Caenorhabditis elegans as a model for innate immunity to pathogens

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1 Blackwell Science, LtdOxford, UKCMICellular Microbiology Blackwell Publishing Ltd, Review ArticleC. elegans as an innate immunity modelm. J. Gravato-Nobre and J. Hodgkin Cellular Microbiology (2005) 7(6), doi: /j x Microreview Caenorhabditis elegans as a model for innate immunity to pathogens Maria João Gravato-Nobre and Jonathan Hodgkin* Genetics Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Summary The amenability of the nematode Caenorhabditis elegans for genetic analysis and other experimentation provides a powerful tool for studying host pathogen interactions. Our current understanding of how C. elegans responds to pathogen challenges is in its infancy, but the discovery that the worm has inducible defence responses, which to some extent parallel those of other organisms, demonstrates the potential of this model organism for the study of innate immunity. Most progress in dissecting the C. elegans antimicrobial response has focused around signal transduction pathways and the expression of genes activated by the worm in response to microbial infections. Introduction All metazoan organisms are exposed to a wide range of microbes and have evolved complex immune defences used to repel infectious agents. In vertebrates, defence against infection is mediated by two highly interdependent immune systems known as innate and adaptive. However, an adaptive immune system, characterized by the creation by somatic rearrangement of a vast repertoire of recognition proteins (immunoglobulins and T-cell receptors), seems to be absent from all invertebrates. Consequently, with exception of vertebrates, most organisms rely exclusively on their innate immune mechanisms in their response against pathogens. Thus, the innate immune response seems to be the most universal and rapidly acting type of immunity. This response depends on the recognition and subsequent elimination of a pathogen Received 16 January, 2005; accepted 28 February, *For correspondence. jonathan.hodgkin@bioch.ox.ac.uk; Tel. (+44) ; Fax (+44) by genomically hard-wired mechanisms. The discovery that the innate defence systems of invertebrates, vertebrates (and even plants, to some extent) share striking similarities has been one of the most remarkable developments in modern immunology. Partly as a result of the simple and conserved mechanisms underpinning their innate immune systems, invertebrates have become popular subjects in the context of organismal immunology. The last decades have witnessed the non-vertebrate host model, Drosophila melanogaster, become a paradigm for the study of host-microbe interactions. Indeed, the fruit fly was important in the discovery of one of the most widely distributed molecules involved in defence, the Toll family of receptors (Lemaitre et al., 1996; Rutschmann et al., 2002). More recently, however, the nematode Caenorhabditis elegans has also been used to provide important insights into how animals perceive and defend themselves against infection. These studies have widened the spectrum of applications of C. elegans as a useful model organism, beyond existing popular fields such as developmental biology, neurobiology and ageing (Riddle et al., 1997). Many aspects of the interaction of C. elegans with pathogens have been described in recent reviews (Millet and Ewbank, 2004; Nicholas and Hodgkin, 2004a; Schulenburg et al., 2004). However, further advances in deciphering the worm immune system, such as the discovery of the signal transducer TIR-1 (Toll and IL-1 receptor) (Couillault et al., 2004; Liberati et al., 2004) and the implication of multiple MAPK cascades in different facets of pathogen response (Kim et al., 2004; Nicholas and Hodgkin, 2004b), provide the occasion for the present review. Here, we briefly introduce the immune system of C. elegans as a convenient model for genetic investigations exploring resistance to pathogen infection and discuss some of the more recently discovered aspects of different molecular mechanisms involved in the worm s defence against pathogens. We will focus particularly on how C. elegans investigations have contributed in establishing or confirming the conserved nature of signal transduction components required for pathogen resistance Blackwell Publishing Ltd

2 742 M. J. Gravato-Nobre and J. Hodgkin Why C. elegans? Meet the model host From a practical standpoint, the worm offers many advantages as a model for research on infectious diseases. These include low cost, simple maintenance, requirement of minimal laboratory space, and easy handling, as well as suitability for automated animal sorting. Its short life cycle (3 days at 25 C) and reproduction by self-fertilization have facilitated the development of advanced genetic methods, and its small and completely sequenced genome (100 Mb) has been exploited in the application of many post-genomic techniques. Moreover, other attributes such as the small size of the organism (1 mm long) and the transparency of its body have greatly facilitated the visualization of both developmental and infection processes. C. elegans is well suited for in vivo monitoring of genes specifically induced on infection, using transgenic lines carrying appropriate GFP-reporters. Lastly, the worm has the useful property of living by feeding on bacterial lawns. This is advantageous in two ways: first, it provides a convenient route for challenge and infection by microbial pathogens, and second, bacterial feeding can also be used for the application of RNA interference (RNAi)-based gene silencing, using Escherichia coli strains expressing double-stranded RNA (dsrna) corresponding to worm genes. Homologues of genes implicated in defence in other organisms can rapidly and cheaply be tested in C. elegans by this means, and new resistance or susceptibility genes can be efficiently identified using wholegenome RNAi screens (Kamath and Ahringer, 2003). Despite these advantages in the use of worms for pathogenesis studies, there are still several drawbacks which reduce the value of C. elegans as a model host for some applications. First, the worm lacks an adaptive immune system. Second, it seems to have few or no specialized cells equivalent to macrophages and neutrophils; the mobile scavenging phagocytic cells found in some other invertebrates such as Drosophila do not have exact counterparts in C. elegans. Scavenger cells called coelomocytes occur in the body cavity, which are capable of endocytosis, but their involvement in phagocytosis of bacteria seems unlikely. Another disadvantage is that worms cannot be reared at 37 C, the temperature required for expression of some virulence factors by some bacterial pathogens. Finally, C. elegans cannot yet be regarded as an experimental system for viruses, other intracellular pathogens, or multicellular parasites, as none of these has been reported to successfully infect the worm to date. Meet the pathogens and their modes of infection A wide and still expanding range of pathogens has been reported to infect or damage C. elegans (Hodgkin et al., 2000; Garsin et al., 2001; Couillault and Ewbank, 2002; Mylonakis et al., 2002; these have been reviewed by Ewbank, 2002; Alegado et al., 2003; Nicholas and Hodgkin, 2004a; Schulenburg et al., 2004; Sifri et al., 2005). A current list of such pathogens, which includes many different bacterial and fungal species, is provided in Table 1. Some of these pathogens, of which Pseudomonas aeruginosa is an example, are common soil organisms and could therefore be met by C. elegans in the wild and countered by general antimicrobial defence mechanisms (after Schulenburg et al., 2004). Others, such as Bacillus thuringiensis and Microbacterium nematophilum seem able to establish more specific interactions with the worm. Boxes indicate pathogens belonging to these two groups. With respect to mode of infection, many organisms are only pathogenic after ingestion by C. elegans, often exerting their effect in the anterior part of the intestine where they may establish an intestinal infection. In order to do this, at least some must manage to pass intact through the pharynx, surviving the action of the pharyngeal grinder, which is remarkably efficient at disrupting microbial cells. Once within the gut they can proliferate, causing distension and in some cases damaging the intestinal epithelium, leading eventually to death (Mylonakis et al., 2002; Kothe et al., 2003). The Gram-negative bacteria P. aeruginosa (PA14) (Mahajan-Miklos et al., 1999), Salmonella typhimurium and other Salmonella enterica serovars (Labrousse et al., 2000), Serratia marcescens (Mallo et al., 2002) and Burkholderia cepacia (O Quinn et al., 2001; Kothe et al., 2003) as well as the Gram-positive bacterium Enterococcus faecalis (Garsin et al., 2001) are examples of pathogens that can establish such intestinal infections. In addition, some pathogens can exert their effect through the production of bacterial toxins and other diffusible factors. This is the situation with P. aeruginosa PAO1 and some B. thuringiensis strains. Because of the chemical and mechanical properties of the cuticle, few bacteria and fungi are able to bind to or degrade the extracellular exoskeleton of the worm. Exceptions to this are the fungus Drechmeria coniospora and the bacteria Yersinia pestis and Yersinia pseudotuberculosis. Whereas the fungus adheres to the surface in the region of the mouth and vulva, and penetrates the worm s body (Jansson, 1994), the plague bacterium Y. pestis, and the closely related species Y. pseudotuberculosis, attach to the cuticle in the head region and form a biofilm, which covers the mouth and prevents feeding (Darby et al., 2002; Joshua et al., 2003). These simple infection or toxicity paradigms using C. elegans have been exploited as a method for defining virulence factors in pathogens, because the test system is so rapid, easy and inexpensive as compared with the use of mammalian hosts. C. elegans has been shown to

3 C. elegans as an innate immunity model 743 Table 1. A list of bacteria and fungi reported to be pathogenic in C. elegans. Infection foci Class of organism Species Pathogenic effect in C. elegans References Intestine Gram-negative bacteria Aeromonas hydrophila Not described 5 Burkholderia cepacia Toxin-mediated 16, 25 Burkholderia mallei Toxin-mediated 10 Burkholderia multivorans Toxin-mediated 25 Burkholderia pseudomallei Toxin-mediated 10, 25 Burkholderia thailandensis Toxin-mediated 25 Burkholderia vietnamiensis Toxin-mediated 25 Erwinia carotovora carotovora Infectious 5 Erwinia chrysanthemi Infectious 5 Photorhabdus luminescens Not described 5 Pseudomonas aeruginosa Toxin-mediated; infectious 6, 9, 20, 28, 29 Pseudomonas fluorescens Infectious 28 Salmonella enteritidis; S. dublin Toxin-mediated; Infectious 1 Salmonella enterica sv typhimurium Toxin-mediated; Infectious 1, 2, 3, 19 Serratia marcescens Infectious 17, 18, 21 Shewanella frigidmarina Not described 5 Shewanella massalia Not described 5 Shewanella oneidensis Not described 5 Xenorhabdus nematophila Not described 5 Escherichia coli Infectious 11 Gram-positive bacteria Agrobacterium tumefaciens Not described 5 Bacillus megaterium Not described 4 Bacillus thuringiensis Toxin-mediated 8, 22 Enterococcus faecalis Infectious 11, 27 Enterococcus faecium Hydrogen peroxide production 23 Staphylococcus aureus Not described 11 Streptococcus pneumoniae Hydrogen peroxide production 11, 13 Streptococcus pyogenes Hydrogen peroxide production 13 Fungus Cryptococcus neoformans Not described 24 Whole body/cuticle Gram-positive bacterium Streptomyces albireticuli Not described 26 Fungus Drechmeria coniospora Infectious 14 Head Gram-negative bacteria Yersinia pestis Biofilm formation 7, 15 Yersinia pseudotuberculosis Biofilm formation 7, 15 Anal region Gram-positive bacterium Microbacterium nematophilum Infectious 12 (1) Aballay et al. (2000); (2) Aballay and Ausubel (2001); (3) Aballay et al. (2003); (4) Andrew and Nicholas (1976); (5) Couillault and Ewbank (2002); (6) Darby et al. (1999); (7) Darby et al. (2002); (8) Devidas and Rehberger (1992); (9) Gallagher and Manoil (2001); (10) Gan et al. (2002); (11) Garsin et al. (2001); (12) Hodgkin et al. (2000); (13) Jansen et al. (2002); (14) Jansson (1994); (15) Joshua et al. (2003); (16) Kothe et al. (2003); (17) Kurz and Ewbank (2000); (18) Kurz et al. (2003); (19) Labrousse et al. (2000); (20) Mahajan-Miklos et al. (1999); (21) Mallo et al. (2002); (22) Marroquin et al. (2000); (23) Moy et al. (2004); (24) Mylonakis et al. (2002); (25) O Quinn et al. (2001); (26) Park et al. (2002); (27) Sifri et al. (2002); (28) Tan et al. (1999a); (29) Tan et al. (1999b). be particularly effective as a model for defining virulence factors in P. aeruginosa, Burkholderia pseudomallei, S. enterica, En. faecalis, Staphylococcus aureus, Streptococcus pneumoniae and Cryptococcus neoformans (Miller et al., 1989; Tan et al., 1999b; Garsin et al., 2001; Mylonakis et al., 2002; Sifri et al., 2002; Tenor et al. 2004). Several of the virulence-related genes that are required for mammalian pathogenesis have proved to be essential for pathogenicity in the worm. LasR in P. aeruginosa and PhoP/Q two-component regulators in Salmonella are examples of such virulence genes (Alegado et al., 2003). The worm immune system Evasion strategy Caenorhabditis elegans is a ubiquitous soil nematode which feeds on bacteria and is continuously exposed to a myriad of chemical, physical and biological dangers. The recognition and avoidance of such detrimental agents, which can be achieved by a change in its behavioural response, would therefore seem crucial to ensure its fitness in the soil. Indeed, C. elegans has a sophisticated navigation system which enables it to perceive and respond to many different physical and chemical cues, resulting in appropriate attraction to nutritious substances or repulsion from noxious factors. Worms are able to distinguish between different food bacteria (Andrew and Nicholas, 1976) and once confronted with pathogens, they can either move away from potentially toxic food, or cease its ingestion. This has been well illustrated in interactions between C. elegans and B. thuringiensis (Schulenburg and Muller, 2004) and also in the response of C. elegans to bacterial lawns of P. aeruginosa (Tan et al., 1999a), S. enterica (Aballay et al., 2000), or Bu. pseudomallei (O Quinn et al., 2001). Surprisingly, in one element of its behavioural avoidance repertoire, C. elegans may evade pathogens through

4 744 M. J. Gravato-Nobre and J. Hodgkin a process mediated by a Toll receptor (Pujol et al., 2001). The nematode has a single homologue of the Drosophila Toll gene, tol-1, which appears to have an essential developmental function because a probable null allele results in late embryonic or larval lethality, with developmental abnormalities. In contrast, mutant worms homozygous for another allele, a partial deletion that affects only the predicted cytoplasmic domain of TOL-1, are viable but are impaired in their ability to avoid a particular strain of Se. marcescens, Db11. It has been suggested that as TOL-1 is expressed in chemosensory neurons, it may contribute to the recognition of a pathogen-associated molecule, which in turn leads to a change in worm behaviour. Further evidence for the possible involvement of the worm s nervous system in defence has emerged recently from the finding that loss-of-function mutations in the DAF- 2 insulin/igf-receptor-like protein (abnormal dauer formation-2), increase resistance in worms exposed to certain Gram-negative and Gram-positive bacteria (Garsin et al., 2003). C. elegans daf-2 gene was previously known for its role in increasing longevity and enhancing resistance to diverse stress factors (Guarente and Kenyon, 2000), and the control of this pathway depends critically on inputs from the nervous system. (Wolkow et al., 2000). Physical barriers As described above, some pathogens are able to adhere to the surface of the worm and penetrate through natural openings such mouth, vulva and anus. The cuticle, being at the interface between the nematodes and its environment, clearly represents a major protective barrier against pathogens. This is further corroborated by the finding that C. elegans mutants with altered body surface antigenicity (srf-2/-3/-5) differ in their susceptibility to M. nematophilum (Hodgkin et al., 2000) and Yersinia spp. (Joshua et al., 2003; Hoflich et al., 2004) and to the trapping fungus Duddingtonia flagrans (Mendoza De Gives et al., 1999). The mouth of the worm is another important route for infection. Microbes entering the mouth encounter the pharynx, which breaks up bacteria and prevents bacterial cells from contacting the intestine. The importance of the pharynx as a major part of the defence mechanism is illustrated by phm-2 mutants, which have a defective pharyngeal grinder and are hypersensitive to P. aeruginosa and S. enterica (Labrousse et al., 2000; Kim et al., 2002; Smith et al., 2002; Tan, 2002). RNA interference: a genetic watchdog for viral pathogens in C. elegans? Although speculative, this section addresses the question of why C. elegans appears to suffer from no diseases caused by viruses. One partial explanation can be suggested from evidence that the worm uses a natural surveillance system, RNAi, for repressing aberrant gene activity such as transposon activation (Vastenhouw and Plasterk, 2001). Viruses as well as transposons are examples of dangerous parasites whose gene expression and replication need to be repressed. One way plants, fungi and animals have found to deal with such molecular parasites is to induce gene silencing at the posttranscriptional level, a process known as posttranscriptional gene silencing (PTGS), quelling or RNAi respectively (Ketting and Plasterk, 2000; Hammond et al., 2001). RNAi is a sequence specific response initially triggered by dsrna which subsequently targets any cytoplasmic RNA species sharing homology with the triggering sequence. RNA viruses replicate their genomes through complementary strands, resulting in dsrna replication intermediates that make them potentially vulnerable to RNAi (Voinnet, 2003). Plant RNA viruses are silenced in a similar way to transposons in C. elegans (Ketting and Plasterk, 2000), so it is conceivable that RNAi, which is particularly potent in C. elegans, may provide antiviral defence. An RNAi-dependent antiviral activity has been shown in Drosophila (Li et al., 2002) but a corresponding role in the worm remains to be established. It is possible that the lack of any known natural viruses for C. elegans simply reflects inadequate searches for such pathogens. However, it is remarkable that no viral diseases have been discovered for any other species of nematode, despite the fact that some of these have been studied in great ecological detail. Signalling pathways regulating immune responses in C. elegans Depending on the pathogen, one or more of at least six different signal transduction cascades appear to be activated on infection and lead to the production of effector molecules able to confine or destroy pathogens, or to other protective cellular responses. These cascades are: a transforming growth factor b-like pathway, an insulinreceptor-like pathway, a programmed cell death (PCD) pathway and finally three MAP kinase pathways (p38 MAP kinase, c-jun amino terminal kinase, and ERK (extracellular signal-regulated kinase) MAP kinase. Interestingly, as described below, there is evidence that these pathways interact, and most of them also respond to stress conditions, suggesting that C. elegans uses elements of stress response as part of its inducible immune defence against pathogens. Most of these pathways also play important roles in various developmental processes, and have been much studied in these contexts. Table 2 summarizes the information gathered from relevant studies, and the different pathways will be discussed in turn below.

5 C. elegans as an innate immunity model 745 Table 2. Regulatory signal transduction networks involved in innate immunity and stress responses. Biological role Signalling pathway Homologue Development Abiotic stresses Pathogen resistance References TGF-b-like Dauer; body size; male tail, axonal guidance Se. marcescens; P. aeruginosa 10, 13 DBL-1 TGF-b-like ligand SMA-6/DAF-4 Type I receptor/type II receptor SMA-2/-3/-4 Smad proteins Insulin-like receptor Insulin/IGF-1 receptor Dauer, longevity; brood size UV, heavy metal; thermotolerance DAF-2 Phosphatidylinositol 3-Kinase AGE-1 3-Phosphoinositide-dependent PDK-1 kinase DAF-16 FOXO family of transcription factor Oxidative stress, detoxification, heat-shock En. faecalis, St. aureus; P. aeruginosa Programmed cell death 131 cells undergo PCD during development S. enterica 1, 2 CED-9 BCL-2W CED-4 Apaf-1, caspase activator CED-3 Caspase Toll/Interleukin-receptor TOL-1 Toll-like receptor Bacterial Avoidance Se. marcescens 12 p38 MAPK TIR-1 SARM Osmotic stress; AWC RAB-1 Ras-related RAB-1A asymmetry D. coniospora; P. aeruginosa; En. faecalis NSY-1 ASK1 MAPKKK P. aeruginosa; S. enterica; En. faecalis; B. thuringiensis SEK-1 MKK3, MKK6 MAPKK PMK-1 p38 MAPK Osmotic/arsenical stress; heavy metal JNK-like MEK-1 MKK7 MAPKK Heavy metals KGB-1 JNK MAPK ERK MAP Kinase LIN-45 MEK-2 ERK MAPKK2 MPK-1 ERK MAPK B-Raf serine/threonine protein kinase Vulval, P12, excretory cell specification; male tail 5 4, 9 3, 7 P. aeruginosa; B. thuringiensis 6, 8 Osmotic regulation M. nematophilum 11 (1) Aballay et al. (2000); (2) Aballay and Ausubel (2001); (3) Aballay et al. (2003); (4) Couillault et al. (2004); (5) Garsin et al. (2003); (6) Huffman et al. (2004); (7) Kim et al. (2002); (8) Kim et al. (2004); (9) Liberati et al. (2004); (10) Mallo et al. (2002); (11) Nicholas and Hodgkin (2004b); (12) Pujol et al. (2001); (13) Tan (2001). AGE-1, ageing alteration; Apaf-1, apoptosis protease activating factor-1; ASK1, apoptosis signalling-regulating kinase-1: CED; cell death abnormality; DAF, dauer formation abnormal; DBL-1, decapentaplegic/bmp-like; ERK, extracellular signal-regulated kinase; FOXO, Forkhead box class O; IGF-1, insulin-like growth factor-1; JNK. C-Jun amino-terminal kinase; KGB-1, kinase, GLHbinding; MAP, mitogen-activated protein; MEK-1, MAP kinase kinase; MKK, MAP kinase kinase; MKK7, MAP kinase kinase; MPK-1, MAP kinase; NSY-1, neuronal symmetry; PMK-1, p38 MAP kinase family; SARM, sterile alpha and HEAT/Armadillo motif protein; SMA, Mothers against decapentaplegic homologue TGF-b, transforming growth factor b; TIR-1, Toll and IL-1 receptor domain protein.

6 746 M. J. Gravato-Nobre and J. Hodgkin A role for TGF-b signalling in C. elegans immunity to Se. marcescens and P. aeruginosa infections has been reported by Mallo et al. and by Tan respectively (Tan, 2001; Mallo et al., 2002). A clear host defence requirement for the TGF-b family ligand DBL-1 was demonstrated by Mallo and collaborators, who observed that the loss of dbl-1 render the worms hypersensitive to infection by Se. marcescens (Mallo et al., 2002). DBL-1 signalling regulates activation of the transducers sma-2, sma-3 and sma- 4. Mutations in all components of this cascade may also contribute to resistance against the Gram-negative bacterium P. aeruginosa (Tan, 2001). In C. elegans, the DBL-1 pathway was first discovered as a result of its role as a regulator of entry into dauer stage (diapause), body-size determination, development of male copulatory structures in the tail and axonal guidance (reviewed in Patterson and Padgett, 2000). The second cascade involved in the worm innate immune response is the DAF-2 insulin/igf-i signalling pathway. Long-lived worms with mutations in daf-2 (a transmembrane tyrosine kinase insulin receptor) and age- 1 (a downstream partner of daf-2) are resistant to Grampositive (En. faecalis and St. aureus) and Gram-negative (P. aeruginosa) bacteria (Garsin et al., 2003). Indirectly, DAF-2 is a negative regulator of DAF-16, a forkhead transcription factor, and the increased pathogen resistance conferred by the daf-2 mutation is completely suppressed in the daf-2; daf-16 double mutant. Microarray technology has been used to show that several DAF-16 targets are probable antimicrobial genes (e.g. the lysozyme genes lys-7 and lys-8), saposin genes including spp.-1, and thaumatin genes, which are known to contribute to immunity in plants (Murphy et al., 2003). Notably, lys-8 seems to be also under the transcriptional control of the TGF-blike receptor pathway (Mallo et al., 2002), illustrating overlap between these two signalling pathways. In addition, other downstream targets of DAF-16 include genes involved in detoxification (e.g. metallothioneins), resistance to oxidative stress (superoxidase dismutase, glutathione-s-transferase, catalase), and in general stress responses (heat shock proteins) (Lee et al., 2003; Murphy et al., 2003). Genes representing the major components of the PCD pathway in C. elegans are involved in immune response. Exposure of C. elegans to S. enterica leads to a persistent infection in the worm intestine and has the unusual effect of stimulating PCD in the germ line (Aballay et al., 2000). The PCD pathway in C. elegans involves: the caspase CED-3, which is bound and activated by CED-4; CED-9, a member of the Bcl-2 family of cell death regulators, which inhibits CED-4, and EGL-1 which can bind to and inhibit CED-9 (reviewed in Kaufmann and Hengartner, 2001). Loss-of-function mutations in the pro-apoptotic genes ced-3/-4 and egl-1 and gain-of-function mutations in the antiapoptotic gene ced-9 result in inhibition of the Salmonella-elicited PCD and decrease longevity in the presence of the bacterium. At present, there is no obvious mechanistic explanation of how germ line PCD can ameliorate the consequences of an intestinal infection. One upstream regulator of this pathogen-induced PCD response was shown to be the p38 MAPK homologue PMK-1 (Aballay and Ausubel, 2001), as it was demonstrated that in the context of Salmonella-elicited PCD, CED-9 functions downstream of PMK-1 (Aballay et al., 2003). In contrast, germ line cell death was found to have no significant role in the response of C. elegans to P. aeruginosa (Aballay and Ausubel, 2001), although the p38 MAP kinase cascade plays an important role in protection against P. aeruginosa infection. It follows that the PCD pathway is not the sole downstream target of the p38 MAP kinase cascade relevant to pathogen defence. The p38 MAP kinase cascade, which functions via the NSY-1/SEK-1/PMK-1 axis, was first shown to play a protective role in the response of C. elegans to infection in studies by Kim et al. (2002). From forward genetic screens these workers isolated mutants hypersensitive to infection by P. aeruginosa, and found that these included alleles of a MAP kinase kinase kinase, nsy-1, and its downstream MAP kinase kinase, sek-1. Inactivation of the predicted target kinase, PMK-1, by RNAi resulted in increased susceptibility to P. aeruginosa infection (Kim et al., 2002). An additional component of this cascade was demonstrated in recent studies by Chuang and Bargmann (2004). Work from this laboratory had previously established the developmental function of NSY-1 and SEK-1 in specifying developmental asymmetry of AWC olfactory neurons (Sagasti et al., 2001), and an additional gene required for this process, initially called nsy-2, was found to encode a protein called TIR-1, which interacts with NSY-1. This molecule had already attracted interest from the laboratories of Ewbank and Ausubel, because it has homology to TOL-1 in the conserved intracellular TIR signalling domain, and belongs to a family implicated in other kinds of immune response. Couillault and coworkers (Couillault et al., 2004) showed by RNAi experiments that TIR-1 is required for the induction of antifungal genes after infection by the pathogen D. coniospora. In addition, reduction of function of tir-1 by RNAi results in enhanced susceptibility to fungi (Couillault et al., 2004) and bacteria (Liberati et al., 2004). The latter group also demonstrated that for the antimicrobial responses, TIR-1 signalling controls activation of PMK-1 (Liberati et al., 2004). The upstream regulation of TIR-1 is context dependent. In the nervous system, it interacts with the calciumcalmodulin protein kinase II (CaMKII), UNC-43, but this protein does not seem to be required for the immune response (Kim et al., 2002), and the identity of upstream regulators for immune activation remain to be established.

7 C. elegans as an innate immunity model 747 In C. elegans, p38 MAPK PMK-1 also mediates resistance to osmotic stress (Solomon et al., 2004) and contributes to protection against pore-forming toxins (Huffman et al., 2004). The latter workers used microarrays to identify differentially regulated genes after exposure to bacteria expressing B. thuringiensis Cry5B poreforming toxin. To distinguish these genes from others that are non-specifically expressed in dying or severely stressed worms, they included control treatments involving exposure to cadmium. Interestingly, both PMK-1 and KGB-1 MAPK pathways were implicated in the response to toxin. The combined action of PMK-1 and KGB-1 resulted in the increased expression of ttm-1 and ttm-2 (toxin-regulated targets of MAPK), which function to increase resistance to pore-forming toxin. TTM-1 is a cation efflux channel that may serve to ameliorate the ionic imbalances caused by pore formation. KGB-1 is a component of a JNK-like MAPK pathway involved in heavy metal stress responses (Mizuno et al., 2004). Moreover, the activity of PMK-1 in innate defence can also be affected by components of c-jun NH2-terminal kinase (JNK)-signalling pathways (Kim et al., 2004). Studying the effects of the ingestion of pathogenic bacterium P. aeruginosa, Kim et al. showed that the MAPK kinase protein MEK-1 (a possible activator of the MAPK proteins JNK-1 and KGB-1) is required for full activation of PMK-1. However, although mek-1 mutants are hypersensitive to the bacterial infection, loss-of-function mutants of a downstream component of the JNK cascade, KGB-1, did not seem to affect susceptibility to this pathogen. In contrast, worms with mutations in the mek-1 gene are hypersensitive to heavy metals as are kgb-1 mutants (Koga et al., 2000; Villanueva et al., 2001) and possibly jnk-1 mutants (Villanueva et al., 2001). Taken together, these data suggest that MEK-1 can regulate innate immunity by activating PMK-1 or instead can mediate stress responses by activating KGB-1, or both. The MAPK phosphatase VHP-1 also modulates both pathways (Kim et al., 2004). The third MAPK cascade implicated in C. elegans immunity, for which an immune role has previously been well established in plants, is the ERK (extracellular signalregulated protein kinase) MAP kinase pathway. The requirement for this cascade in worm defence was found in research on the interaction between C. elegans and M. nematophilum (Nicholas and Hodgkin, 2004b). This Gram-positive bacterium is able to colonize the rectum and post-anal cuticle of infected worms and induce a pronounced swelling of the surrounding hypodermal cells. Other than this, the most obvious symptom of the infection is constipation. Nicholas and Hodgkin found that when MPK-1 ERK MAPK activity is reduced by mutations in central components of the cascade, the tail swelling response is abolished and severe constipation is induced after infection, resulting in growth arrest, sterility and death. The C. elegans ERK MAP kinase cascade serves numerous and varied functions, acting both in development and in non-developmental contexts. These include among others: vulval specification, development of the male spicules (Chamberlin and Sternberg, 1994), specification of the fate of the ventral cord neurectoblast, P12 (Jiang and Sternberg, 1998), specification of the excretory duct cell (Yochem et al., 1997), regulation of fluid balance (Huang and Stern, 2004), perception and transmission of sensory signals (Hirotsu et al., 2000). In most contexts the cascade is activated by the LET-60 Ras protein, but surprisingly the response to M. nematophilum does not seem to be affected by loss-of-function or gain-of-function in Ras, suggesting that a different input may be involved. The six signalling pathways discussed so far appear to contribute in different ways to protect worms against pathogens, and also appear to be activated in a pathogenspecific way. However, it remains to be seen whether these responses are triggered by specific recognition of pathogen molecules, rather than being more general reactions to the different kinds of organismal stress that may result from different kinds of infection. Some studies have begun to address this question, for example by comparing the effects of Cry5B toxin with the effects of cadmium poisoning as a general stress (Huffman et al., 2004). Also, Aballay et al. (2003) showed that some S. enterica mutants with altered lipopolysaccharide (LPS) failed to elicit germ line PCD, despite accumulating to high titres in the intestinal lumen, which suggests that specific recognition of LPS may be occurring. Antimicrobial and detoxifying effector molecules The studies discussed above reveal that C. elegans possesses inducible immune defences, which presumably act by the production of effector molecules that directly destroy and/or inhibit the growth of pathogens, or counteract their effects on host cells. The worm has multiple families of genes that are candidates for these roles, acting either as antimicrobial factors or as protective proteins. Examples of inducible components of the worm s immune system were demonstrated in exploring the response to Se. marcescens (Mallo et al., 2002). Genes encoding the lysozymes lys-1/-7/-8 and a lipase (ZK6.7) were seen to be upregulated on infection. Furthermore, transgenic worms that overexpress lys-1 were more resistant to infection by Se. marcescens. Lysozymes degrade bacterial peptidoglycan and are known to contribute to immunity in invertebrates and vertebrates (Leippe, 1999). Another group of potential effector molecules include caenopores (amoebapore-like enzymes) or saposin-like proteins. Amoebapores are lipid-degrading enzymes and

8 748 M. J. Gravato-Nobre and J. Hodgkin pore-forming peptides implicated in innate immunity in mammals. Because of their capacity to form ion-channels in the membranes of the target cells these peptides possess antibacterial activity (Banyai and Patthy, 1998). Twenty proteins containing saposin domains are present in C. elegans genome, encoded by genes spp-1 spp-20. As noted above, spp-1 is under the control of the forkhead transcription factor DAF-16, a component of the insulinlike receptor pathway (Murphy et al., 2003). The YGGYG family of antimicrobial peptides is a new group of effector molecules recently identified in the worm. Microarrays showed expression of two related peptides to be upregulated after infection by D. coniospora and Se. marcescens (Couillault et al., 2004). One of these was the NLP-29 (neuropeptide-like protein), previously regarded as a possible neurotransmitter (Nathoo et al., 2001). The other, cnc-2 (caenacin-2: Caenorhabditis bacteriocin-2) had not previously been characterized but was found to have a primary structure similar to that of NLP-29. C. elegans genome has another five peptides closely related to CNC-2, which along with this peptide have since been named caenacins. Additionally, four other members of these peptide-encoding genes were also induced specifically after D. coniospora infection, nlp-31, nlp-33, cnc-4 and cnc-6. Chemically synthesized NLP-31 showed potent antifungal activity against D. coniospora, Neurospora crassa and Aspergillus fumigatus, and some residual antibacterial effect against the Gram-negative E. coli and the Gram-positive Micrococcus luteus. NLP-31 expression is partly dependent on TIR-1, the immunetransducer molecule described earlier (Couillault et al., 2004). Six ABF peptides, ABF-1 to ABF-6 were identified in the worm based on their homology to the ASABF (for Ascaris suum antibacterial factor). These peptides share certain structural features with insect defensins and are most closely related to the molluscan mysticin (Kato and Komatsu, 1996). ABF-1 and ABF-2 are constitutively expressed in the pharynx, and may play a role in defending the pharyngeal tissue against microbial infection (Kato et al., 2002). Although none of the ASABF homologues have yet been shown to be upregulated in response to infection in C. elegans, a number of those in A. suum are induced after injection of heat-killed bacteria into the pseudocoelom of this nematode (Pillai et al., 2003). It seems likely that these peptides represent just the tip of the iceberg, with respect to antimicrobial and protective factors produced by C. elegans. Many of the genes seen to be upregulated in microarray experiments such as those of Mallo et al., 2002 and Huffman et al., 2004 encode proteins with no obvious similarities or functions, but these could well be contributing to defence in novel ways, and should repay more detailed investigation. Conclusions A premise of this review is that C. elegans is able to discriminate different kinds of pathogenic attack and possesses inducible defence systems to protect itself. One way to survive such challenges is an evasion/avoidance strategy, because the worm has the capacity to move away from noxious environments and towards more accommodating surroundings. Alternatively, the worm can use its innate immune system to promote activation of defence-related molecules and cellular changes. The various MAPK signalling cascades induced on infection represent perhaps the most ancient of evolutionarily conserved pathways of immunity, being present from plants to invertebrates and mammals (Asai et al., 2002). Three classes of MAPKs function as mediators of both immune signalling and stress in the worms. It has also emerged that the worm s innate immune response exhibits considerable specificity, depending on the nature of the pathogen and the conditions of infection. In addition, C. elegans immune system is under the control of complex regulatory networks, which can maintain homeostasis while accurately distinguishing pathogenic infections from harmless exposures, and producing the most appropriate response for each kind of pathogen. Studies on C. elegans offer the potential to provide a very thorough description of innate immunity and its integration into the general development and physiology of the organism. 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