Microbiota-Derived Lactate Activates Production of Reactive Oxygen Species by the Intestinal NADPH Oxidase Nox and Shortens Drosophila Lifespan.

Commensal microbes colonize the gut epithelia of virtually all animals and provide several benefits to their hosts. Changes in commensal populations can lead to dysbiosis, which is associated with numerous pathologies and decreased lifespan. Peptidoglycan recognition proteins (PGRPs) are important regulators of the commensal microbiota and intestinal homeostasis. Here, we found that a null mutation in Drosophila PGRP-SD was associated with overgrowth of Lactobacillus plantarum in the fly gut and a shortened lifespan. L. plantarum-derived lactic acid triggered the activation of the intestinal NADPH oxidase Nox and the generation of reactive oxygen species (ROS). In turn, ROS production promoted intestinal damage, increased proliferation of intestinal stem cells, and dysplasia. Nox-mediated ROS production required lactate oxidation by the host intestinal lactate dehydrogenase, revealing a host-commensal metabolic crosstalk that is probably broadly conserved. Our findings outline a mechanism whereby host immune dysfunction leads to commensal dysbiosis that in turn promotes age-related pathologies.

A novel short-type peptidoglycan recognition protein with unique polysaccharide recognition specificity in sea cucumber, Apostichopus japonicus.

Pattern recognition receptors (PRRs) are the first line of immune defense in invertebrates against pathogen infection; they recognize pathogens and transmit signals to downstream immune pathways. Among these, peptidoglycan recognition proteins (PGRPs) are an important family in invertebrates that generally comprise of complicated isoforms. A comprehensive understanding of PGRPs in evolutionarily and economically important marine invertebrates, such as the sea cucumber, Apostichopus japonicus, is crucial. Previous studies have identified two PGRPs in sea cucumber, AjPGRP-S and AjPGRP-S1, and another novel short-type PGRP, AjPGRP-S3, was additionally identified here. The full-length cDNA sequence of AjPGRP-S3 was obtained here by PCR-RACE, followed by which showed its gene expression analyses by in situ hybridization that showed it to be relatively highly expressed in coelomocytes and tube feet. Based on an analysis of the recombinant protein, rAjPGRP-S3, a board-spectrum pathogen recognition ability was noted that covered diverse Gram-negative and -positive bacteria, and fungi. Moreover, according to the results of yeast two-hybridization, it was suggested that rAJPGRP-S3 interacted with multiple immune-related factors, including proteins involved in the complement system, extracellular matrix, vesicle trafficking, and antioxidant system. These findings prove the important functions of AjPGRP-S3 in the transduction of pathogen signals to downstream immune effectors and help explore the functional differences in the AjPGRP isoforms.

Molecular characterization of peptidoglycan recognition proteins from Mytilus coruscus.

Mytilus shows great immune resistance to various bacteria from the living waters, indicating a complex immune recognition mechanism against various microbes. Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Therefore, eight PGRPs were identified from the gill transcriptome of Mytilus coruscus. The sequence features, expression pattern in various organs and larval development stages, and microbes induced expression profiles of these Mytilus PGRPs were determined. Our data revealed the constitutive expression of PGRPs in various organs with relative higher expression level in immune-related organs. The expression of PGRPs is developmentally regulated, and most PGRPs are undetectable in larvae stages. The expression level of most PGRPs was significantly increased with in vivo microbial challenges, showing strong response to Gram-positive strain in gill and digestive gland, strong response to Gram-negative strain in hemocytes, and relative weaker response to fungus in the three tested organs. In addition, the function analysis of the representative recombinant expressed PGRP (rMcPGRP-2) confirmed the antimicrobial and agglutination activities, showing the immune-related importance of PGRP in Mytilus. Our work suggests that Mytilus PGRPs can act as pattern recognition receptors to recognize the invading microorganisms and the antimicrobial effectors during the innate immune response of Mytilus.

Peptidoglycan recognition protein 6 (PGRP6) from Asian corn borer, Ostrinia furnacalis (Guenée) serve as a pattern recognition receptor in innate immune response.

Peptidoglycan recognition proteins (PGRPs) recognize invading microbes via detecting peptidoglycans from microbial cell walls. PGRPs are highly conserved from insects to vertebrates and all play roles during the immune defensive response. Ten putative PGRPs have been identified through transcriptome analysis in the Asian corn borer, Ostrinia furnacalis (Guenée). Whereas, the biochemical functions of most of them have not yet been elucidated. In this study, we found PGRP6 messenger RNA exhibited extremely high expression levels in the midgut, and its transcript level increased dramatically upon bacterial infection. Moreover, the enzyme-linked immunosorbent assay indicated recombinant PGRP6 exhibited a strong binding affinity to peptidoglycans from Micrococcus luteus and Bacillus subtilis, which could agglutinate M. luteus and yeast Pichia pastoris. Additionally, we demonstrated that PGRP6 was involved in the pathway of antimicrobial peptides synthesis, but could not enhance encapsulation and melanization of hemocytes. Overall, our results indicated that O. furnacalis PGRP6 serves as a pattern recognition receptor and detects peptidoglycans from microbes to initiate the immune response.

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 Vibrioalginolyticus 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.

A role of peptidoglycan recognition protein in mediating insecticide detoxification in Glyphodes pyloalis.

Glyphodes pyloalis Walker has become one of the most significant mulberry pests, and it has caused serious economic losses in major mulberry growing regions in China. Peptidoglycan recognition proteins (PGRPs) are responsible for initiating and regulating immune signalling pathways in insects. However, their roles responding to chemical pesticides is still less known. This study aimed to investigate the possible detoxication function of GpPGRP-S2 and GpPGRP-S3 in G. pyloalis in response to chlorfenapyr and phoxim. The chlorfenapyr and phoxim treatment significantly induced the expression level of GpPGRP-S3 at 48 h. In addition, the expression levels of GpPGRP-S2 and GpPGRP-S3 in the chlorfenapyr/phoxim treatment group were significantly higher in midgut than those in the control group at 48 h. The results of the survival experiment showed that silencing either GpPGRP-S2 or GpPGRP-S3 would not influence the survival rate of G. pyloalis which treated with phoxim, however, silencing GpPGRP-S2 or GpPGRP-S3 would cause G. pyloalis to be more easily killed by chlorfenapyr. The expression of carboxylesterase GpCXE1 was significantly induced by chlorfenapyr/phoxim treatment, while it was suppressed once silenced GpPGRP-S2 followed with chlorfenapyr treatment or silenced GpPGRP-S3 followed with phoxim treatment. These results might suggest that under the chlorfenapyr/phoxim treatment condition, the connection between GpPGRPs and detoxification genes in insect was induced to maintain physiological homeostasis; and these results may further enrich the mechanisms of insects challenged by insecticides.

PGRP-LB: An Inside View into the Mechanism of the Amidase Reaction.

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.

RETRACTED: Dietary oligosaccharides attenuate DSS-induced colitis in mice, induce PGlyRP3 expression, and inhibit NF-κB and MEK/ERK signaling.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editors. The Editors of Cellular Immunology have been informed by Elsevier that the article had been submitted to another journal while under consideration at "Cellular Immunology", which is a case of double submission. Based on the above infringement and its deleterious impact on the mutual trust necessary for the evaluation of scientific work - the corresponding authors had stated that the article was not submitted to another journal - it was decided to retract this article.

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.

Transcriptome analysis of air-breathing land slug, Incilaria fruhstorferi reveals functional insights into growth, immunity, and reproduction.

BACKGROUND: Incilaria (= Meghimatium) fruhstorferi is an air-breathing land slug found in restricted habitats of Japan, Taiwan and selected provinces of South Korea (Jeju, Chuncheon, Busan, and Deokjeokdo). The species is on a decline due to depletion of forest cover, predation by natural enemies, and collection. To facilitate the conservation of the species, it is important to decide on a number of traits related to growth, immunity and reproduction addressing fitness advantage of the species. RESULTS: The visceral mass transcriptome of I. fruhstorferi was enabled using the Illumina HiSeq 4000 sequencing platform. According to BUSCO (Benchmarking Universal Single-Copy Orthologs) method, the transcriptome was considered complete with 91.8% of ortholog genes present (Single: 70.7%; Duplicated: 21.1%). A total of 96.79% of the raw read sequences were processed as clean reads. TransDecoder identified 197,271 contigs that contained candidate-coding regions. Of a total of 50,230 unigenes, 34,470 (68.62% of the total unigenes) annotated to homologous proteins in the Protostome database (PANM-DB). The GO term and KEGG pathway analysis indicated genes involved in metabolism, phosphatidylinositol signalling system, aminobenzoate degradation, and T-cell receptor signalling pathway. Many genes associated with molluscan innate immunity were categorized under pathogen recognition receptor, TLR signalling pathway, MyD88 dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related. The reproduction-associated unigenes showed homology to protein fem-1, spermatogenesis-associated protein, sperm associated antigen, and testis expressed sequences, among others. In addition, we identified key growth-related genes categorized under somatotrophic axis, muscle growth, chitinases and collagens. A total of 4822 Simple Sequence Repeats (SSRs) were also identified from the unigene sequences of I. fruhstorferi. CONCLUSIONS: This is the first available genomic information for non-model land slug, I. fruhstorferi focusing on genes related to growth, immunity, and reproduction, with additional focus on microsatellites and repeating elements. The transcriptome provides access to greater number of traits of unknown relevance in the species that could be exploited for in-depth analyses of evolutionary plasticity and making informed choices during conservation planning. This would be appropriate for understanding the dynamics of the species on a priority basis considering the ecological, health, and social benefits.

The peptidoglycan recognition protein PGRP-LE regulates the Drosophila immune response against the pathogen Photorhabdus.

Photorhabdus bacteria are potent pathogens of insects and humans. To elucidate the infection strategies Photorhabdus employs to subvert the host innate immune response, it is critical to use model organisms that permit the genetic dissection of the dynamics involved in host-pathogen interactions. Here, we employed the fruit fly Drosophila melanogaster to interrogate the role of the immune deficiency (Imd) pathway receptor peptidoglycan recognition protein LE (PGRP-LE) in the regulation of the fly's response to the insect pathogen Photorhabdus luminescens and the insect/human pathogen P. asymbiotica. We show that PGRP-LE is upregulated in response to injection of Photorhabdus bacteria in background control flies, and that loss-of-function PGRP-LE mutant flies are more sensitive specifically to P. luminescens infection and harbor a higher bacterial burden of this species compared to background controls. Also, our results indicate that the absence of functional PGRP-LE alters the transcriptional pathway activity of Imd and Jnk signaling upon infection with P. asymbiotica, while infection with P. luminescens modifies the activity of Jak/Stat signaling. These findings denote the participation of the PGRP-LE receptor in the response of D. melanogaster to Photorhabdus challenge and contribute to a better understanding of pathogen detection and host immune regulation against virulent microbial invaders.

A peptidoglycan recognition protein involved in immune recognition and immune defenses in Ruditapes philippinarum.

Peptidoglycan recognition proteins (PGRPs) are important pattern recognition receptors in the innate immune system of invertebrates. In the study, a short PGRP (designed as RpPGRP) was identified and characterized from the manila clam Ruditapes philippinarum. The open reading frame of RpPGRP encoded a polypeptide of 249-amino acids with a calculated molecular mass of 27.2 kDa and an isoelectric point of 6.62. Multiple alignments and phylogenetic analysis strongly suggested that RpPGRP was a new member of the PGRP superfamily. In non-stimulated clams, RpPGRP exhibited different tissue expression pattern, and highly expressed in hepatopancreas and hemocytes. Expression of RpPGRP transcripts was significantly up-regulated in hemocytes of clams post Vibrio anguillarum or Micrococcus luteus challenge. The recombinant RpPGRP (rRpPGRP) exhibited high affinity to PGN, LPS and zymosan in a concentration-dependent manner. With a broad spectrum of bacterial binding activities, rRpPGRP exhibited strong agglutination activity to Escherichia coli, Vibrio splendidus, V. anguillarum and M. luteus. Furthermore, rRpPGRP exhibited Zn2+-dependent amidase activity and catalyzed the degradation of insoluble PGN. Especially, rRpPGRP exhibited significant antibacterial activity against E. coli and M. luteus. Moreover, the biofilm formation of E. coli could be inhibited after rRpPGRP incubation in the presence of Zn2+. This inhibitory effect of rRpPGRP might attribute to its amide bactericidal activity. Taken together, rRpPGRP played important roles in PGRP-mediated immune defense mechanisms, especially by recognizing antigens and eliminating bacteria.

Molecular cloning and functional characterization of a short peptidoglycan recognition protein from triangle-shell pearl mussel (Hyriopsis cumingii).

Peptidoglycan (PGN) is an important target of recognition in invertebrate innate immunity. PGN recognition proteins (PGRPs) are responsible for PGN recognition. In this study, we cloned and functionally analyzed a short PGRP (HcPGRP2) from the triangle-shell pearl mussel Hyriopsis cumingii. The full-length cDNA sequence of HcPGRP2 gene was 1185 bp containing an open reading frame of 882 bp encoding a 293 amino acid protein. HcPGRP2 was predicted to have two SH3b domains and a conserved C-terminal PGRP domain. Quantitative real-time RT-PCR showed that HcPGRP2 was expressed in all examined tissues and its expression was induced most significantly by Staphylococcus aureus and Vibrio parahaemolyticus in the hepatopancreas and gills. RNA interference by siRNA results revealed that HcPGRP2 was involved in the regulation of whey acidic protein, theromacin, and defensin expression. As a pattern-recognition receptor, recombinant HcPGRP2 (rHcPGRP2) protein can bind and agglutinate (Ca2+ dependent) all tested bacteria. rHcPGRP2 exhibited specific binding to PGN but not to lipopolysaccharide. Moreover, rHcPGRP2 inhibited the growth activities of S. aureus and V. parahaemolyticus in vitro and accelerated the clearance of V. parahaemolyticus in vivo. Overall, our results indicated that HcPGRP2 may play an important role in the antibacterial immune mechanisms of H. cumingii.

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 gene expression profiles and functional analysis of peptidoglycan recognition protein 2 from rock bream (Oplegnathus fasciatus).

Peptidoglycan recognition protein 2 (PGRP2) is a Zn2+-dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6-53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida, Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn2+. These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection.

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.

Regulation of the Peptidoglycan Amidase PGLYRP2 in Epithelial Cells by Interleukin-36γ.

Interleukin-36 (IL-36) cytokines are important regulators of mucosal homeostasis and inflammation. We have previously established that oral epithelial cells upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis Here, we have established that IL-36γ can stimulate the gene expression of mechanistically distinct antimicrobial proteins, including the peptidoglycan amidase PGLYRP2, in oral epithelial cells (e.g., TIGK cells). PGLYRP2 gene expression was not stimulated by either IL-17 or IL-22, thus demonstrating selectivity in the regulation of PGLYRP2 by IL-36γ. The IL-36γ-inducible expression of PGLYRP2 was shown to be mediated by IRAK1- and p38 mitogen-activated protein (MAP) kinase-dependent signaling. Furthermore, our finding that IL-36γ-inducible PGLYRP2 expression was reduced in proliferating TIGK cells but increased in terminally differentiating cells suggests that control of PGLYRP2 expression is associated with the maturation of the oral epithelium. PGLYRP2 expression in TIGK cells can also be directly stimulated by oral bacteria. However, the extracellular gingipain proteases (Kgp and RgpA/B) produced by P. gingivalis, which are critical virulence factors, can antagonize PGLYRP2 expression. Thus, the expression of IL-36γ by oral epithelial cells in response to P. gingivalis might enable the subsequent autocrine stimulation of PGLYRP2 expression. In summary, our data identify how IL-36γ may promote oral mucosal homeostasis by regulating PGLYRP2 expression.

Peptidoglycan recognition protein of Solen grandis (SgPGRP-S1) mediates immune recognition and bacteria clearance.

Peptidoglycan recognition proteins (PGRPs) are indispensable molecules in innate immunity due to their prominent function in sensing and eliminating invading microorganisms. In the present study, a short type PGRP from razor clam Solen grandis (SgPGRP-S1) was recombinantly expressed and purified to investigate its potential function in innate immunity. As a pattern recognition receptor, recombinant SgPGRP-S1 (rSgPGRP-S1) specifically bind Lys-type and Dap-type peptidoglycan in vitro, but not lipopolysaccharide or ß-glucan. The peptidoglycan binding ability of rSgPGRP-S1 resulted in significant agglutination activity against Gram-negative Escherichia coli and Listonella anguillarum, as well as Gram-positive Micrococcus luteus. Furthermore, rSgPGRP-S1 was bactericidal, significantly suppressing the growth of both E. coli and Gram-positive Staphylococcus aureus. The protein also exhibited strong amidase activity and degraded bacterial peptidoglycan in the presence of Zn2+, suggesting amidase activity might contribute to SgPGRP-S1 antibacterial activity. These results indicate SgPGRP-S1 is multifunctional in innate immunity, mediating both immune recognition and bacteria elimination.

Molecular characterization, expression and functional analysis of peptidoglycan recognition protein-SC2 from rock bream, Oplegnathus fasciatus.

Peptidoglycan recognition proteins are members of the family of pattern recognition receptors (PRRs), that play important roles in the recognition of peptidoglycan and various biological processes. In this study, we have characterized peptidoglycan recognition protein-SC2 (PGRP-SC2) in rock bream (Oplegnathus fasciatus) (RbPGRP-SC2) and analysed its expression in various tissues after pathogen challenge. A sequence alignment revealed that the residues essential to zinc binding of the deduced protein were highly conserved among all the organisms. Phylogenetic analysis revealed that RbPGRP-SC2 is most closely related to the large yellow croaker PGRP-SC2. RbPGRP-SC2 was ubiquitously expressed in all tissues analysed, predominantly distributed in muscle and skin. After challenge with microbial pathogens (Edwardsiella piscicida), Streptococcus iniae or red seabream iridovirus [RSIV]), RbPGRP-SC2 was up-regulated in all the tissues examined, especially in liver. We produced recombinant RbPGRP-SC2 (rRbPGRP-SC2) using an Escherichia coli expression system. The rRbPGRP-SC2 had agglutination activity towards both Gram-negative (E. piscicida) and Gram-positive bacteria (S. iniae). In addition, rRbPGRP-SC2 induced leukocyte apoptosis and promoted leukocyte phagocytosis. These results suggest that the RbPGRP-SC2 plays an important role in the immune system and in maintaining cellular homeostasis of rock bream.

Characterization and expression analysis of a peptidoglycan recognition protein gene, SmPGRP2 in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

Peptidoglycan recognition receptor proteins (PGRPs), a group of pattern recognition receptors (PRRs), can recognize peptidoglycan (PGN) of the bacteria cell wall and play an important role in host immune defense against pathogen infection. They are highly structurally conserved through evolution, but with different function in innate immunity between invertebrates and vertebrates. In teleost fish, several PGRPs have been characterized recently. They have both amidase activity and bactericidal activity and are involved in indirectly killing bacteria and regulating multiple signaling pathways. However, the knowledge of PGRPs in mucosal immunity of teleost fish is still limited. In this study, we identified a PGRPs gene (SmPGRP2) of turbot and investigated its expression patterns in mucosal tissues after challenge with Gram-positive bacteria Streptococcus iniae and Gram-negative bacteria Vibrio anguillarum. Phylogenetic analysis showed the strongest relationship of turbot PGRP to halibut, which was consistent with their phylogenetic relationships. In addition, SmPGRP2 was ubiquitously expressed in turbot tissues, and constitutive expression levels were higher in classical immune tissues (including liver, spleen, and head-kidney) than mucosal tissues (intestine, gill and skin). After bacterial challenge, the expression of SmPGRP2 was induced and showed a general trend of up-regulation in mucosal tissues, except in intestine following V. anguillarum infection. These different expression patterns varied depending on both pathogen and tissue type, suggesting its distinct roles in the host immune response to bacterial pathogen.