Dana J. Philpott

CARD4/Nod1 mediates NF‐κB and JNK activation by invasive Shigella flexneri

Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF‐κB and c‐Jun N‐terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll‐like receptors (TLRs), which mediate LPS responsiveness from the outside‐in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance‐like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF‐κB induction. Dominant‐negative versions of CARD4 block activation of NF‐κB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease‐resistance proteins.

Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition

Toll‐like receptor 2 (TLR2) has been shown to recognize several classes of pathogen‐associated molecular patterns including peptidoglycan (PG). However, studies linking PG with TLR2 recognition have relied mainly on the use of commercial Staphylococcus aureus PG and have not addressed TLR2 recognition of other PG types. Using highly purified PGs from eight bacteria (Escherichia coli, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Helicobacter pylori, Bacillus subtilis, Listeria monocytogenes, Streptococcus pneumoniae and S. aureus), we show that these PGs are not sensed through TLR2, TLR2/1 or TLR2/6. PG sensing is lost after removal of lipoproteins or lipoteichoic acids (LTAs) from Gram‐negative and Gram‐positive cell walls, respectively. Accordingly, purified LTAs are sensed synergistically through TLR2/1. Finally, we show that elicited peritoneal murine macrophages do not produce tumour necrosis factor‐α or interleukin‐6 in response to purified PGs, suggesting that PG detection is more likely to occur intracellularly (through Nod1/Nod2) rather than from the extracellular compartment.

Recognition of Staphylococcus aureus by the Innate Immune System

SUMMARY The gram-positive bacterium Staphylococcus aureus is a major pathogen responsible for a variety of diseases ranging from minor skin infections to life-threatening conditions such as sepsis. Cell wall-associated and secreted proteins (e.g., protein A, hemolysins, and phenol-soluble modulin) and cell wall components (e.g., peptidoglycan and alanylated lipoteichoic acid) have been shown to be inflammatory, and these staphylococcal components may contribute to sepsis. On the host side, many host factors have been implicated in the innate detection of staphylococcal components. One class of pattern recognition molecules, Toll-like receptor 2, has been shown to function as the transmembrane component involved in the detection of staphylococcal lipoteichoic acid and phenol-soluble modulin and is involved in the synthesis of inflammatory cytokines by monocytes/macrophages in response to these components. Nod2 (nucleotide-binding oligomerization domain 2) is the intracellular sensor for muramyl dipeptide, the minimal bioactive structure of peptidoglycan, and it may contribute to the innate immune defense against S. aureus. The staphylococcal virulence factor protein A was recently shown to interact directly with tumor necrosis factor receptor 1 in airway epithelium and to reproduce the effects of tumor necrosis factor alpha. Finally, peptidoglycan recognition protein L is an amidase that inactivates the proinflammatory activities of peptidoglycan. However, peptidoglycan recognition protein L probably plays a minor role in the innate immune response to S. aureus. Thus, several innate immunity receptors may be implicated in host defense against S. aureus.

Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation.

The innate immune system is the most ancestral and ubiquitous system of defence against microbial infection. The microbial sensing proteins involved in innate immunity recognize conserved and often structural components of microorganisms. One class of these pattern‐recognition molecules, the Toll‐like receptors (TLRs), are involved in detection of microbes in the extracellular compartment whereas a newly discovered family of proteins, the NBS‐LRR proteins (for nucleotide‐binding site and leucine‐rich repeat), are involved in intracellular recognition of microbes and their products. NBS‐LRR proteins are characterized by three structural domains: a C‐terminal leucine‐rich repeat (LRR) domain able to sense a microbial motif, an intermediary nucleotide binding site (NBS) essential for the oligomerization of the molecule that is necessary for the signal transduction induced by different N‐terminal effector motifs, such as a pyrin domain (PYD), a caspase‐activating and recruitment domain (CARD) or a baculovirus inhibitor of apoptosis protein repeat (BIR) domain. Two of these family members, Nod1 and Nod2, play a role in the regulation of pro‐inflammatory pathways through NF‐κB induced by bacterial ligands. Recently, it was shown that Nod2 recognizes a specific peptidoglycan motif from bacteria, muramyl dipeptide (MDP). A surprising number of human genetic disorders have been linked to NBS‐LRR proteins. For example, mutations in Nod2, which render the molecule insensitive to MDP and unable to induce NF‐κB activation when stimulated, are associated with susceptibility to a chronic intestinal inflammatory disorder, Crohns disease. Conversely, mutations in the NBS region of Nod2 induce a constitutive activation of NF‐κB and are responsible for Blau syndrome, another auto‐inflammatory disease. Nalp3, which is an NBS‐LRR protein with an N‐terminal Pyrin domain, is also implicated in rare auto‐inflammatory disorders. In conclusion, NBS‐LRR molecules appear as a new family of intracellular receptors of innate immunity able to detect specific bacterial compounds and induce inflammatory response; the dysregulation of these processes due to mutations in the genes encoding these proteins is involved in numerous auto‐inflammatory disorders.

Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists

Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide‐binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro‐inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc‐L‐Ala‐γ‐D‐Glu‐meso‐diaminopimelic acid, MtriDAP) and NOD2 (MurNAc‐L‐Ala‐D‐isoGln, MDP; MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro‐inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro‐ and anti‐inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub‐active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll‐like receptor 4 to instruct the onset of adaptive immune responses.

NOD proteins: regulators of inflammation in health and disease

Entry of bacteria into host cells is an important virulence mechanism. Through peptidoglycan recognition, the nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2 enable detection of intracellular bacteria and promote their clearance through initiation of a pro-inflammatory transcriptional programme and other host defence pathways, including autophagy. Recent findings have expanded the scope of the cellular compartments monitored by NOD1 and NOD2 and have elucidated the signalling pathways that are triggered downstream of NOD activation. In vivo, NOD1 and NOD2 have complex roles, both during bacterial infection and at homeostasis. The association of alleles that encode constitutively active or constitutively inactive forms of NOD2 with different diseases highlights this complexity and indicates that a balanced level of NOD signalling is crucial for the maintenance of immune homeostasis.

A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N-acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA, in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-β response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system.

Are the PE‐PGRS proteins of Mycobacterium tuberculosis variable surface antigens?

Mycobacterium tuberculosis H37Rv contains 67 PE‐PGRS genes, with multiple tandem repetitive sequences, encoding closely related proteins that are exceptionally rich in glycine and alanine. As no functional information was available, 10 of these genes were selected and shown to be expressed in vitro by reverse transcription–polymerase chain reaction (RT–PCR). Antibodies against five PE‐PGRS proteins, raised in mice by DNA vaccination, detected single proteins when the same plasmid constructs used for immunization were expressed in epithelial cells or in reticulocyte extracts, confirming that the PE‐PGRS proteins are antigenic. As expected from the conserved repetitive structure, the antibodies cross‐reacted with more than one PE‐PGRS protein, suggesting that different proteins share common epitopes. PE‐PGRS proteins were detected by West‐ern blotting in five different mycobacterial species (M. tuberculosis, M. bovis BCG, M. smegmatis, M. marinum and M. gordonae) and 11 clinical isolates of M. tuberculosis. Whole‐genome comparisons of M. tuberculosis predicted allelic diversity in the PE‐PGRS family, and this was confirmed by immunoblot studies as size variants were detected in clinical strains. Subcellular fractionation studies and immunoelectron microscopy localized many PE‐PGRS proteins in the cell wall and cell membrane of M. tuberculosis. The data suggest that some PE‐PGRS proteins are variable surface antigens.

Conversion of PtdIns(4,5)P2 into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology

Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram‐negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4‐phosphatase that specifically dephosphorylates phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] into phosphatidylinositol 5‐monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P2 into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site.

The Ubiquitin-Editing Enzyme A20 Restricts Nucleotide-Binding Oligomerization Domain Containing 2-Triggered Signals

Muramyl dipeptide (MDP), a product of bacterial cell-wall peptidoglycan, activates innate immune cells by stimulating nucleotide-binding oligomerization domain containing 2 (NOD2) -dependent activation of the transcription factor NFkappaB and transcription of proinflammatory genes. A20 is a ubiquitin-modifying enzyme that restricts tumor necrosis factor (TNF) receptor and Toll-like receptor (TLR) -induced signals. We now show that MDP induces ubiquitylation of receptor- interacting protein 2 (RIP2) in primary macrophages. A20-deficient cells exhibit dramatically amplified responses to MDP, including increased RIP2 ubiquitylation, prolonged NFkappaB signaling, and increased production of proinflammatory cytokines. In addition, in vivo responses to MDP are exaggerated in A20-deficient mice and in chimeric mice bearing A20-deficient hematopoietic cells. These exaggerated responses occur independently of the TLR adaptors MyD88 and TRIF as well as TNF signals. These findings indicate that A20 directly restricts NOD2 induced signals in vitro and in vivo, and provide new insights into how these signals are physiologically restricted.