Lys-type Peptidoglycan Research Articles

Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan.

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.

Infection-induced molecular pattern recognition proteins in larvae of Protaetia brevitarsis seulensis (Coleoptera: Cetoniidae)

We cloned and sequenced full-length peptidoglycan recognition protein (PGRP)-like cDNAs, named PS PGRP-SA(a)-like, PS PGRP-SA(b)-like, PS PGRP-SB1-like and PS PGRP-SC-like, from Protaetia brevitarsis seulensis. The amino acid sequences of PS PGRPs share 32.03-47.93% homology with those of PGRP family members in insects and mammals, including humans. We identified a conserved consensus sequence for amidase activity (His; H-Tyr; Y-His; H-Thr; T-Cys; C) and residues for binding peptidoglycan (PGN), one of the major bacterial cell wall components, including Asp (D) and Phe (F) for Lys-type PGN; and Gly(G), Trp (W) and Arg (R) for DAP-type PGN. The topological structures of PS PGRP-SA(a)-like, PS PGRP-SA(b)-like and PS PGRP-SC-like proteins are structurally similar to those of Drosophila melanogaster PGRP-SA, which has three α-helices and six β-strands. The β-strands are located in a central region and helix α1 on the back and peripheral α2 and α3 helices are on the front. The three α-helices and six β-strands are also present in PS PGRP-SB1-like, but the topological structure differs from that of typical PGRP. Significantly increased levels of PS PGRP-SA (a)-like and PS PGRP-SA (b)-like mRNA were recorded when Gram-positive bacteria or yeast cells were injected into larvae. PS PGRP-SB1-like mRNA levels were up-regulated by infection by all three pathogens; however, expression of PS PGRP-SC-like mRNA was increased 20- or 30-fold only shortly after injection with Gram-negative bacteria.

Two Amphioxus ApeC-Containing Proteins Bind to Microbes and Inhibit the TRAF6 Pathway.

The apextrin C-terminal (ApeC) domain is a class of newly discovered protein domains with an origin dating back to prokaryotes. ApeC-containing proteins (ACPs) have been found in various marine and aquatic invertebrates, but their functions and the underlying mechanisms are largely unknown. Early studies suggested that amphioxus ACP1 and ACP2 bind to bacterial cell walls and have a role in immunity. Here we identified another two amphioxus ACPs (ACP3 and ACP5), which belong to the same phylogenetic clade with ACP1/2, but show distinct expression patterns and sequence divergence (40-50% sequence identities). Both ACP3 and ACP5 were mainly expressed in the intestine and hepatic cecum, and could be up-regulated after bacterial challenge. Both prokaryotic-expressed recombinant ACP3 and ACP5 could bind with several species of bacteria and yeasts, showing agglutinating activity but no microbicidal activity. ELISA assays suggested that their ApeC domains could interact with peptidoglycan (PGN), but not with lipoteichoic acid (LTA), lipopolysaccharides (LPS) and zymosan A. Furthermore, they can only bind to Lys-type PGN from Staphylococcus aureus, but not to DAP-type PGN from Bacillus subtilis and not to moieties of PGN such as MDPs, NAMs and NAGs. This recognition spectrum is different from that of ACP1/2. We also found that when expressed in mammalian cells, ACP3 could interact with TRAF6 via a conserved non-ApeC region, which inhibited the ubiquitination of TRAF6 and hence suppressed downstream NF-κB activation. This work helped define a novel subfamily of ACPs, which have conserved structures, and have related yet diversified molecular functions. Its members have dual roles, with ApeC as a lectin and a conserved unknown region as a signal transduction regulator. These findings expand our understanding of the ACP functions and may guide future research on the role of ACPs in different animal clades.

Renal Function: Guardian of Immune Homeostasis

Microbial cell components from the intestinal microbiota spread systemically in a healthy host, posing the threat of inappropriate immune activation. In this issue of Immunity, Troha etal. identify a key role for renal function in the clearance of circulating Lys-type peptidoglycan, revealing a mechanism that keeps these inflammatory triggers in check to maintain systemic immune homeostasis.

Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA 2 expression.

Phospholipase A2 (PLA2 ) hydrolyzes fatty acids from phospholipids at the sn-2 position. Two intracellular PLA2 s, iPLA2 A and iPLA2 B, have been found in Spodoptera exigua. Both are calcium-independent cellular PLA2 . Their orthologs have been found in other insects. These two iPLA2 s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA2 expression. Both iPLA 2 s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA2 s. Both iPLA2 s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor-κB- and Relish-responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA2 s in response to Gram-positive bacteria containing Lys-type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA2 s in response to infection with Gram-negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA2 ) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA2 expression.

Enterococcus faecium secreted antigen A generates muropeptides to enhance host immunity and limit bacterial pathogenesis.

We discovered that Enterococcus faecium (E. faecium), a ubiquitous commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) were sufficient to enhance intestinal barrier function and pathogen tolerance, but the precise biochemical mechanism was unknown. Here we show E. faecium has unique peptidoglycan composition and remodeling activity through SagA, which generates smaller muropeptides that more effectively activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells. Our structural and biochemical studies show that SagA is a NlpC/p60-endopeptidase that preferentially hydrolyzes crosslinked Lys-type peptidoglycan fragments. SagA secretion and NlpC/p60-endopeptidase activity was required for enhancing probiotic bacteria activity against Clostridium difficile pathogenesis in vivo. Our results demonstrate that the peptidoglycan composition and hydrolase activity of specific microbiota species can activate host immune pathways and enhance tolerance to pathogens.

Characterization of the ligand binding of PGRP-L in half-smooth tongue sole ( Cynoglossus semilaevis ) by molecular dynamics and free energy calculation

Background: Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors of the host innate immune system that are involved in the immune defense against bacterial pathogens. PGRPs have been characterized in several fish species. The PGN-binding ability is important for the function of PGRPs. However, the PGRP-PGN interaction mechanism in fish remains unclear. In the present study, the 3-D model of a long PGRP of half-smooth tongue sole (Cynoglossus semilaevis) (csPGRP-L), a marine teleost with great economic value, was constructed through the comparative modeling method, and the key amino acids involved in the interaction with Lys-type PGNs and Dap-type PGNs were analyzed by molecular dynamics and molecular docking methods. Results: csPGRP-L possessed a typical PGRP structure, consisting of five β-sheets and four α-helices. Molecular docking showed that the van der Waals forces had a slightly larger contribution than Coulombic interaction in the csPGRP-L-PGN complex. Moreover, the binding energies of csPGRP-L-PGNs computed by MM-PBSA method revealed that csPGRP-L might selectively bind both types of MTP-PGNs and MPP-PGNs. In addition, the binding energy of each residue of csPGRP-L was also calculated, revealing that the residues involved in the interaction with Lys-type PGNs were different from that with Dap-type PGNs. Conclusions: The 3-D structure of csPGRP-L possessed typical PGRP structure and might selectively bind both types of MTP- and MPP-PGNs, which provided useful insights to understanding the functions of fish PGRPs.

Role of NF-kβ factor Rel2 during Plasmodium falciparum and bacterial infection in Anopheles dirus.

BackgroundAnopheles mosquitoes transmit malaria which is one of the world’s most threatening diseases. Anopheles dirus (sensu stricto) is among the main vectors of malaria in South East Asia. The mosquito innate immune response is the first line of defence against malaria parasites during its development. The immune deficiency (IMD) pathway, a conserved immune signaling pathway, influences anti-Plasmodium falciparum activity in Anopheles gambiae, An. stephensi and An. albimanus. The aim of the study was to determine the role of Rel2, an IMD pathway-controlled NF-kappaβ transcription factor, in An. dirus.MethodsRACE (Rapid amplification of cDNA ends) was performed on the Rel2 gene. Double-stranded Rel2 was constructed and injected into the thorax of female mosquitoes. The injected mosquitoes were fed on a P. falciparum gametocyte culture and dissected on day 7–9 post-feeding in order to count the oocysts. A survival analysis was conducted by exposing the dsRNA injected mosquitoes to Gram-positive and Gram-negative bacteria.ResultsThis study demonstrated that the Rel2 gene in An. dirus has two isoforms, short length and full length. RNA interference-mediated gene silencing of Rel2 showed that the latter is involved in protection against P. falciparum, Gram-positive bacteria (Micrococcus luteus) with Lys-type peptidoglycan and Gram-negative bacteria (Escherichia coli) with DAP-type peptidoglycan.ConclusionThis study suggested that there are similarities in the splicing events and functionality of the Rel2 gene, between the Anopheles species. Among all the important anophelines, the immunity of only a few has been thoroughly investigated. In order to develop novel vector-based control strategies and restrict malaria transmission, the immune pathways of these important vectors should be thoroughly investigated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1810-0) contains supplementary material, which is available to authorized users.

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-κB and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-κB, as well as in regulating innate immune genes.

Solution Structure and Peptidoglycan Interaction of Rv0899, a Virulence Factor from Mycobacterium Tuberculosis

Rv0899 is a membrane protein encoded by an operon that is required for fast ammonia secretion, rapid pH neutralization and growth of M. tuberculosis in acidic environments. The gene is restricted to pathogenic mycobacteria and, thus, is an attractive candidate for the development of anti-tuberculosis chemotherapy. Rv0899 contains three independently structured domains. The N-terminus (Rv0899-M; residues 1-80) includes a membrane-anchoring sequence of 20 hydrophobic amino acids (residues 28-50) that is required for membrane translocation. The central region (Rv0899-B; residues 81-194) contains two consecutive repeats of the BON (Bacterial OsmY and Nodulation) domain (pfam04972), a conserved, putative lipid binding sequence that is found in some bacterial osmotic shock protection proteins. Finally, the C-terminus (Rv0899-C; residues 195-326) contains an OmpA-like domain (pfam00691), a periplasmic peptidoglycan-binding structure found in several types of bacterial membrane proteins. Using NMR spectroscopy, we determined the high-resolution structures of Rv0899-B and Rv0899-C. Rv0899-B does not form a transmembrane beta barrel and has an undocumented mixed alpha/beta fold. Furthermore, we found Rv0899-B has a specific zinc binding site and associates with lipid vesicles in a pH dependent manner. The structure of Rv0899-C adopts the characteristic OmpA-like family fold. It exhibits pH-dependent conformational dynamics, with significant disorder at neutral pH and a more ordered structure at acidic pH. Rv0899-C associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved Rv0899-C residues, including two key arginines, establish the specificity for DAP- but not Lys-type peptidoglycan. The three-dimensional structure of Rv0899 suggests alternative possible modes of membrane association and provides a molecular basis for the screening of small molecules with anti-TB potential.

Molecular Structure and Peptidoglycan Recognition of Mycobacterium tuberculosis ArfA (Rv0899)

Mycobacterium tuberculosis ArfA (Rv0899) is a membrane protein encoded by an operon that is required for supporting bacterial growth in acidic environments. Its C-terminal domain (C domain) shares significant sequence homology with the OmpA-like family of peptidoglycan-binding domains, suggesting that its physiological function in acid stress protection may be related to its interaction with the mycobacterial cell wall. Previously, we showed that ArfA forms three independently structured modules, and we reported the structure of its central domain (B domain). Here, we describe the high-resolution structure and dynamics of the C domain, we identify ArfA as a peptidoglycan-binding protein and we elucidate the molecular basis for its specific recognition of diaminopimelate-type peptidoglycan. The C domain of ArfA adopts the characteristic fold of the OmpA-like family. It exhibits pH-dependent conformational dynamics (with significant heterogeneity at neutral pH and a more ordered structure at acidic pH), which could be related to its acid stress response. The C domain associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved ArfA residues, including two key arginines, establish the specificity for diaminopimelate- but not Lys-type peptidoglycan. ArfA is the first peptidoglycan-binding protein to be identified in M. tuberculosis. Its functions in acid stress protection and peptidoglycan binding suggest a link between the acid stress response and the physicochemical properties of the mycobacterial cell wall.

Drosophila Immunity: Analysis of PGRP-SB1 Expression, Enzymatic Activity and Function

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?

Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes of the freshwater crayfish, Pacifastacus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type peptidoglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPHs are involved in this activation possibly by forming a complex with LGBP and without a PGRP.

Peptidoglycan recognition protein (PGRP) from eri-silkworm, Samia cynthia ricini; protein purification and induction of the gene expression

Peptidoglycan recognition protein (PGRP) was isolated from immunized hemolymph of the wild silkworm, Samia cynthia ricini, detecting the biding activity with 125I-labeled peptidoglycan (PGN). The binding specificity of PGRP was tested by competitive inhibition of the binding to 125I-labeled-PGN by a large excess amount of non-labeled-PGN or other glucans. The binding to labeled uncross-linked Lys-type PGN from Micrococcus luteus was strongly inhibited by non-labeled-PGN of the same structure and meso-diaminopimelic acid (DAP)-type cross-linked PGN from Bacillus cell wall, but only a little by cross-linked PGN from M. luteus cell wall. The PGRP cDNA encodes a 193 amino acid open reading frame. The deduced amino acid sequence had 62 to 91% identities to known lepidopteran PGRPs, but less than 40% to Drosophila PGRPs. The PGRP gene constitutively expressed at a low level in naive fat body, and strongly induced by an injection of DAP-type cross-linked and Lys-type uncross-linked PGNs, but only weakly by Lys-type cross-linked PGN from M. luteus. The silkworm possibly distinguish between PGNs based on the structure of cross-linking peptide, but has less if any preference for the diamino acid residue of the stem peptide.

Structural Basis for Preferential Recognition of Diaminopimelic Acid-type Peptidoglycan by a Subset of Peptidoglycan Recognition Proteins

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.

The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila

From a forward genetic screen for phagocytosis mutants in Drosophila melanogaster, we identified a mutation that affects peptidoglycan recognition protein (PGRP) SC1a and impairs the ability to phagocytose the bacteria Staphylococcus aureus, but not Escherichia coli and Bacillus subtilis. Because of the differences in peptidoglycan peptide linkages in these bacteria, our data suggest that PGRP-SC1a is necessary for recognition of the Lys-type peptidoglycan typical of most Gram(+) bacteria. PGRP-SC1a mutants also fail to activate the Toll/NF-kappaB signaling pathway and are compromised for survival after S. aureus infection. This mutant phenotype is the first found for an N-acetylmuramoyl-l-alanine amidase PGRP that cleaves peptidoglycan at the lactylamide bond between the glycan backbone and the crosslinking stem peptides. By generating transgenic rescue flies that express either wild-type or a noncatalytic cysteine-serine mutant PGRP-SC1a, we find that PGRP-SC1a amidase activity is not necessary for Toll signaling, but is essential for uptake of S. aureus into the host phagocytes and for survival after S. aureus infection. Furthermore, we find that the PGRP-SC1a amidase activity can be substituted by exogenous addition of free peptidoglycan, suggesting that the presence of peptidoglycan cleavage products is more important than the generation of cleaved peptidoglycan on the bacterial surface for PGRP-SC1a mediated phagocytosis.

Peptidoglycan recognition by the Drosophila Imd pathway

The structural requirements for recognition of peptidoglycan (PGN) by PGRP-LC and activation of the Drosophila IMD pathway are not yet clear. In order to examine this question more carefully, the activity of peptidoglycan from different types of bacteria was compared in cell-based and whole animal assays. Drosophila S2* cells, but not adult flies, responded to Lys-type Micrococcus luteus PGN, but with significantly less potency compared to Dap-type Escherichia coli PGN, while intact Lys-type PGN from Staphylococcus aureus was inactive. After treatment with lysostaphin, which digests the cross-bridging peptides, S. aureus PGN weakly stimulated the IMD pathway, similar to M. luteus PGN. Further digestion with mutanolysin, which creates monomeric PGN fragments, abolished the activity of S. aureus PGN. On the other hand, monomeric E. coli PGN, generated by mutanolysin digestion, was still active but required different isoforms of PGRP-LC for recognition. Polymeric PGN required only PGRP-LCx, while monomeric E. coli PGN required both the PGRP-LCa and PGRP-LCx isoforms. These results suggest that the recognition by PGRP-LCx alone requires polymeric PGN, and that polymeric Dap-type PGN is a more potent PGRP-LCx agonist, compared to Lys-type PGN. These results also suggest that the heteromeric PGRP-LCa/LCx receptor complex recognizes monomeric Dap-type, but not Lys-type, PGN.

Peptidoglycan recognition by the <I>Drosophila</I> Imd pathway

The structural requirements for recognition of peptidoglycan (PGN) by PGRP-LC and activation of the Drosophila IMD pathway are not yet clear. In order to examine this question more carefully, the activity of peptidoglycan from different types of bacteria was compared in cell-based and whole animal assays. Drosophila S2* cells, but not adult flies, responded to Lys-type Micrococcus luteus PGN, but with significantly less potency compared to Dap-type Escherichia coli PGN, while intact Lys-type PGN from Staphylococcus aureus was inactive. After treatment with lysostaphin, which digests the cross-bridging peptides, S. aureus PGN weakly stimulated the IMD pathway, similar to M. luteus PGN. Further digestion with mutanolysin, which creates monomeric PGN fragments, abolished the activity of S. aureus PGN. On the other hand, monomeric E. coli PGN, generated by mutanolysin digestion, was still active but required different isoforms of PGRP-LC for recognition. Polymeric PGN required only PGRP-LCx, while monomeric E. coli PGN required both the PGRP-LCa and PGRP-LCx isoforms. These results suggest that the recognition by PGRP-LCx alone requires polymeric PGN, and that polymeric Dap-type PGN is a more potent PGRP-LCx agonist, compared to Lys-type PGN. These results also suggest that the heteromeric PGRP-LCa/LCx receptor complex recognizes monomeric Dap-type, but not Lys-type, PGN.