Mammalian peptidoglycan recognition proteins kill bacteria by activating two-component systems and modulate microbiome and inflammation

Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and function in antibacterial immunity. We have revealed a novel mechanism of bacterial killing by innate immune system, in which mammalian PGRPs bind to bacterial cell wall or outer membrane and exploit bacterial stress defense response to kill bacteria. PGRPs enter Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins exported out of bacterial cells. This activation results in membrane depolarization, production of hydroxyl radicals, and cessation of intracellular peptidoglycan, protein, RNA, and DNA synthesis, which are responsible for bacterial death. PGRPs also bind to the outer membrane in Escherichia coli and activate functionally homologous CpxA-CpxR two-component system, which also results in bacterial death. We excluded other potential bactericidal mechanisms, such as inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan, and membrane permeabilization. In vivo, mammalian PGRPs are expressed in polymorphonuclear leukocytes, skin, salivary glands, oral cavity, intestinal tract, eyes, and liver. They control acquisition and maintenance of beneficial normal gut microflora, which protects the host from enhanced inflammation, tissue damage, and colitis.     

哺乳动物固有免疫中肽聚糖识别蛋白研究进展

肽聚糖识别蛋白(PGRPs或PGLYRPs)是重要的识别细菌肽聚糖的模式识别受体之一,且从昆虫到哺乳动物高度保守.哺乳动物有4个PGRPs,它们分别表达于不同的细胞/组织.PGLYRP1、PGLYRP3和PGLYRP4具有抑杀细菌,而PGLYRP2水解肽聚糖的特性,最新研究表明PGLYRP2还具有促炎症的特性,其促炎症...     

Peptidoglycan recognition proteins: on and off switches for innate immunity

Summary: Insects rely on innate immune mechanisms to defend themselves against microbes. The inducible anti‐microbial peptides constitute an important arm of this defense. In Drosophila, the Toll and the Imd pathways are the major routes to induce the peptides, and it has become clear that to a certain extent, these pathways can discriminate between different microbes and mount an appropriate response to eliminate the intruder. This review discusses the proteins responsible for this discriminatory recognition, the peptidoglycan recognition proteins (PGRPs). The serum protein PGRP‐SA triggers a humoral cascade of proteases upon infection by certain gram‐positive bacteria to activate the Toll pathway. The membrane‐bound receptor PGRP‐LC activates the Imd pathway in response to certain gram‐ negative bacteria or their peptidoglycans. Other PGRPs have enzymatic activity, cleaving lactylamide bonds in peptidoglycan to eliminate its immunogenicity, thus turning off the immune response. The PGRP family is conserved from insects to man. Short mammalian PGRP variants are synthesized in neutrophils and stored in granules. These PGRPs seem to influence the survival of phagocytosed non‐pathogenic bacteria. Long PGRP variants are expressed in the liver and secreted into the bloodstream where their peptidoglycan‐degrading activity might serve scavenger functions.     

Short and long peptidoglycan recognition proteins (PGRPs) in zebrafish, with findings of multiple PGRP homologs in teleost fish

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (tn: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response.     

Zebrafish peptidoglycan recognition proteins are bactericidal amidases essential for defense against bacterial infections

Peptidoglycan recognition proteins (PGRPs) are structurally conserved through evolution, but their functions in innate immunity are different in invertebrates and vertebrates. We asked what the functions of PGRPs in fish are and whether they are indispensable for defense against infection because fish are the first vertebrates that developed adaptive immunity, but they still rely solely on innate immunity during early development of embryos. We identified and cloned three zebrafish PGRPs and showed that they are highly expressed in eggs, developing embryos, and adult tissues that contact external environment. Zebrafish PGRPs have both peptidoglycan-lytic amidase activity and broad-spectrum bactericidal activity, which is a unique feature. Furthermore, we demonstrated that in the developing zebrafish embryo, one of these PGRPs is essential for defense and survival during bacterial infections. These data demonstrate an absolute requirement for innate immunity in defense against infections in fish embryos and for a PGRP protein for survival in vertebrates.     

Crystal structure of Drosophila PGRP-SD suggests binding to DAP-type but not lysine-type peptidoglycan

In Drosophila the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathways. The Toll signaling pathway responds mainly to Gram-positive bacterial and fungal infection while the Imd pathway mediates the response to Gram-negative bacteria. Microbial recognition upstream of Toll involves, at least in part, peptidoglycan recognition proteins (PGRPs). The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and Gram-negative binding protein 1 (GNBP1) that cooperate to detect the presence of lysine-type peptidoglycan in the host. Recently it has been shown that a loss-of-function mutation in peptidoglycan recognition protein SD (PGRP-SD) severely exacerbates the PGRP-SA and GNBP1 mutant phenotypes. Here we have solved the crystal structure of PGRP-SD at 1.5A resolution. Comparison with available structures of PGRPs in complex with their peptidoglycan (PGN) ligand strongly suggests a diaminopimelic acid (DAP) specificity for PGRP-SD. This result is supported by pull-down assays with insoluble PGNs. In addition we show that Toll pathway activation after infection by DAP-type PGN containing bacteria is clearly reduced in PGRP-SD mutant flies. Our hypothesis is that the role of PGRP-SD is the recognition of DAP-type PGNs responsible for the activation of the Toll pathway by Gram-negative bacteria.     

Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection

Peptidoglycan recognition proteins (PGRPs) are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings support the role of PGRP-LB as a negative regulator of the Imd pathway and brought to light a synergy of PGRP-SCs with PGRP-LB in the systemic response. Flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to innocuous gut infections. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. Analysis of the immune phenotype of flies lacking all catalytic PGRPs and the Imd regulator Pirk reveals that the Imd-mediated immune response is highly constrained by the existence of multiple negative feedbacks.     

Toll-like receptor gene family and TIR-domain adapters in Danio rerio

The toll-like family of receptors (TLR) is an ancient pattern recognition receptor family, conserved from insects to mammals. We have identified in zebrafish (Danio rerio) 19 putative TLR variants, the orthologs of mammalian TLR2-5, 7-9, a fish specific receptor type group and three putative splice variants. One receptor is very close to mammalian TLR1, 6 and 10 and seems to be their common ancestor. However, in contrast to the pufferfish, Fugu rubripes, we found two receptors homologous to TLR4, showing that lack of TLR4 is not general for fish. In addition, we identified two members close to mammalian TLR8 and five members close to FuguTLR21 and goldfish TLR, a TLR group which now has only been found in fish. By RT-PCR we showed that all TLR are widely expressed in adult tissues, but also at different stages of development. All these TLRs contain very conserved toll/interleukin-1 receptor (TIR) domains able to interact with TIR-domain of adapter molecules. We demonstrate here that TIR-domain containing adapters MyD88 and SARM are present in zebrafish, showing that TLR adapter molecules are highly conserved in evolution.     

Sensing infection in Drosophila: Toll and beyond

Drosophila has evolved a potent immune system that is somewhat adapted to the nature of infections through the selective activation of either one of two NF-kappa B-like signalling pathways, the Toll and IMD (Immune deficiency) pathways. In contrast to the mammalian system, the Toll receptor does not act as a pattern recognition receptor (PRR) but as a cytokine receptor. The sensing of microbial infections is achieved by at least four PRRs that belong to two distinct families: the peptidoglycan recognition proteins (PGRPs) and the Gram-negative binding proteins (GNBPs)/beta-glucan recognition proteins (beta GRPs).     

Primitive complement system--recognition and activation

The complement system, composed of more than 30 serum and cell surface components, is collaborating in recognition and elimination of pathogens as a part of both the innate and acquired immune systems. The two collagenous lectins, mannose-binding lectin (MBL) and ficolins, are one of the pattern recognition molecules acting in innate immunity and upon recognition of the pathogens, they trigger the activation of the lectin complement pathway through attached serine proteases (MASPs). A similar lectin-base complement system, consisting of the lectin-protease complex and C3, is present in ascidians, our closest invertebrate relatives and functions in an opsonic manner. On the other hand, ongoing genome projects in both vertebrates and invertebrates revealed that most domains used by mammalian complement components are found in both protostomes and deuterostomes. However, the unique combinations of them as found in mammalian complement components are present only in deuterostomes, indicating the deuterostome origin of the complement system. Unexpectedly, the complement system of an invertebrate deuterostome, ascidian, shows a similar level of complexity as that of mammals, suggesting that expansion of complement genes by gene duplications occurred independently both in the ascidian and vertebrate lineages. Although most characteristic domain structures of the mammalian complement components are found in ascidians, detailed evolutionary analysis casts doubt on their mutual reactivity in several points. Thus, another integrative step seems to have been required to establish the modern complement system of higher vertebrates.     

Expression and functional characterization of PGRP6 splice variants in grass carp Ctenopharyngodon idella

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved pattern recognition receptors from insects to mammals, recognize bacterial PGN and function in antibacterial innate immunity. The existence of alternative splicing is a common feature for PGRP family. Here the splicing pattern from the splicing at the 5′ end of PGRP6 gene was identified in a teleost fish, the grass carp (Ctenopharyngodon idella). Four splice variants of grass carp PGRP6 were designated as gcPGRP6a, gcPGRP6b, gcPGRP6c and gcPGRP6d, respectively. Real-time PCR revealed the different expression of these variants in fish individuals and CIK cell line in response to stimulation with different microbial ligands. Immunofluorescence microscopy and Western blotting showed that the splice variants are intracellular protein. Cell lysates from Epithelioma papulosum cyprini (EPC) cells transfected with gcPGRP6 splice variants are able to bind microbial PAMPs including Lys-type PGN from Staphylococcus aureus, DAP-type PGN from Bacillus subtilis, glucan, mannan, and microorganisms including Streptococcus dysgalactiae, Flavobacterium columnare and Saccharomyces cerevisiae. Moreover, overexpression of gcPGRP6 variants inhibited earlier stage growth of intracellular bacteria. The data also identified a specific role for gcPGRP6c variant in the positive regulation of cytolytic molecule perforin, and for gcPGRP6a, gcPGRP6b and gcPGRP6c variants in positive regulation of antimicrobial peptides (AMPs). However, the gcPGRP6d variant, which encoded basically only the PGRP domain, failed to induce the expression of perforin and AMPs. It is suggested that fish PGRP6 splice variants have common and variant-specific function in innate immune response.     

Expression and functional characterization of PGRP6 splice variants in grass carp Ctenopharyngodon idella

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved pattern recognition receptors from insects to mammals, recognize bacterial PGN and function in antibacterial innate immunity. The existence of alternative splicing is a common feature for PGRP family. Here the splicing pattern from the splicing at the 5′ end of PGRP6 gene was identified in a teleost fish, the grass carp (Ctenopharyngodon idella). Four splice variants of grass carp PGRP6 were designated as gcPGRP6a, gcPGRP6b, gcPGRP6c and gcPGRP6d, respectively. Real-time PCR revealed the different expression of these variants in fish individuals and CIK cell line in response to stimulation with different microbial ligands. Immunofluorescence microscopy and Western blotting showed that the splice variants are intracellular protein. Cell lysates from Epithelioma papulosum cyprini (EPC) cells transfected with gcPGRP6 splice variants are able to bind microbial PAMPs including Lys-type PGN from Staphylococcus aureus, DAP-type PGN from Bacillus subtilis, glucan, mannan, and microorganisms including Streptococcus dysgalactiae, Flavobacterium columnare and Saccharomyces cerevisiae. Moreover, overexpression of gcPGRP6 variants inhibited earlier stage growth of intracellular bacteria. The data also identified a specific role for gcPGRP6c variant in the positive regulation of cytolytic molecule perforin, and for gcPGRP6a, gcPGRP6b and gcPGRP6c variants in positive regulation of antimicrobial peptides (AMPs). However, the gcPGRP6d variant, which encoded basically only the PGRP domain, failed to induce the expression of perforin and AMPs. It is suggested that fish PGRP6 splice variants have common and variant-specific function in innate immune response.     

肽聚糖识别蛋白

天然免疫系统通过一系列高度保守的模式识别受体识别病原体相关分子模式.肽聚糖识别蛋白家族是重要的模式识别受体,从昆虫到人类均高度保守,可识别肽聚糖和含肽聚糖的细菌,在天然免疫和获得性免疫应答中发挥重要的识别和调节功能.     

Gene silencing and overexpression of porcine peptidoglycan recognition protein long isoforms: involvement in beta-defensin-1 expression

Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased by Salmonella infection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 have N-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide beta-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged with Listeria monocytogenes results in downregulation of beta-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of beta-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.     

Crystal structure of peptidoglycan recognition protein LB from Drosophila melanogaster

The family of peptidoglycan recognition proteins (PGRPs) are associated with the recognition of the peptidoglycan of microbes and subsequent activation of signaling pathways for immune response. Here the crystal structure of Drosophila PGRP-LB is determined at a resolution of 2.0 A and shows an active-site cleft with a zinc cage. Poor conservation of surface residues at the cleft predicts a widely varying individual specificity of PGRPs for molecular patterns on microbial cell walls. At the back of this cleft is a putatively conserved distinctive groove. The location and mainly hydrophobic nature of the groove indicate that the back face serves for subsequent signaling after clustering of PGRP molecules by binding to polymeric cell wall components.     

The Toll-receptor family and control of innate immunity

Innate immune recognition is mediated by a system of germline-encoded receptors that recognize conserved molecular patterns that are associated with microbial pathogens. These receptors are coupled to signal transduction pathways that control expression of a variety of inducible immune-response genes. Toll receptors and the associated signaling pathways of nuclear factor kappaB may represent the most ancient host defense system found in mammals, insects and plants.     

Peptidoglycan: a critical activator of the mammalian immune system during infection and homeostasis

Peptidoglycan is a conserved structural component of the bacterial cell wall with molecular motifs unique to bacteria. The mammalian immune system takes advantage of these properties and has evolved to recognize this microbial associated molecular pattern. Mammals have four secreted peptidoglycan recognition proteins, PGLYRP-1-4, as well as two intracellular sensors of peptidoglycan, Nod1 and Nod2. Recognition of peptidoglycan is important in initiating and shaping the immune response under both homeostatic and infection conditions. During infection, peptidoglycan recognition drives both cell-autonomous and whole-organism defense responses. Here, we examine recent advances in the understanding of how peptidoglycan recognition shapes mammalian immune responses in these diverse contexts.