Dipika Gupta

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.

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.

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.

Micrococci and Peptidoglycan Activate TLR2→MyD88→IRAK→TRAF→NIK→IKK→NF-κB Signal Transduction Pathway That Induces Transcription of Interleukin-8

ABSTRACT This study was done to elucidate the signal transduction pathway of interleukin-8 (IL-8) induction by gram-positive bacteria. Bacteria (micrococci) and peptidoglycan (PGN) induced transcription of IL-8 in HEK293 cells expressing Toll-like receptor 2 (TLR2) and CD14 but not in those expressing TLR1 or TLR4. A mutation within the NF-κB site in the IL-8 promoter abrogated transcriptional induction of IL-8 by the two stimulants. Dominant negative myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), NFκB-inducing kinase (NIK), and IκB kinase (IKK) mutant forms completely inhibited micrococcus- and PGN-induced activation of NF-κB and expression of the gene for IL-8. Induction of NF-κB was partially inhibited by dominant negative tumor necrosis factor receptor-associated kinase 6 (TRAF6) but not TRAF2, whereas induction of IL-8 gene was partially inhibited by both TRAF6 and TRAF2. These data indicate that micrococci and PGN induce TLR2-dependent activation of the gene for IL-8 and that this activation requires MyD88, IRAK, NIK, IKK, and NF-κB and may also utilize TRAF6 and, to a lesser extent, TRAF2.

Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems

Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the 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 that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.

Review: Mammalian peptidoglycan recognition proteins (PGRPs) in innate immunity:

Peptidoglycan recognition proteins (PGRPs or PGLYRPs) are innate immunity proteins that are conserved from insects to mammals, recognize bacterial peptidoglycan, and function in antibacterial immunity and inflammation. Mammals have four PGRPs — PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4. They are secreted proteins expressed in polymorphonuclear leukocytes (PGLYRP1), liver (PGLYRP2), or on body surfaces, mucous membranes, and in secretions (saliva, sweat) (PGLYRP3 and PGLYRP4). All PGRPs recognize bacterial peptidoglycan. Three PGRPs, PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal for both Gram-positive and Gram-negative bacteria and have no enzymatic activity, whereas PGLYRP2 is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial cell wall peptidoglycan. Peptidoglycan recognition proteins influence host— pathogen interactions not only through their antibacterial or peptidoglycan-hydrolytic properties, but also through their pro-inflammatory and anti-inflammatory properties that are independent of their hydrolytic and antibacterial activities. The PGRPs likely play a role both in antibacterial defenses and several inflammatory diseases. They modulate local inflammatory responses in tissues (such as arthritic joints) and there is evidence for association of PGRPs with inflammatory diseases, such as psoriasis.

Peptidoglycan recognition in innate immunity

The innate immune system recognizes micro-organisms through a series of pattern recognition receptors that are highly conserved in evolution. Peptidoglycan (PGN) is a unique and essential component of the cell wall of virtually all bacteria, is not present in eukaryotes, and is an excellent target for the innate immune system. Indeed, higher eukaryotes, including mammals, have several PGN recognition molecules, including CD14, Toll-like receptor 2 (TLR2), nucleotide oligomerization domain (Nod)-containing proteins, a family of peptidoglycan recognition proteins (PGRPs), and PGN-lytic enzymes (lysozyme and amidase). These molecules induce host responses to micro-organisms, degrade PGN, or have direct antimicrobial effects.

Human Peptidoglycan Recognition Proteins Require Zinc to Kill Both Gram-Positive and Gram-Negative Bacteria and Are Synergistic with Antibacterial Peptides

Mammals have four peptidoglycan recognition proteins (PGRPs or PGLYRPs), which are secreted innate immunity pattern recognition molecules with effector functions. In this study, we demonstrate that human PGLYRP-1, PGLYRP-3, PGLYRP-4, and PGLYRP-3:4 have Zn2+-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria at physiologic Zn2+ concentrations found in serum, sweat, saliva, and other body fluids. The requirement for Zn2+ can only be partially replaced by Ca2+ for killing of Gram-positive bacteria but not for killing of Gram-negative bacteria. The bactericidal activity of PGLYRPs is salt insensitive and requires N-glycosylation of PGLYRPs. The LD99 of PGLYRPs for Gram-positive and Gram-negative bacteria is 0.3–1.7 μM, and killing of bacteria by PGLYRPs, in contrast to killing by antibacterial peptides, does not involve permeabilization of cytoplasmic membrane. PGLYRPs and antibacterial peptides (phospholipase A2, α- and β-defensins, and bactericidal permeability-increasing protein), at subbactericidal concentrations, synergistically kill Gram-positive and Gram-negative bacteria. These results demonstrate that PGLYRPs are a novel class of recognition and effector molecules with broad Zn2+-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria that are synergistic with antibacterial peptides.