Roman Dziarski

Peptidoglycan- and Lipoteichoic Acid-induced Cell Activation Is Mediated by Toll-like Receptor 2

The life-threatening complications of sepsis in humans are elicited by infection with Gram-negative as well as Gram-positive bacteria. Recently, lipopolysaccharide (LPS), a major biologically active agent of Gram-negative bacteria, was shown to mediate cellular activation by a member of the human Toll-like receptor family, Toll-like receptor (TLR) 2. Here we investigate the mechanism of cellular activation by soluble peptidoglycan (sPGN) and lipoteichoic acid (LTA), main stimulatory components of Gram-positive bacteria. Like LPS, sPGN and LTA bind to the glycosylphosphatidylinositol-anchored membrane protein CD14 and induce activation of the transcription factor NF-κB in host cells like macrophages. We show that whole Gram-positive bacteria, sPGN and LTA induce the activation of NF-κB in HEK293 cells expressing TLR2 but not in cells expressing TLR1 or TLR4. The sPGN- and LTA-induced NF-κB activation was not inhibited by polymyxin B, an antibiotic that binds and neutralizes LPS. Coexpression together with membrane CD14 enhances sPGN signal transmission through TLR2. In contrast to LPS signaling, activation of TLR2 by sPGN and LTA does not require serum. These findings identify TLR2 as a signal transducer for sPGN and LTA in addition to LPS.

A Subset of Toll-Like Receptor Ligands Induces Cross-presentation by Bone Marrow-Derived Dendritic Cells

Dendritic cells (DCs) are capable of cross-presenting exogenous Ag to CD8+ CTLs. Detection of microbial products by Toll-like receptors (TLRs) leads to activation of DCs and subsequent orchestration of an adaptive immune response. We hypothesized that microbial TLR ligands could activate DCs to cross-present Ag to CTLs. Using DCs and CTLs in an in vitro cross-presentation system, we show that a subset of microbial TLR ligands, namely ligands of TLR3 (poly(inosinic-cytidylic) acid) and TLR9 (immunostimulatory CpG DNA), induces cross-presentation. In contrast to presentation of Ag to CD4+ T cells by immature DCs, TLR-induced cross-presentation is mediated by mature DCs, is independent of endosomal acidification, and relies on cytosolic Ag processing machinery.

Peptidoglycan recognition proteins: pleiotropic sensors and effectors of antimicrobial defences

Peptidoglycan recognition proteins (PGRPs) are innate immunity molecules that are present in most invertebrate and vertebrate animals. All PGRPs function in antimicrobial defence and are homologous to the prokaryotic peptidoglycan-lytic type 2 amidases. However, only some PGRPs have the catalytic activity that protects the host from excessive inflammation, and most PGRPs have diversified to carry out other host-defence functions. Insect and mammalian PGRPs defend host cells against infection through very different mechanisms. Insect PGRPs activate signal transduction pathways in host cells or trigger proteolytic cascades in the haemolymph, both of which generate antimicrobial effectors. By contrast, mammalian PGRPs are directly bactericidal. Here, we review these contrasting modes of action.

Peptidoglycan recognition proteins are a new class of human bactericidal proteins

Skin and mucous membranes come in contact with external environment and protect tissues from infections by producing antimicrobial peptides. We report that human peptidoglycan recognition proteins 3 and 4 (PGLYRP3 and PGLYRP4) are secreted as 89-115-kDa disulfide-linked homo- and heterodimers and are bactericidal against several pathogenic and nonpathogenic transient, but not normal flora, Gram-positive bacteria. PGLYRP3 and PGLYRP4 are also bacteriostatic toward all other tested bacteria, which include Gram-negative bacteria and normal flora Gram-positive bacteria. PGLYRP3 and PGLYRP4 are also active in vivo and protect mice against experimental lung infection. In contrast to antimicrobial peptides, PGLYRPs kill bacteria by interacting with their cell wall peptidoglycan, rather than permeabilizing their membranes. PGLYRP3 and PGLYRP4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach, and intestine. Thus, we have identified the function of mammalian PGLYRP3 and PGLYRP4, and show that they are a new class of bactericidal and bacteriostatic proteins that have different structures, mechanism of actions, and expression patterns than antimicrobial peptides.

Staphylococcus aureus peptidoglycan is a toll-like receptor 2 activator: a reevaluation.

ABSTRACT Since the ability of peptidoglycan (PGN) to activate Toll-like receptor 2 (TLR2) was recently questioned, we reevaluated activation of TLR2 by PGN. Polymeric soluble or insoluble Staphylococcus aureus PGN, repurified by sodium dodecyl sulfate or phenol extraction, activated TLR2 at 0.1 to 1 or 10 μg/ml, respectively, and induced tumor necrosis factor alpha production. The TLR2 activation by PGN, but not by lipoteichoic acid, was abolished by muramidase digestion. We conclude that polymeric S. aureus PGN is a TLR2 activator.

The peptidoglycan recognition proteins (PGRPs)

SummaryPeptidoglycan recognition proteins (PGRPs) are innate immunity molecules present in insects, mollusks, echinoderms, and vertebrates, but not in nematodes or plants. PGRPs have at least one carboxy-terminal PGRP domain (approximately 165 amino acids long), which is homologous to bacteriophage and bacterial type 2 amidases. Insects have up to 19 PGRPs, classified into short (S) and long (L) forms. The short forms are present in the hemolymph, cuticle, and fat-body cells, and sometimes in epidermal cells in the gut and hemocytes, whereas the long forms are mainly expressed in hemocytes. The expression of insect PGRPs is often upregulated by exposure to bacteria. Insect PGRPs activate the Toll or immune deficiency (Imd) signal transduction pathways or induce proteolytic cascades that generate antimicrobial products, induce phagocytosis, hydrolyze peptidoglycan, and protect insects against infections. Mammals have four PGRPs, which are secreted; it is not clear whether any are directly orthologous to the insect PGRPs. One mammalian PGRP, PGLYRP-2, is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and reduces its proinflammatory activity; PGLYRP-2 is secreted from the liver into the blood and is also induced by bacteria in epithelial cells. The three remaining mammalian PGRPs are bactericidal proteins that are secreted as disulfide-linked homo- and hetero-dimers. PGLYRP-1 is expressed primarily in polymorphonuclear leukocyte granules and PGLYRP-3 and PGLYRP-4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach, and intestine. These three proteins kill bacteria by interacting with cell wall peptidoglycan, rather than permeabilizing bacterial membranes as other antibacterial peptides do. Direct bactericidal activity of these PGRPs either evolved in the vertebrate (or mammalian) lineage or is yet to be discovered in insects.

Mammalian Peptidoglycan Recognition Protein Binds Peptidoglycan with High Affinity, Is Expressed in Neutrophils, and Inhibits Bacterial Growth

Peptidoglycan recognition protein (PGRP) is conserved from insects to mammals. In insects, PGRP recognizes bacterial cell wall peptidoglycan (PGN) and activates prophenoloxidase cascade, a part of the insect antimicrobial defense system. Because mammals do not have the prophenoloxidase cascade, its function in mammals is unknown. However, it was suggested that an identical protein (Tag7) was a tumor necrosis factor-like cytokine. Therefore, the aim of this study was to identify the function of PGRP in mammals. Mouse PGRP bound to PGN with fast kinetics and nanomolar affinity (K d = 13 nm). The binding was specific for polymeric PGN or Gram-positive bacteria with unmodified PGN, and PGRP did not bind to other cell wall components or Gram-negative bacteria. PGRP mRNA and protein were expressed in neutrophils and bone marrow cells, but not in spleen cells, mononuclear cells, T or B lymphocytes, NK cells, thymocytes, monocytes, and macrophages. PGRP was not a PGN-lytic or a bacteriolytic enzyme, but it inhibited the growth of Gram-positive but not Gram-negative bacteria. PGRP inhibited phagocytosis of Gram-positive bacteria by macrophages, induction of oxidative burst by Gram-positive bacteria in neutrophils, and induction of cytokine production by PGN in macrophages. PGRP had no tumor necrosis factor-like cytotoxicity for mammalian cells, and it was not chemotactic on its own or in combination with PGN. Therefore, mammalian PGRP binds to PGN and Gram-positive bacteria with nanomolar affinity, is expressed in neutrophils, and inhibits growth of bacteria.

MD-2 Enables Toll-Like Receptor 2 (TLR2)-Mediated Responses to Lipopolysaccharide and Enhances TLR2-Mediated Responses to Gram-Positive and Gram-Negative Bacteria and Their Cell Wall Components

MD-2 is associated with Toll-like receptor 4 (TLR4) on the cell surface and enables TLR4 to respond to LPS. We tested whether MD-2 enhances or enables the responses of both TLR2 and TLR4 to Gram-negative and Gram-positive bacteria and their components. TLR2 without MD-2 did not efficiently respond to highly purified LPS and LPS partial structures. MD-2 enabled TLR2 to respond to nonactivating protein-free LPS, LPS mutants, or lipid A and enhanced TLR2-mediated responses to both Gram-negative and Gram-positive bacteria and their LPS, peptidoglycan, and lipoteichoic acid components. MD-2 enabled TLR4 to respond to a wide variety of LPS partial structures, Gram-negative bacteria, and Gram-positive lipoteichoic acid, but not to Gram-positive bacteria, peptidoglycan, and lipopeptide. MD-2 physically associated with TLR2, but this association was weaker than with TLR4. MD-2 enhanced expression of both TLR2 and TLR4, and TLR2 and TLR4 enhanced expression of MD-2. Thus, MD-2 enables both TLR4 and TLR2 to respond with high sensitivity to a broad range of LPS structures and to lipoteichoic acid, and, moreover, MD-2 enhances the responses of TLR2 to Gram-positive bacteria and peptidoglycan, to which the TLR4-MD-2 complex is unresponsive.

Chemokines Are the Main Proinflammatory Mediators in Human Monocytes Activated by Staphylococcus aureus, Peptidoglycan, and Endotoxin

It is widely believed that the cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 are the main proinflammatory mediators induced in the host by bacteria and their cell wall components. To test this hypothesis, we compared the level of expression of 600 genes activated in human monocytes byStaphylococcus aureus, peptidoglycan, endotoxin, and interferon-γ. These stimulants induced expression of over 120 genes, as identified by cDNA arrays. The highest activated genes for proinflammatory mediators induced by all three bacterial stimulants were chemokine genes (IL-8 and macrophage inflammatory protein (MIP)-1α), whereas cytokine genes (TNF-α, IL-1, and IL-6) were induced to a lower extent. Genes for other chemokines (MIP-2α, MIP-1β, and monocyte chemoattractant protein-1) were also induced higher than the cytokine genes by peptidoglycan, and as high or higher than the cytokine genes by S. aureus and endotoxin. This high induction of chemokine genes was confirmed by quantitative RNase protection assay, and high secretion of chemokines was confirmed by enzyme-linked immunosorbent assays. Although genes for chemokines were the highest and genes for cytokines were the second highest induced genes by all three bacterial stimulants, each stimulus induced a unique pattern of gene expression. By contrast, expression of a completely different gene pattern was induced by a nonbacterial stimulus, interferon-γ. These results establish chemokines as the main mediators induced by both Gram-positive and Gram-negative bacteria and are consistent with the highly inflammatory nature of bacterial infections.

Peptidoglycan recognition proteins: modulators of the microbiome and inflammation

All animals, including humans, live in symbiotic association with microorganisms. The immune system accommodates host colonization by the microbiota, maintains microbiota–host homeostasis and defends against pathogens. This Review analyses how one family of antibacterial pattern recognition molecules — the peptidoglycan recognition proteins — has evolved a fascinating variety of mechanisms to control host interactions with mutualistic, commensal and parasitic microorganisms to benefit both invertebrate and vertebrate hosts.