proteins and their stability; in this regard it seems appropriate to activate the components of the innate immunity. The aim of this study was to increase the expression level of native peptidoglycan recognition protein genes in HeLa cells using genome-editing technology with synergistic activation mediators (CRISPR/Cas9-SAM) and evaluate antichlamydial effect of PGLYRP. We demonstrated activation of the chlamydial two-component gene system (ctcB-ctcC), which played a key role in the mechanism of action of the peptidoglycan recognition proteins. We generated the HeLa cell line transduced with lentiviruses encoding CRISPR/Cas9-SAM activation system with increased PGLYRP gene expression. It was shown that activation of the own peptidoglycan recognition proteins gene expression in the cell line caused inhibition of the chlamydial infection development. The proposed approach makes it possible to use the capabilities of innate immunity to combat infectious diseases caused by Gram-positive and Gram-negative bacteria.

Genome-Wide Analysis of Peptidoglycan Recognition Protein Genes in Fig Wasps (Hymenoptera, Chalcidoidea).

Simple SummaryInsects live in a complex and diverse environment, threatened by a variety of microorganisms, and the innate immunity of which plays an important role in defending the invasion of pathogens. From an evolutionary perspective, different living environments and lifestyles drive the different evolutionary patterns of immune systems of insects. Fig wasps are closely associated with the fig syconia, divided into pollinators and non-pollinators according to whether they pollinate the figs. The pollinators are all herbivorous, and fulfil their development within the fig syconia, presenting different lifestyles and diets to non-pollinators, which lead to the chances of exposure to the pathogens varying greatly. The recognition of pathogens is the first step in innate immunity. Therefore, we focused on the different evolutionary patterns of peptidoglycan recognition protein genes between pollinators and non-pollinators, and found that the number of peptidoglycan recognition protein genes was significantly smaller than that of non-pollinators, and the initiation of Toll pathway of pollinators was simpler than that of non-pollinators. All the results suggested a streamlined innate immune recognition system of pollinators, and this information will provide more insights into the adaptive evolution of innate immunity in insects of host specificity.The innate immunity is the most important defense against pathogen of insects, and the peptidoglycan recognition proteins (PGRPs) play an important role in the processes of immune recognition and initiation of Toll, IMD and other signal pathways. In fig wasps, pollinators and non-pollinators present different evolutionary histories and lifestyles, even though both are closely associated with fig syconia, which may indicate their different patterns in the evolution of PGRPs. By manual annotation, we got all the PGRP genes of 12 fig wasp species, containing seven pollinators and five non-pollinators, and investigated their putative different evolutionary patterns. We found that the number of PGRP genes in pollinators was significantly lower than in non-pollinators, and the number of catalytic PGRP presented a declining trend in pollinators. More importantly, PGRP-SA is associated with initiating the Toll pathway, as well as gram-negative bacteria-binding proteins (GNBPs), which were completely lost in pollinators, which led us to speculate that the initiation of Toll pathway was simpler in pollinators than in non-pollinators. We concluded that fig pollinators owned a more streamlined innate immune recognition system than non-pollinators. Our results provide molecular evidence for the adaptive evolution of innate immunity in insects of host specificity.

Gene identification and functional analysis of peptidoglycan recognition protein from the spotted sea bass (Lateolabrax maculatus)

Peptidoglycan recognition proteins (PGRPs), which are structurally conserved innate immune molecules in invertebrate and vertebrate animals, play the important roles in regulation of innate immune responses. In this paper, three PGRP genes of spotted sea bass, Lateolabrax maculatus, were cloned, designated as Ssb-PGRP2, Ssb-PGRP-L2 and Ssb-PGRP-SC2, respectively. Sequence analysis showed that the deduced amino acid sequences of Ssb-PGRP2, Ssb-PGRP-L2 and Ssb-PGRP-SC2 proteins contained respectively 468, 482 and 167 amino acid residues, and had the typical structural features of PGRPs, i.e. conserved PGRP domain and Zn2+ binding domain including four specific amino acid residues which were required for amidase activity. q-PCR analysis of total mRNA showed that the mRNA expression of three PGRP genes were detected in all the examined tissues and the expression patterns of Ssb-PGRP2, Ssb-PGRP-L2 and Ssb-PGRP-SC2 were different. After injected with LPS, Poly (I:C) and Edwardsiella tarda, there was a clear time-dependent expression pattern for each of the three PGRP genes in head kidney, spleen, intestine and gill of the spotted sea bass. In our study, three recombinant proteins corresponding to the three members of the peptidoglycan recognition protein family were expressed and purified. Moreover, all of the three recombinant PGRP proteins significantly inhibited bacterial survival and growth, and expressed bactericidal effects on Vibrio harveyi, Staphylococcus aureus and Edwardsiella tarda. In particular, it was firstly verified that their antimicrobial activity presented the superimposed effect. Overall, these findings indicated that three PGRP genes of spotted sea bass were at least involved in host defense against bacterial infections.

Numerous peptidoglycan recognition protein genes expressed in the bacteriome of the green rice leafhopper Nephotettix cincticeps (Hemiptera, Cicadellidae)

Peptidoglycan recognition proteins (PGRPs) contribute to the immune response of vertebrates and higher invertebrates. Animals have a varying number of PGRP genes: 4 in human and mouse, 13 in Drosophila, and 0 in an aphid. Transcriptome analyses revealed that numerous PGRP genes (> 300 in an RNA-Seq analysis) were expressed in the green rice leafhopper Nephotettix cincticeps (Uhler) (Hemiptera, Cicadellidae). PGRP genes were mostly expressed in the bacteriomes, in which two bacterial endosymbionts, Sulcia and Nasuia, are harbored. E. coli inoculation upregulated antimicrobial peptide gene expression in the leafhopper, whereas PGRP gene expression was not affected by the bacterial challenge. High and constant expression of PGRP genes in the bacteriomes and the lack of an immune response against invading bacteria suggest that these PGRPs play a hitherto unknown role in the bacteriomes, implicating a relationship with symbiotic association in N. cincticeps.

Identification of cells expressing two peptidoglycan recognition proteins in the gill of the vent mussel, Bathymodiolus septemdierum

In symbiotic systems in which symbionts are transmitted horizontally, hosts must accept symbionts from the environment while defending themselves against invading pathogenic microorganisms. How they distinguish pathogens from symbionts and how the latter evade host immune defences are not clearly understood. Recognition of foreign materials is one of the most critical steps in stimulating immune responses, and pattern recognition receptors (PRRs) play vital roles in this process. In this study, we focused on a group of highly conserved PRRs, peptidoglycan recognition proteins (PGRPs), in the deep-sea mussel, Bathymodiolus septemdierum, which harbours chemosynthetic bacteria in their gill epithelial cells. We isolated B. septemdierum PGRP genes BsPGRP-S and BsPGRP-L, which encode a short- and a long-type PGRP, respectively. The short-type PGRP has a signal peptide and was expressed in the asymbiotic goblet mucous cells in the gill epithelium, whereas the long-type PGRP was predicted to include a transmembrane domain and was expressed in gill bacteriocytes. Based on these findings, we hypothesize that the secreted and transmembrane PGRPs are engaged in host defence against pathogenic bacteria and/or in the regulation of symbiosis via different cellular localizations and mechanisms.

Characterization of immune-related PGRP gene expression and phenoloxidase activity in Cry1Ac-susceptible and -resistant Plutella xylostella (L.)

Peptidoglycan recognition proteins (PGRPs) are important recognition receptors which play a critical role in signal identification and transmission in Toll or immune deficiency (IMD) pathways, particularly when pathogens evade and circumvent reactive oxygen species. Antimicrobial peptides (AMPs) synthesis can be activated by these signals to further eliminate pathogens. In this study, we cloned and characterized three different PGRP genes in Plutella xylostella strains, DBM1Ac-S, DBM1Ac-R and a field strain (DBMF). The results showed that PGRP1 belongs to the PGRP-SA family, PGRP2 to PGRP-LB, and PGRP3 to PGRP-LF. Moreover, PGRP1 expressed the highest transcript level, followed by PGRP3 and PGRP2, in two tissues including the gut and the larval carcass tissues of the DBM1Ac-S strain. Furthermore, altered expression levels of PGRP1-3 genes were detected in both gut and carcass tissues. Moreover, the DBM1Ac-R strain had the highest phenol oxidase (PO) activity among these three strains. The characterization of PGRP gene expression and PO activity in DBM1Ac-S, DBM1Ac-R and DBM-F provides insights into their important physiological roles in the immune system of P. xylostella exposed to Bt Cry1Ac toxin.

Molecular and functional characterization of a short-type peptidoglycan recognition protein, PGRP-S in the amphibian Xenopus laevis

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the function of frog PGRPs. In this study, a short-type PGRP (termed Xl-PGRP-S) gene was identified in the African clawed frog, Xenopus laevis. The predicted protein of Xl-PGRP-S contains several structural features known in PGRPs, including a typical PGRP domain and two closely spaced conserved cysteines. Xl-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in muscle. As a typical PRR, Xl-PGRP-S is inducible after peptidoglycan (PGN) stimulation, and has an ability to bind PGN. In addition, Xl-PGRP-S has been proven to have Zn2+-dependent amidase activity and antibacterial activity against Edwardsiella tarda. The present study represents the first discovery on the function of frog PGRPs, thus contributing to a better understanding of the functional evolution of PGRPs in early tetrapods.

Molecular and functional characterization of ApPGRP from Anatolica polita in the immune response to Escherichia coli

Peptidoglycan recognition proteins (PGRPs) widely distributed in invertebrates and vertebrates are pattern-recognition molecules in innate immunity. In the present study, a novel short PGRP gene, designated as ApPGRP was identified from Anatolica polita. The deduced amino acid sequence of ApPGRP is composed of 196 residues and contains a conserved PGRP domain at the C-terminus. The phylogenetic tree showed that ApPGRP shared high homology of amino acid sequence with TcPGRP2 from Tribolium castaneum. The recombinant protein of ApPGRP exhibited binding activity toward Escherichia coli and DAP-type PGN from E. coli. Quantitative real time PCR (RT-qPCR) analysis indicated that the relative expression level of ApPGRP was up-regulated significantly after E. coli and DAP-type PGN challenge in the fifth instar larvae of A. polita. Moreover, the expressions of three antimicrobial peptides (AMPs) (ApAttacin 1, ApAttacin 2 and ApColeoptericin) were significantly increased after E. coli and DAP-type PGN challenge. RNA interference (RNAi) experiments showed that the expressions of three AMPs in the larvae of A. polita were significantly decreased after injection of ApPGRP dsRNA. Furthermore, the expressions of three AMPs in the larvae injected were still significantly decreased after E. coli challenge compared to the control samples without dsRNA injection. The predicted 3D structure showed that the ApPGRP could form the protein core of five β-sheet and three ɑ-helices, which would be involved in specific PGN recognition. These results suggested that ApPGRP may play an important role in the immune response to E. coli infection and function as a receptor for antimicrobial peptide gene induction in Anatolica polita.

A novel peptidoglycan recognition protein involved in the prophenoloxidase activation system and antimicrobial peptide production in Antheraea pernyi

Pattern recognition receptors (PRRs) are employed in insects to defend against infectious pathogens by triggering various immune responses. Peptidoglycan recognition proteins (PGRPs), a vital family of PRRs, are widely distributed and highly conserved from vertebrates to invertebrates. To date, five PGRP genes have been identified in Antheraea pernyi, but their biochemical roles still remain unknown. In this study, we focused on the immune functions of PGRP-SA in A. pernyi (ApPGRP-SA), which was confirmed to be immune-related according to its significantly up-regulated expression level post microbial injection. In addition, the binding properties of ApPGRP-SA were investigated using a recombinant protein produced in a prokaryotic expression system, revealing that rApPGRP-SA displayed a multi-binding ability to various microbes, including the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and fungus Candida albicans, together with their surface pathogen associated molecular patterns (PAMPs). Further studies showed that after recognition, the mixture of rApPGRP-SA/PAMP remarkably stimulated prophenoloxidase (PPO) activation in the hemolymph of A. pernyi in vitro, while the ds-PGRP-SA-treated hemolymph exhibited a lower sensitivity to PAMPs in comparison to the native sample. Moreover, the transcriptional level of the three antimicrobial peptides was also decreased in PGRP-SA knock-down larvae in response to immune-challenge. In summary, we conclude that ApPGRP-SA is a novel identified PGRP in A. pernyi that might act as a broad-spectrum pattern recognition receptor and is involved in the PPO activation system as well as antimicrobial peptide production.