- Email: [email protected]
Battling enteroinvasive bacteria: Nod1 comes to the rescue
Update
TRENDS in Microbiology
uncovered a remarkable interaction and it will be exciting to determine whether E2 is a passive hitchhiker that tags along with a convenient cellular protein, or rather a hijacker that captures this protein and modifies or makes use of its function for its own devices. References 1 Skiadopoulos, M.H. and McBride, A.A. (1998) Bovine papillomavirus type 1 genomes and the E2 transactivator protein are closely associated with mitotic chromatin. J. Virol. 72, 2079–2088 2 Lehman, C.W. and Botchan, M.R. (1998) Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 95, 4338–4343 3 Bastien, N. and McBride, A.A. (2000) Interaction of the papillomavirus E2 with mitotic chromosomes. Virology 270, 124–134 4 Ilves, I. et al. (1999) Long-term episomal maintenance of bovine papillomavirus type 1 plasmids is determined by attachment to host chromosomes, which is mediated by the viral E2 protein and its binding sites. J. Virol. 73, 4404–4412 5 You, J. et al. (2004) Interaction of the bovine papillomavirus e2 protein with brd4 tethers the viral DNA to host mitotic chromosomes. Cell 117, 349–360 6 Piirsoo, M. et al. (1996) Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator. EMBO J. 15, 1–11 7 Florence, B. and Faller, D.V. (2001) You bet-cha: a novel family of transcriptional regulators. Front. Biosci. 6, D1008–D1018 8 Dey, A. et al. (2000) A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition. Mol. Cell. Biol. 20, 6537–6549 9 Dey, A. et al. (2003) The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc. Natl. Acad. Sci. U. S. A. 100, 8758–8763
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10 Maruyama, T. et al. (2002) A Mammalian bromodomain protein, brd4, interacts with replication factor C and inhibits progression to S phase. Mol. Cell. Biol. 22, 6509–6520 11 Jiang, Y.W. et al. (1998) Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways. Proc. Natl. Acad. Sci. U. S. A. 95, 8538–8543 12 Houzelstein, D. et al. (2002) Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4. Mol. Cell. Biol. 22, 3794–3802 13 Abroi, A. et al. (2004) Analysis of chromatin attachment and partitioning functions of bovine papillomavirus type 1 E2 protein. J. Virol. 78, 2100–2113 14 Wu, H. et al. (2000) The DNA segregation mechanism of Epstein-Barr virus nuclear antigen 1. EMBO Rep. 1, 140–144 15 Krithivas, A. et al. (2002) Protein interactions targeting the latencyassociated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus to cell chromosomes. J. Virol. 76, 11596–11604 16 Platt, G.M. et al. (1999) Latent nuclear antigen of Kaposi’s sarcoma-associated herpesvirus interacts with RING3, a homolog of the Drosophila female sterile homeotic (fsh) gene. J. Virol. 73, 9789–9795 17 Cotter, M.A. and Robertson, E.S. (1999) The latency-associated nuclear antigen tethers the Kaposi’s sarcoma- associated herpesvirus genome to host chromosomes in body cavity-based lymphoma cells. Virology 264, 254–264 18 Van Tine, B.A. et al. (2004) Human papillomavirus (HPV) originbinding protein associates with mitotic spindles to enable viral DNA partitioning. Proc. Natl. Acad. Sci. U. S. A. 101, 4030–4035
0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.10.002
Battling enteroinvasive bacteria: Nod1 comes to the rescue Mathias Chamaillard, Naohiro Inohara and Gabriel Nun˜ez Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA
Recognition of pathogenic bacteria by mammalian hosts is largely mediated by membrane-bound Toll-like receptors (TLRs). Recently, a family of cytosolic proteins, termed NODs, with homology to plant disease-resistance gene products has been implicated in sensing microbes within the cytosol. The role of NOD family members in host defense is largely unknown. However, a recent report revealed that Nod1 is a crucial sensor for certain enteroinvasive bacteria that avoid TLR signaling. This finding suggests that Nod1 plays an important role in the initial recognition of pathogenic bacteria at epithelial surfaces, such as the gut, where innate immune responses to commensal bacteria must be avoided. Bacterial pathogens that infect the gastrointestinal tract, including Salmonella, invasive Escherichia coli, Listeria, Corresponding author: Gabriel Nun˜ez ([email protected]).
www.sciencedirect.com
Shigella and Yersinia spp., are a major cause of morbidity and mortality worldwide. These bacteria actively induce their own uptake by epithelial cells that line the intestinal mucosa [1]. Successful elimination of enteroinvasive bacteria depends on the rapid recognition of the invading pathogen, which is crucial for the development of an immune response against the bacterium. The initial detection of invasive bacteria occurs at the mucosal surface, however, the mechanism of bacterial recognition by host epithelial cells remains poorly understood. In animals, detection of microbial pathogens by the innate immune system relies on specialized host receptors that recognize molecules expressed exclusively by microbes [2]. These molecular signatures (specific for microbes) are termed pathogen-associated molecular patterns (PAMPs) and are detected by specific host pattern-recognition molecules (PRMs) [2]. Classical PAMPs include components of the bacterial cell envelope, such as
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lipopolysaccharide (LPS), peptidoglycan (PGN), lipoproteins, flagellin and bacterial DNA. Because the structure of each PAMP is highly conserved and invariant in microorganisms of the same class, the animal can recognize most or all microbes with a limited number of PRMs. The activation of PRMs initiates innate immunity and provides signals for the successful development of adaptive immunity against the invading pathogen. There is compelling evidence that Toll-like receptors (TLRs) are major PRMs that are involved in the detection of microbial pathogens by professional antigen-presenting cells (APCs), including macrophages and dendritic cells [2]. TLRs are transmembrane proteins with ectodomains containing extracellular leucine-rich repeats (LRRs) that are essential for PAMP recognition. The role of TLRs in the recognition of invasive bacteria by intestinal epithelial cells is less clear. Because the intestinal mucosa is exposed to a large concentration of commensal bacteria that express a wide array of PAMPs, it is crucial that innate recognition and inflammatory responses are avoided. How the host epithelia avoid recognition of commensal bacteria to prevent harmful inflammation at intestinal sites is not well understood. In contrast to airway epithelial cells in the lung, there is mounting evidence that intestinal epithelial cells are hyporesponsive to several PAMPs [3]. The lack of TLR signaling in the intestinal epithelium could be explained at least in part by low expression levels of functional TLRs, such as TLR2 and TLR4 [4–6]. If TLRs cannot sense commensal bacteria, how might the intestinal epithelium detect invasive bacteria? A recent article [7], together with previous results from other investigators [8], suggest that Nod1 (an intracellular PRR) plays an important role in the recognition of invasive bacteria by intestinal epithelial. The NOD protein family Several experiments have revealed that host cells, including epithelial cells, can sense bacterial components in the cytosol, but the recognition system has remained elusive [9,10]. Recent studies, however, have identified a large family of cytosolic proteins (named NODs) that has been implicated in the detection of bacterial components in the cytosol. Originally the NOD protein family was identified by searching genomic databases for the presence of proteins with structural homology to Apaf-1, an important regulator of caspase activation and apoptosis [11]. The human NOD protein family contains more than 20 members, including Nod1, Nod2, cryopyrin and Ipaf [12]. These proteins exhibit remarkable structural homology to a class of proteins that are encoded by plant disease-resistance (R) genes. Plant R proteins recognize distinct effector molecules from invading pathogens and mediate a defense response resulting in plant disease resistance [13]. In plants, deficient recognition of pathogen products by R proteins leads to pathogen overgrowth and disease [13]. The great majority of mammalian and plant NODs contain an N-terminal effector domain, a centrally located nucleotide-binding oligomerization domain (NOD) and C-terminal LRRs [12]. Depending on the particular N-terminal effector domain, the protein can have a variety of functions. For example, Nod1, Nod2 and www.sciencedirect.com
Vol.12 No.12 December 2004
Ipaf contain a caspase-recruitment domain (CARD), whereas cryopyrin and another 13 additional proteins have a pyrin domain (PD) as an N-terminal effector domain. The LRRs serve as a ligand-sensing domain. Nod1 and Nod2 are PRMs and recognize conserved PGN structures Nod1 and Nod2 were the first mammalian members of the family to be identified and their functions are more clearly understood. Nod1 and Nod2 activate nuclear factor (NF)-kB through interaction with the downstream factor RICK (also known as RIP2) [11,14,15]. Nod1 and Nod2 recognize PGN-related molecules. PGN is the major component of the cell wall of Gram-positive bacteria, whereas in Gram-negative bacteria it resides as a thin layer in the periplasmic space. PGN is composed of glycan chains of two alternating amino sugars, N-acetylglucosamine (GlcNac) and N-acetylmuramic acid (MurNac) crosslinked to each other by short peptides. Small PGN molecules containing the amino acid meso-diaminopimelic acid (meso-DAP) are produced by intracellular hydrolases or released during bacterial growth and are sensed by Nod1 [16–18]. The minimal moiety in PGN recognized by Nod2 is muramyl dipeptide MurNAc-L-Ala-D-isoGln (MDP), whereas Nod1 detects g-D-glutamyl-meso-DAP (iE-DAP), a dipeptide primarily found in PGN from Gramnegative bacteria but also in certain Gram-positive bacteria, including Listeria monocytogenes and Bacillus spp. [18–20]. Macrophages that are isolated from mice deficient in Nod1 do not respond to iE-DAP but are responsive to LPS or MDP [18]. Thus, Nod1 and Nod2, and possibly other NOD family proteins, recognize PAMPs and act as PRMs. Possible role of Nod1 in the recognition of enteroinvasive bacteria Nod1 is expressed in macrophages and epithelial cells, including those lining the intestinal mucosa [11]. A crucial role for Nod1 in sensing of bacteria by epithelial cells was first suggested by Girardin et al. [8] who showed that NF-kB activation, induced by Shigella flexneri in 293 embryonic kidney cells, was inhibited by overexpression of dominant-negative Nod1. Furthermore, Nod1 is required for primary intestinal cells to respond to natural muropeptides secreted by Gram-negative bacteria [16]. Recently, Kim et al. [7] generated intestinal Caco-2 cell lines that have been stably transfected with dominantnegative Nod1 and assessed the response to invasive E. coli 029:NM. It was shown that E. coli 029:NM-induced NF-kB activation was greatly inhibited by dominantnegative Nod1. Furthermore, induction of NF-kB-dependent genes, such as CXC5 and CXCL8 (two neutrophil chemoattractants), was inhibited by dominant-negative Nod1. The inhibitory effect was specific, in that dominantnegative Nod1 did not inhibit NF-kB activation that had been induced by interleukin (IL)-1a or flagellin [7]. The latter results are consistent with previous studies that showed that NF-kB activation mediated by Nod1 is not affected by dominant-negative forms of MyD88, tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 or TRAF6 [11]. Collectively, the work by Kim et al. [7] and
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Vol.12 No.12 December 2004
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Lumen TLR
i-EDAP
TLR and NOD-stimulating Bacteria
?
TLR-stimulating Bacteria NOD-stimulating Bacteria
NOD1
TLR-stimulating molecule NOD-stimulating molecule
NF-κB
NF-κB Neutrophils Proinflammatory cytokines Chemoattractants
M-cell
Dendritic cell Macrophages Lamina propria TRENDS in Microbiology
Figure 1. Host sensing of enteropathogenic bacteria. Enteroinvasive bacteria are sensed by specific cells (intestinal epithelial cells, M cells, macrophages and dendritic cells) located in the intestinal mucosa. Resident and invasive bacteria and their molecules released into the intestinal lumen could be recognized by host cells. Sensing of bacteria and their products is mediated by surface Toll-like receptors (TLRs) and cytosolic Nod1 receptors. Intestinal epithelial cells lack functional TLR2 and TLR4 but they might express TLR5 at the basolateral surface [21]. Thus, some entero-invasive flagellate bacteria might stimulate epithelial cells through both TLR5 and Nod1 (depicted in red), whereas other invasive bacteria might activate Nod1 but not TLRs (depicted in green). Flagellate Gram-positive bacteria lacking Nod1-stimulating molecules are expected to trigger TLRs but not Nod1 signaling (depicted in blue). Soluble TLR- and Nod1-stimulating products are found in the intestinal contents but their role in host defense is unknown. Certain TLRs might be also localized to intracellular compartments (e.g. Golgi apparatus for TLR4 [22]), but the relevance of intracellular TLR signaling in the intestinal mucosa remains elusive.
others [16] suggest an important role for Nod1 in the detection of invasive bacteria in intestinal epithelial cells. A cautionary note is that the studies to date have relied on intestinal cell lines, and understanding the role of Nod1 in innate recognition of enteroinvasive bacteria awaits further investigation in vivo. In contrast to most TLRs, Nod1 is an intracellular protein, and thus, recognition of bacteria through Nod1 would signal potentially harmful invasion in the intestinal tissue and the need for a host immune response against the pathogen. Commensal bacteria, which do not typically invade the epithelium, are not expected to be sensed by Nod1 (Figure 1). Nonetheless, DAP-containing bacteria that are resident in the intestine can release Nod1 stimulatory molecules that might enter the cytosol and induce Nod1 signaling in epithelial cells (Figure 1). However, the physiological relevance of these soluble Nod1 stimulatory molecules in intestinal immunity is presently unclear.
Concluding remarks Members of the NOD protein family have been recently implicated in cytosolic recognition of conserved microbial structures and upon stimulation they activate a defense program against the invading pathogen. Recent studies suggest that Nod1, which is expressed in intestinal epithelial cells, plays a crucial role in the response to enteroinvasive bacteria through the activation of NF-kB and proinflammatory genes. Future studies should clarify the role of Nod1 during bacterial infection in vivo. www.sciencedirect.com
Acknowledgements Work on Nod proteins in our laboratories is supported by grants from the National Institutes of Health (Gabriel Nun˜ez and Naohiro Inohara) and Broad Medical Research Program (Gabriel Nun˜ez). Mathias Chamaillard is supported by a postdoctoral fellowship from the Crohn’s and Colitis Foundation of America.
References 1 Cossart, P. and Sansonetti, P.J. (2004) Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304, 242–248 2 Medzhitov, R. (2001) Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145 3 Abreu, M.T. et al. (2003) TLR signaling at the intestinal epithelial interface. J. Endotoxin Res. 9, 322–330 4 Hausmann, M. et al. (2002) Toll-like receptors 2 and 4 are upregulated during intestinal inflammation. Gastroenterology 122, 1987–2000 5 Melmed, G. et al. (2003) Human intestinal epithelial cells are broadly unresponsive to Toll-like receptor 2-dependent bacterial ligands: implications for host–microbial interactions in the gut. J. Immunol. 170, 1406–1415 6 Abreu, M.T. et al. (2001) Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J. Immunol. 167, 1609–1616 7 Kim, J.G. et al. (2004) Nod1 is an essential signal transducer in intestinal epithelial cells infected with bacteria that avoid recognition by toll-like receptors. Infect. Immun. 72, 1487–1495 8 Girardin, S.E. et al. (2001) CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri. EMBO Rep. 2, 736–742 9 O’Riordan, M. et al. (2002) Innate recognition of bacteria by a macrophage cytosolic surveillance pathway. Proc. Natl. Acad. Sci. U. S. A. 99, 13861–13866 10 Philpott, D.J. et al. (2000) Invasive Shigella flexneri activates
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NF-kappa B through a lipopolysaccharide-dependent innate intracellular response and leads to IL-8 expression in epithelial cells. J. Immunol. 165, 903–914 Inohara, N. et al. (1999) Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J. Biol. Chem. 274, 14560–14567 Inohara, N. and Nunez, G. (2003) NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3, 371–382 Dangl, J.L. and Jones, J.D. (2001) Plant pathogens and integrated defence responses to infection. Nature 411, 826–833 Kobayashi, K. et al. (2002) RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature 416, 194–199 Ogura, Y. et al. (2001) Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J. Biol. Chem. 276, 4812–4818 Girardin, S.E. et al. (2003) Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300, 1584–1587 Girardin, S.E. et al. (2003) Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J. Biol. Chem. 278, 41702–41708
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18 Chamaillard, M. et al. (2003) An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4, 702–707 19 Girardin, S.E. et al. (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, 8869–8872 20 Inohara, N. et al. (2003) Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J. Biol. Chem. 278, 5509–5512 21 Gewirtz, A.T. et al. (2001) Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J. Immunol. 167, 1882–1885 22 Hornef, M.W. et al. (2002) Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells. J. Exp. Med. 195, 559–570
0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.10.001
Microbial Genomics
It’s a cold world out there (but the prospects are hot) Barbara Methe´, Karen E. Nelson and Claire M. Fraser The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
A significant portion of the Earth’s biosphere is cold and marine in nature and exposed permanently to temperatures below 58C. For the most part, these frigid regions of the world are colonized by cold-adapted microorganisms, which makes the study of these psychrophiles or ‘coldloving’ microbes an important step in deciphering global microbial diversity [1,2]. Psychrophilic bacteria are generally defined as having an optimum growth temperature of greater than 158C; and a maximum growth temperature of less than 208C [3]. Although the application of genomics to the investigation of polar environments is in its formative stages, one area in which genomics is already creating an impact in polar biology investigations is through the analysis of genome sequences from psychrophilic bacteria and archaea. The genesis of this sequence information is also creating the tantalizing ability to compare these cold-adapted microbes with their mesophilic (middle-temperature-loving) and thermophilic (hot-loving) counterparts. The ability to conduct these genomic comparisons has important implications across diverse areas of both basic and applied science. For example, these genomic comparisons can facilitate a better understanding of the evolution of cold-adaptation in bacteria, while at the same time elucidating probable metabolic pathways and metabolic potential, thereby increasing our comprehension of their physiological roles in biogeochemical cycling. Microbes that live in extreme environments frequently exhibit unique and interesting metabolic properties or adaptations that make them attractive candidates for biotechnological applications. Therefore, these analyses are crucial for (i) the discovery of novel proteins and secondary metabolites, (ii) the Corresponding author: Claire M. Fraser ([email protected]).
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employment of microorganisms as potential agents of wastewater treatment and in the bioremediation of toxic pollutants in cold environments (including temperate environments that experience a winter season) and (iii) the use of cold-adapted enzymes in industrial processes. Cold-adapted enzymes (e.g. lipases and proteases) are already being used in industry, in areas as diverse as leather tanning, food processing and in laundry detergent for cold-water washing of clothes [4]. The discovery of new and innovative applications for coldadapted enzymes in industrial processes has hot prospects. The recent publication of the genome of the psychrophilic d-proteobacterium Desulfotalea psychrophila [5] represents an initial step in the efforts to decipher adaptations to cold. D. psychrophila is a sulfate-reducing bacterium that was isolated from permanently cold Artic sediments. This bacterium grows optimally at 108C but is capable of growth at temperatures below 08C, and is thought to contribute to the global cycles of carbon and sulfur. Its genome consists of one large circular chromosome (3.5 Mbp) and two small plasmids of 121.6 kb and 14.6 kb in size. A TRAP-T (tripartite ATP-independent periplasmic transporter) transport system was identified as the major route of uptake for C4-carboxylates; many genes of the TCA (tricarboxylic acid) cycle and a twin arginine transport (TAT) secretion system were also identified. Of additional interest was the apparent lack of homologues in D. psychrophila to several c-type cytochromes (c553, c3, ncc) that have been found in other sulfate-reducing lineages of the d-proteobacteria. The D. psychrophila genome is the first psychrophilic bacterium to be sequenced, although several other genome projects are known to be under way, including those for Colwellia psychrerythraea, a member
TRENDS in Microbiology
uncovered a remarkable interaction and it will be exciting to determine whether E2 is a passive hitchhiker that tags along with a convenient cellular protein, or rather a hijacker that captures this protein and modifies or makes use of its function for its own devices. References 1 Skiadopoulos, M.H. and McBride, A.A. (1998) Bovine papillomavirus type 1 genomes and the E2 transactivator protein are closely associated with mitotic chromatin. J. Virol. 72, 2079–2088 2 Lehman, C.W. and Botchan, M.R. (1998) Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 95, 4338–4343 3 Bastien, N. and McBride, A.A. (2000) Interaction of the papillomavirus E2 with mitotic chromosomes. Virology 270, 124–134 4 Ilves, I. et al. (1999) Long-term episomal maintenance of bovine papillomavirus type 1 plasmids is determined by attachment to host chromosomes, which is mediated by the viral E2 protein and its binding sites. J. Virol. 73, 4404–4412 5 You, J. et al. (2004) Interaction of the bovine papillomavirus e2 protein with brd4 tethers the viral DNA to host mitotic chromosomes. Cell 117, 349–360 6 Piirsoo, M. et al. (1996) Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator. EMBO J. 15, 1–11 7 Florence, B. and Faller, D.V. (2001) You bet-cha: a novel family of transcriptional regulators. Front. Biosci. 6, D1008–D1018 8 Dey, A. et al. (2000) A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition. Mol. Cell. Biol. 20, 6537–6549 9 Dey, A. et al. (2003) The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc. Natl. Acad. Sci. U. S. A. 100, 8758–8763
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10 Maruyama, T. et al. (2002) A Mammalian bromodomain protein, brd4, interacts with replication factor C and inhibits progression to S phase. Mol. Cell. Biol. 22, 6509–6520 11 Jiang, Y.W. et al. (1998) Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways. Proc. Natl. Acad. Sci. U. S. A. 95, 8538–8543 12 Houzelstein, D. et al. (2002) Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4. Mol. Cell. Biol. 22, 3794–3802 13 Abroi, A. et al. (2004) Analysis of chromatin attachment and partitioning functions of bovine papillomavirus type 1 E2 protein. J. Virol. 78, 2100–2113 14 Wu, H. et al. (2000) The DNA segregation mechanism of Epstein-Barr virus nuclear antigen 1. EMBO Rep. 1, 140–144 15 Krithivas, A. et al. (2002) Protein interactions targeting the latencyassociated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus to cell chromosomes. J. Virol. 76, 11596–11604 16 Platt, G.M. et al. (1999) Latent nuclear antigen of Kaposi’s sarcoma-associated herpesvirus interacts with RING3, a homolog of the Drosophila female sterile homeotic (fsh) gene. J. Virol. 73, 9789–9795 17 Cotter, M.A. and Robertson, E.S. (1999) The latency-associated nuclear antigen tethers the Kaposi’s sarcoma- associated herpesvirus genome to host chromosomes in body cavity-based lymphoma cells. Virology 264, 254–264 18 Van Tine, B.A. et al. (2004) Human papillomavirus (HPV) originbinding protein associates with mitotic spindles to enable viral DNA partitioning. Proc. Natl. Acad. Sci. U. S. A. 101, 4030–4035
0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.10.002
Battling enteroinvasive bacteria: Nod1 comes to the rescue Mathias Chamaillard, Naohiro Inohara and Gabriel Nun˜ez Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA
Recognition of pathogenic bacteria by mammalian hosts is largely mediated by membrane-bound Toll-like receptors (TLRs). Recently, a family of cytosolic proteins, termed NODs, with homology to plant disease-resistance gene products has been implicated in sensing microbes within the cytosol. The role of NOD family members in host defense is largely unknown. However, a recent report revealed that Nod1 is a crucial sensor for certain enteroinvasive bacteria that avoid TLR signaling. This finding suggests that Nod1 plays an important role in the initial recognition of pathogenic bacteria at epithelial surfaces, such as the gut, where innate immune responses to commensal bacteria must be avoided. Bacterial pathogens that infect the gastrointestinal tract, including Salmonella, invasive Escherichia coli, Listeria, Corresponding author: Gabriel Nun˜ez ([email protected]).
www.sciencedirect.com
Shigella and Yersinia spp., are a major cause of morbidity and mortality worldwide. These bacteria actively induce their own uptake by epithelial cells that line the intestinal mucosa [1]. Successful elimination of enteroinvasive bacteria depends on the rapid recognition of the invading pathogen, which is crucial for the development of an immune response against the bacterium. The initial detection of invasive bacteria occurs at the mucosal surface, however, the mechanism of bacterial recognition by host epithelial cells remains poorly understood. In animals, detection of microbial pathogens by the innate immune system relies on specialized host receptors that recognize molecules expressed exclusively by microbes [2]. These molecular signatures (specific for microbes) are termed pathogen-associated molecular patterns (PAMPs) and are detected by specific host pattern-recognition molecules (PRMs) [2]. Classical PAMPs include components of the bacterial cell envelope, such as
530
Update
TRENDS in Microbiology
lipopolysaccharide (LPS), peptidoglycan (PGN), lipoproteins, flagellin and bacterial DNA. Because the structure of each PAMP is highly conserved and invariant in microorganisms of the same class, the animal can recognize most or all microbes with a limited number of PRMs. The activation of PRMs initiates innate immunity and provides signals for the successful development of adaptive immunity against the invading pathogen. There is compelling evidence that Toll-like receptors (TLRs) are major PRMs that are involved in the detection of microbial pathogens by professional antigen-presenting cells (APCs), including macrophages and dendritic cells [2]. TLRs are transmembrane proteins with ectodomains containing extracellular leucine-rich repeats (LRRs) that are essential for PAMP recognition. The role of TLRs in the recognition of invasive bacteria by intestinal epithelial cells is less clear. Because the intestinal mucosa is exposed to a large concentration of commensal bacteria that express a wide array of PAMPs, it is crucial that innate recognition and inflammatory responses are avoided. How the host epithelia avoid recognition of commensal bacteria to prevent harmful inflammation at intestinal sites is not well understood. In contrast to airway epithelial cells in the lung, there is mounting evidence that intestinal epithelial cells are hyporesponsive to several PAMPs [3]. The lack of TLR signaling in the intestinal epithelium could be explained at least in part by low expression levels of functional TLRs, such as TLR2 and TLR4 [4–6]. If TLRs cannot sense commensal bacteria, how might the intestinal epithelium detect invasive bacteria? A recent article [7], together with previous results from other investigators [8], suggest that Nod1 (an intracellular PRR) plays an important role in the recognition of invasive bacteria by intestinal epithelial. The NOD protein family Several experiments have revealed that host cells, including epithelial cells, can sense bacterial components in the cytosol, but the recognition system has remained elusive [9,10]. Recent studies, however, have identified a large family of cytosolic proteins (named NODs) that has been implicated in the detection of bacterial components in the cytosol. Originally the NOD protein family was identified by searching genomic databases for the presence of proteins with structural homology to Apaf-1, an important regulator of caspase activation and apoptosis [11]. The human NOD protein family contains more than 20 members, including Nod1, Nod2, cryopyrin and Ipaf [12]. These proteins exhibit remarkable structural homology to a class of proteins that are encoded by plant disease-resistance (R) genes. Plant R proteins recognize distinct effector molecules from invading pathogens and mediate a defense response resulting in plant disease resistance [13]. In plants, deficient recognition of pathogen products by R proteins leads to pathogen overgrowth and disease [13]. The great majority of mammalian and plant NODs contain an N-terminal effector domain, a centrally located nucleotide-binding oligomerization domain (NOD) and C-terminal LRRs [12]. Depending on the particular N-terminal effector domain, the protein can have a variety of functions. For example, Nod1, Nod2 and www.sciencedirect.com
Vol.12 No.12 December 2004
Ipaf contain a caspase-recruitment domain (CARD), whereas cryopyrin and another 13 additional proteins have a pyrin domain (PD) as an N-terminal effector domain. The LRRs serve as a ligand-sensing domain. Nod1 and Nod2 are PRMs and recognize conserved PGN structures Nod1 and Nod2 were the first mammalian members of the family to be identified and their functions are more clearly understood. Nod1 and Nod2 activate nuclear factor (NF)-kB through interaction with the downstream factor RICK (also known as RIP2) [11,14,15]. Nod1 and Nod2 recognize PGN-related molecules. PGN is the major component of the cell wall of Gram-positive bacteria, whereas in Gram-negative bacteria it resides as a thin layer in the periplasmic space. PGN is composed of glycan chains of two alternating amino sugars, N-acetylglucosamine (GlcNac) and N-acetylmuramic acid (MurNac) crosslinked to each other by short peptides. Small PGN molecules containing the amino acid meso-diaminopimelic acid (meso-DAP) are produced by intracellular hydrolases or released during bacterial growth and are sensed by Nod1 [16–18]. The minimal moiety in PGN recognized by Nod2 is muramyl dipeptide MurNAc-L-Ala-D-isoGln (MDP), whereas Nod1 detects g-D-glutamyl-meso-DAP (iE-DAP), a dipeptide primarily found in PGN from Gramnegative bacteria but also in certain Gram-positive bacteria, including Listeria monocytogenes and Bacillus spp. [18–20]. Macrophages that are isolated from mice deficient in Nod1 do not respond to iE-DAP but are responsive to LPS or MDP [18]. Thus, Nod1 and Nod2, and possibly other NOD family proteins, recognize PAMPs and act as PRMs. Possible role of Nod1 in the recognition of enteroinvasive bacteria Nod1 is expressed in macrophages and epithelial cells, including those lining the intestinal mucosa [11]. A crucial role for Nod1 in sensing of bacteria by epithelial cells was first suggested by Girardin et al. [8] who showed that NF-kB activation, induced by Shigella flexneri in 293 embryonic kidney cells, was inhibited by overexpression of dominant-negative Nod1. Furthermore, Nod1 is required for primary intestinal cells to respond to natural muropeptides secreted by Gram-negative bacteria [16]. Recently, Kim et al. [7] generated intestinal Caco-2 cell lines that have been stably transfected with dominantnegative Nod1 and assessed the response to invasive E. coli 029:NM. It was shown that E. coli 029:NM-induced NF-kB activation was greatly inhibited by dominantnegative Nod1. Furthermore, induction of NF-kB-dependent genes, such as CXC5 and CXCL8 (two neutrophil chemoattractants), was inhibited by dominant-negative Nod1. The inhibitory effect was specific, in that dominantnegative Nod1 did not inhibit NF-kB activation that had been induced by interleukin (IL)-1a or flagellin [7]. The latter results are consistent with previous studies that showed that NF-kB activation mediated by Nod1 is not affected by dominant-negative forms of MyD88, tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 or TRAF6 [11]. Collectively, the work by Kim et al. [7] and
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Figure 1. Host sensing of enteropathogenic bacteria. Enteroinvasive bacteria are sensed by specific cells (intestinal epithelial cells, M cells, macrophages and dendritic cells) located in the intestinal mucosa. Resident and invasive bacteria and their molecules released into the intestinal lumen could be recognized by host cells. Sensing of bacteria and their products is mediated by surface Toll-like receptors (TLRs) and cytosolic Nod1 receptors. Intestinal epithelial cells lack functional TLR2 and TLR4 but they might express TLR5 at the basolateral surface [21]. Thus, some entero-invasive flagellate bacteria might stimulate epithelial cells through both TLR5 and Nod1 (depicted in red), whereas other invasive bacteria might activate Nod1 but not TLRs (depicted in green). Flagellate Gram-positive bacteria lacking Nod1-stimulating molecules are expected to trigger TLRs but not Nod1 signaling (depicted in blue). Soluble TLR- and Nod1-stimulating products are found in the intestinal contents but their role in host defense is unknown. Certain TLRs might be also localized to intracellular compartments (e.g. Golgi apparatus for TLR4 [22]), but the relevance of intracellular TLR signaling in the intestinal mucosa remains elusive.
others [16] suggest an important role for Nod1 in the detection of invasive bacteria in intestinal epithelial cells. A cautionary note is that the studies to date have relied on intestinal cell lines, and understanding the role of Nod1 in innate recognition of enteroinvasive bacteria awaits further investigation in vivo. In contrast to most TLRs, Nod1 is an intracellular protein, and thus, recognition of bacteria through Nod1 would signal potentially harmful invasion in the intestinal tissue and the need for a host immune response against the pathogen. Commensal bacteria, which do not typically invade the epithelium, are not expected to be sensed by Nod1 (Figure 1). Nonetheless, DAP-containing bacteria that are resident in the intestine can release Nod1 stimulatory molecules that might enter the cytosol and induce Nod1 signaling in epithelial cells (Figure 1). However, the physiological relevance of these soluble Nod1 stimulatory molecules in intestinal immunity is presently unclear.
Concluding remarks Members of the NOD protein family have been recently implicated in cytosolic recognition of conserved microbial structures and upon stimulation they activate a defense program against the invading pathogen. Recent studies suggest that Nod1, which is expressed in intestinal epithelial cells, plays a crucial role in the response to enteroinvasive bacteria through the activation of NF-kB and proinflammatory genes. Future studies should clarify the role of Nod1 during bacterial infection in vivo. www.sciencedirect.com
Acknowledgements Work on Nod proteins in our laboratories is supported by grants from the National Institutes of Health (Gabriel Nun˜ez and Naohiro Inohara) and Broad Medical Research Program (Gabriel Nun˜ez). Mathias Chamaillard is supported by a postdoctoral fellowship from the Crohn’s and Colitis Foundation of America.
References 1 Cossart, P. and Sansonetti, P.J. (2004) Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304, 242–248 2 Medzhitov, R. (2001) Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145 3 Abreu, M.T. et al. (2003) TLR signaling at the intestinal epithelial interface. J. Endotoxin Res. 9, 322–330 4 Hausmann, M. et al. (2002) Toll-like receptors 2 and 4 are upregulated during intestinal inflammation. Gastroenterology 122, 1987–2000 5 Melmed, G. et al. (2003) Human intestinal epithelial cells are broadly unresponsive to Toll-like receptor 2-dependent bacterial ligands: implications for host–microbial interactions in the gut. J. Immunol. 170, 1406–1415 6 Abreu, M.T. et al. (2001) Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J. Immunol. 167, 1609–1616 7 Kim, J.G. et al. (2004) Nod1 is an essential signal transducer in intestinal epithelial cells infected with bacteria that avoid recognition by toll-like receptors. Infect. Immun. 72, 1487–1495 8 Girardin, S.E. et al. (2001) CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri. EMBO Rep. 2, 736–742 9 O’Riordan, M. et al. (2002) Innate recognition of bacteria by a macrophage cytosolic surveillance pathway. Proc. Natl. Acad. Sci. U. S. A. 99, 13861–13866 10 Philpott, D.J. et al. (2000) Invasive Shigella flexneri activates
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NF-kappa B through a lipopolysaccharide-dependent innate intracellular response and leads to IL-8 expression in epithelial cells. J. Immunol. 165, 903–914 Inohara, N. et al. (1999) Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J. Biol. Chem. 274, 14560–14567 Inohara, N. and Nunez, G. (2003) NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3, 371–382 Dangl, J.L. and Jones, J.D. (2001) Plant pathogens and integrated defence responses to infection. Nature 411, 826–833 Kobayashi, K. et al. (2002) RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature 416, 194–199 Ogura, Y. et al. (2001) Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J. Biol. Chem. 276, 4812–4818 Girardin, S.E. et al. (2003) Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300, 1584–1587 Girardin, S.E. et al. (2003) Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J. Biol. Chem. 278, 41702–41708
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18 Chamaillard, M. et al. (2003) An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4, 702–707 19 Girardin, S.E. et al. (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, 8869–8872 20 Inohara, N. et al. (2003) Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J. Biol. Chem. 278, 5509–5512 21 Gewirtz, A.T. et al. (2001) Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J. Immunol. 167, 1882–1885 22 Hornef, M.W. et al. (2002) Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells. J. Exp. Med. 195, 559–570
0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.10.001
Microbial Genomics
It’s a cold world out there (but the prospects are hot) Barbara Methe´, Karen E. Nelson and Claire M. Fraser The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
A significant portion of the Earth’s biosphere is cold and marine in nature and exposed permanently to temperatures below 58C. For the most part, these frigid regions of the world are colonized by cold-adapted microorganisms, which makes the study of these psychrophiles or ‘coldloving’ microbes an important step in deciphering global microbial diversity [1,2]. Psychrophilic bacteria are generally defined as having an optimum growth temperature of greater than 158C; and a maximum growth temperature of less than 208C [3]. Although the application of genomics to the investigation of polar environments is in its formative stages, one area in which genomics is already creating an impact in polar biology investigations is through the analysis of genome sequences from psychrophilic bacteria and archaea. The genesis of this sequence information is also creating the tantalizing ability to compare these cold-adapted microbes with their mesophilic (middle-temperature-loving) and thermophilic (hot-loving) counterparts. The ability to conduct these genomic comparisons has important implications across diverse areas of both basic and applied science. For example, these genomic comparisons can facilitate a better understanding of the evolution of cold-adaptation in bacteria, while at the same time elucidating probable metabolic pathways and metabolic potential, thereby increasing our comprehension of their physiological roles in biogeochemical cycling. Microbes that live in extreme environments frequently exhibit unique and interesting metabolic properties or adaptations that make them attractive candidates for biotechnological applications. Therefore, these analyses are crucial for (i) the discovery of novel proteins and secondary metabolites, (ii) the Corresponding author: Claire M. Fraser ([email protected]).
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employment of microorganisms as potential agents of wastewater treatment and in the bioremediation of toxic pollutants in cold environments (including temperate environments that experience a winter season) and (iii) the use of cold-adapted enzymes in industrial processes. Cold-adapted enzymes (e.g. lipases and proteases) are already being used in industry, in areas as diverse as leather tanning, food processing and in laundry detergent for cold-water washing of clothes [4]. The discovery of new and innovative applications for coldadapted enzymes in industrial processes has hot prospects. The recent publication of the genome of the psychrophilic d-proteobacterium Desulfotalea psychrophila [5] represents an initial step in the efforts to decipher adaptations to cold. D. psychrophila is a sulfate-reducing bacterium that was isolated from permanently cold Artic sediments. This bacterium grows optimally at 108C but is capable of growth at temperatures below 08C, and is thought to contribute to the global cycles of carbon and sulfur. Its genome consists of one large circular chromosome (3.5 Mbp) and two small plasmids of 121.6 kb and 14.6 kb in size. A TRAP-T (tripartite ATP-independent periplasmic transporter) transport system was identified as the major route of uptake for C4-carboxylates; many genes of the TCA (tricarboxylic acid) cycle and a twin arginine transport (TAT) secretion system were also identified. Of additional interest was the apparent lack of homologues in D. psychrophila to several c-type cytochromes (c553, c3, ncc) that have been found in other sulfate-reducing lineages of the d-proteobacteria. The D. psychrophila genome is the first psychrophilic bacterium to be sequenced, although several other genome projects are known to be under way, including those for Colwellia psychrerythraea, a member