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Laboratory of Respiratory Biology,1 Laboratory of Molecular Toxicology, National Institutes of Environmental Health Sciences, NIH, 111 T.W. Alexander Dr., Durham, North Carolina 27709,3 Duke University Medical Center, Durham, North Carolina 27707,2 Nicholas School of the Environment and Earth Sciences, Duke University, Durham, North Carolina 277084
Received 6 November 2006/ Returned for modification 16 January 2007/ Accepted 16 May 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The nematode C. elegans is susceptible to many of the pathogens that infect humans, including gram-positive Staphylococcus aureus and gram-negative Serratia marcescens and Pseudomonas aeruginosa (28, 31, 45, 49, 52). Like most pathogens that infect C. elegans, these three bacteria colonize the digestive tract and ultimately kill the nematode. In contrast, other bacteria such as Escherichia coli and Bacillus subtilis are usually not toxic to C. elegans (13). Many of the host genes involved in pathogen defense in mammals also function in host defense of C. elegans (3, 10, 35, 46). Similarly, many virulence genes used by pathogens to infect C. elegans are also required for mammalian infection (3, 8, 31, 39, 48, 50, 53-55, 57). Thus, C. elegans has proven to be a useful and relatively simple model with which to study innate immunity.
The C. elegans innate immune response consists of the production of numerous antimicrobial proteins, many of which are conserved in higher organisms. Many candidate antimicrobial genes have been identified in the C. elegans genome (17, 40, 46). Previous data have demonstrated that the expression of some of these genes is induced upon pathogen infection (7, 32, 41, 47, 56). Moreover, some of these putative antimicrobial genes are regulated by signaling pathways involved in pathogen defense in nematodes and mammals (7, 21, 36, 38, 56). To better understand how these genes function in host defense, we have undertaken a study of a large, representative sample of candidate antimicrobial genes encoded in the C. elegans genome (Table 1).
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GFP expression patterns were visualized using differential interference contrast optics and fluorescence microscopy. To quantitate total nematode fluorescence, the COPAS Biosort (Union Biometrica) was used. All 14 gfp strains expressed green fluorescent protein (GFP) that was visible by microscopy; however, only nine strains were bright enough to score reliably using the COPAS Biosort. Thus, GFP localization data are presented for all 14 GFP fusions, but COPAS Biosort data are only presented for the nine brightest gfp fusions. As a control for the RNA interference (RNAi) experiments, a gfp fusion under the control of the non-immune-regulated scm promoter was used (S. Alper and C. Kenyon, unpublished data). To determine whether the gfp fusions were expressed in chemosensory neurons, the animals harboring the gfp fusions were stained by dye filling using DiI, which labels some chemosensory neurons (18). In cases where previous expression data were available (either as a gfp fusion or in situ data), our expression data were consistent with those data (7, 24, 32; http://www.wormbase.org).
RNAi of antimicrobial::gfp nematodes. RNAi was performed in liquid culture in a 96-well format, largely as described previously (2). Briefly, frozen stocks of E. coli RNAi bacteria were inoculated into LB medium supplemented with 80 µg/ml ampicillin plus 10 µg/ml tetracycline. After incubation overnight at 37°C, double-stranded RNA (dsRNA) production was induced by the addition of 2 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) (final concentration). After four hours, the bacteria were recovered by centrifugation and suspended in 1/4 of the volume of nematode growth (NG) medium supplemented with 80 µg/ml ampicillin and 2 mM IPTG. This medium (50 µl) was added to the wells in a 96-well plate. Nematode eggs (10 µl) isolated by bleaching (100 to 200 eggs) (59) were then added to the wells. Plates were covered with Breathe Easy film (USA Scientific) and assayed three days later on the COPAS Biosort. The control RNAi strain and the daf-2(RNAi) strain were from reference 9; all other RNAi strains were from MRC Geneservice (23). The identities of the bacterial RNAi constructs were verified by DNA sequencing.
Fluorescence was assayed using the COPAS Biosort (Union Biometrica), which reports time of flight (nematode length) and green fluorescence (GFP-induced fluorescence) for each individual animal. Three types of data analysis were performed after an initial filtering of the data to remove obvious outliers. First, overlay plots of fluorescence versus time of flight were generated to visualize expression of test RNAi-treated animals compared with control RNAi-treated animals (as depicted in Fig. 2A). Second, using boxplots as depicted in Fig. 2C, fluorescence was compared between animals of similar sizes (typically measuring 200 to 400 in arbitrary COPAS Biosort time-of-flight units). Third, mean expression values as a percent of control mean RNAi bacteria treatment were calculated for each sample, as depicted in Fig. 3. The scripts used for data analysis are available upon request.
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Real-time RT-PCR to measure antimicrobial RNA production in nematodes exposed to pathogens. CF512 [fer-15(b26) II; fem-1(hc17) IV] (12) eggs were isolated by bleaching and then plated on NG medium plates seeded with E. coli strain OP50. Eggs were allowed to hatch and were subsequently grown for two days at 25°C, a temperature at which these nematodes are sterile. Nematodes were then collected in M9 medium, washed with M9 supplemented with 10 µg/ml tetracycline, washed several times with M9 without supplement, and then plated. In the gram-negative experiments, synchronized CF512 animals were plated onto NG plates containing either E. coli strain OP50, S. marcescens strain IGX2 (11, 28), or P. aeruginosa strain PA14 (31, 44, 52). For the gram-positive experiments, the synchronized CF512 animals were plated onto brain heart infusion plates containing either the B. subtilis strain PY79 (60) or the S. aureus strain NCTC 8325 (22, 49) (except for the control experiment shown in Fig. 5C, in which S. aureus was grown on NG medium plates). After the indicated incubation (24 h in Fig. 5 or 12, 24, 36, or 48 h in Fig. S1 in the supplemental material), nematodes were processed and RNA production was measured by real-time RT-PCR as described above for the mutants. In these experiments, two additional duf-141 family members were examined by real-time RT-PCR that were not investigated in our GFP experiments.
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RESULTS |
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NLP-29 and SPP-1 (4, 7) have demonstrated antimicrobial activity. The three lysozyme genes, spp-7, and the two abf genes are homologs of genes with demonstrated antimicrobial function. In contrast, other candidate antimicrobial genes that we have chosen to examine are in families induced by pathogen infection, but their precise antimicrobial function is poorly understood. These include clec-85, a member of the C-type lectin family (32). C. elegans C-type lectins are hypothesized to play a role in pathogen recognition or clearance (40). Another family of genes whose precise function is unknown but which is induced by infection encodes a protein family containing Pfam domain PF02408, formerly known as the domain of unknown function number 141 (we refer to this family as the duf-141 family). Several members of this family are also reported to be regulated by the insulin signaling pathway (32, 38, 41). We fused gfp to the promoter for five of these genes. For simplicity, we refer to all 14 gfp fusions as antimicrobial::gfp fusions, although the precise functions of members of the duf-141 family are still uncertain.
C. elegans antimicrobial genes are expressed in tissues exposed to the environment. In the presence of the standard laboratory E. coli strain OP50 (59), thirteen of these fourteen antimicrobial::gfp fusions were expressed in the C. elegans intestine, a position where they would come into contact with ingested pathogens (Fig. 1A to G and I and Table 1). The pattern of this expression was largely uniform throughout the intestine, except in the case of spp-7, in which gfp expression was much stronger in the posterior intestine than in the anterior. spp-7 was also strongly expressed in several cells in the pharynx (primarily in the pm7 pharyngeal muscle cells), another organ in the digestive tract (Fig. 1I and J and Table 1). Although the intensity of gfp expression in the digestive tract was by far the strongest expression in all these antimicrobial::gfp strains, expression was also observed in other cells. This includes some head and tail neurons, including chemosensory neurons that are exposed to the environment, and the rectal gland cells, which are thought to secrete substances into the digestive tract (http://www.wormatlas.org) (Fig. 1D, F, and K to M and Table 1).
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C. elegans antimicrobial genes are regulated by known immune signaling pathways. Interestingly, all the antimicrobial::gfp fusions that we generated were expressed in the presence of the nonpathogenic strain of E. coli, OP50, often quite strongly. This raised the possibility that although OP50 is normally not toxic to C. elegans, it might be inducing an immune response. To test this hypothesis and to investigate the regulation of antimicrobial production in the nematode, we inhibited known immune response pathways using RNAi and examined the effect on antimicrobial production. To do this, we used E. coli that expressed dsRNA corresponding to genes in three known C. elegans immune signaling pathways: tir-1 and nsy-1 in the SARM-MAPKKK pathway, dbl-1 in the TGF-ß pathway, and daf-2 and daf-16 in the insulin signaling pathway. tir-1, nsy-1, dbl-1, and daf-16 mutant animals are all more susceptible to infection and are predicted to have a weaker antimicrobial response. daf-2 mutants, on the other hand, are more resistant to pathogens and are expected to have a stronger antimicrobial response. E. coli expressing these dsRNAs were fed to nematodes that carried the antimicrobial::gfp fusions, and fluorescence was measured to determine the effect on antimicrobial::gfp production.
As a control, E. coli expressing daf-16 dsRNA were fed to animals carrying the lys-7::gfp fusion. Previous microarray data indicated that daf-16+ is required for expression of lys-7 (38). In keeping with this result, we found that inhibition of daf-16 by RNAi inhibited lys-7::gfp expression (Fig. 2A and C). In daf-2 mutant animals, daf-16 is constitutively active and lys-7 is overexpressed (38). When daf-2 was inhibited by RNAi, lys-7::gfp expression increased (Fig. 2B and C). Thus, as expected, we can observe opposing effects of daf-2 and daf-16 on lys-7 expression.
Inhibition of tir-1 by RNAi inhibits the expression of two antimicrobial genes, nlp-29 and nlp-31 (7). When we used RNAi to inhibit tir-1 or nsy-1 in animals bearing the clec-85::gfp fusion, we found that expression of clec-85::gfp was strongly inhibited (Fig. 2D to F). Thus, wild-type tir-1 and nsy-1 are required for proper expression of this candidate antimicrobial gene, in keeping with their proposed role in host defense.
We next examined the effect of inhibition of each of the three different immune signaling pathways on nine of our antimicrobial gene fusions and on a control gfp fused to a promoter not involved in immunity. The other five antimicrobial::gfp fusions, while bright enough to assay by fluorescence microscopy, were too weak to assay on the COPAS Biosort used to quantitate nematode fluorescence and were therefore not used in this assay. Inhibition of each immune pathway affected a different subset of antimicrobial genes (Fig. 3). For example, inhibition of nsy-1 by RNAi led to a strong reduction in expression of lys-1, lys-8, clec-85, and dod-22, as assayed by reduced GFP fluorescence, but had a much more moderate or no effect on expression of the other gfp fusions. Interestingly, inhibition of tir-1, which is thought to act upstream of nsy-1 (5, 29), had an almost identical effect (compare Fig. 3A to B), causing strong inhibition of lys-1, lys-8, clec-85, and dod-22 expression and a much more moderate effect on the other genes. We also found that nlp-29::gfp fluorescence exhibited a moderate decrease when either nsy-1 or tir-1 were inhibited (data not shown), consistent with previous work (7). However, the level of this fluorescence was very close to background on the COPAS Biosort.
Inhibition of dbl-1 by RNAi had a different effect on antimicrobial gene expression. clec-85 expression was strongly decreased when dbl-1 was inhibited by RNAi (Fig. 3C). Inhibition of dbl-1 by RNAi also caused a much more moderate decrease in expression of the three lysozyme genes and dod-22 (Fig. 3C). Thus, the pattern of genes regulated by dlb-1 overlaps with but is distinct from those regulated by nsy-1 and tir-1.
Inhibition of daf-16 by RNAi had a very different effect, causing the strongest inhibition of lys-7::gfp expression and weaker inhibition of several other genes including lys-8 and clec-85 (Fig. 3D).
To verify the RNAi data generated with our antimicrobial::gfp fusions, we examined RNA production in available mutant animals using real-time RT-PCR. Wild-type and mutant animals were grown in liquid culture in the presence of E. coli, and RNA was isolated. Real-time RT-PCR was then used to examine antimicrobial RNA levels in different wild-type or mutant animals. In general, antimicrobial RNA production was decreased in mutant nematodes in a pattern analogous to that seen in the RNAi experiments using the antimicrobial::gfp animals (Fig. 4). For lys-1, lys-8, clec-85, nlp-29, and the duf-141 genes dod-22 and K08D8.5, RNA levels were decreased in animals harboring mutations in either of two alleles of nsy-1. Expression of the two defensin-like genes abf-1 and abf-3 and the two saposin-containing genes spp-1and spp-7 was not decreased. The pattern of genes affected by the tir-1 mutation was also similar to the pattern of those regulated by tir-1(RNAi), with lys-1, lys-8, and clec-85 expression all strongly reduced in the tir-1 mutant background.
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The daf-16 mutant animals exhibited a strong decrease in lys-7 expression, which was consistent with the RNAi data, as well as decreased expression of several other genes, particularly the three duf-141 family genes (Fig. 4D). lys-8 and spp-1 RNA levels exhibited much more moderate decreases in daf-16 mutant animals. Interestingly, several duf-141 family members that were positively regulated by daf-16+ in both assays were reported to be negatively regulated in previous microarray experiments (33, 38, 56). This is likely due to the fact that our experiments were carried out in an otherwise wild-type background where DAF-16 is minimally active, whereas the prior experiments were performed in a daf-2 mutant background in which DAF-16 is hyperactive.
Different pathogens induce expression of different antimicrobial genes. To explore the effects of different pathogenic bacteria on C. elegans antimicrobial gene expression, we exposed animals to the gram-negative pathogens S. marcescens or P. aeruginosa (using E. coli as a control) and the gram-positive pathogen S. aureus (using B. subtilis as a control) and then examined antimicrobial RNA levels using real-time RT-PCR. Exposure to either S. marcescens or P. aeruginosa resulted in similar but not identical patterns of antimicrobial gene induction (Fig. 5A). Some of the strongest genes induced by both gram-negative bacteria were members of the duf-141 gene family, in particular dod-22, F55G11.7, and K08D8.5. Expression of nlp-29 and clec-85 was also induced by both pathogens, although more weakly. In contrast, the expression of several genes, including lys-7 and spp-1, was actually stronger on E. coli than on the pathogens. The two gram-negative pathogens did not have identical effects, since several other genes such as lys-1 and the duf-141 gene F55G11.4 were induced by P. aeruginosa but not S. marcescens. In fact, expression of the duf-141 gene F10A3.4 was moderately induced on P. aeruginosa but repressed on S. marcescens. Experiments in which gene inductions by pathogen were monitored over time suggest that the differences in gene induction by P. aeruginosa and S. marcescens are largely due to a difference in magnitude of the response, although different temporal patterns of induction were observed as well (see Fig. S1 in the supplemental material).
In contrast, the gram-positive pathogen S. aureus induced expression of a different set of genes (Fig. 5B). Again, the strongest effects were on the duf-141 gene family, although the duf-141 family members induced by S. aureus (F10A3.4 and F54B11.11) were distinct from those induced by the gram-negative pathogens. Several other genes exhibited weak induction by S. aureus (spp-1, lys-7, abf-3, and nlp-29) and quite a few were down-regulated on S. aureus, including several genes that were induced by gram-negative bacteria.
Because gram-positive infection experiments of C. elegans are typically performed on richer media than are gram-negative infection experiments, we also examined the expression of the duf-141 gene family in nematodes exposed to S. aureus grown on NG medium (the less-rich medium used to grow gram-negative pathogens) and used E. coli as a control. The pattern of duf-141 genes induced under these nonstandard gram-positive infection conditions was similar but not identical to that observed when S. aureus was compared to B. subtilis on rich medium but was distinct from the pattern of induction caused by gram-negative pathogens (Fig. 5C).
The pathogen-mediated induction of C. elegans antimicrobial genes is regulated by known immune signaling pathways. To determine the effect of known immune signaling pathways on pathogen-mediated antimicrobial gene expression, we examined the effects of three different gram-negative bacteria (E. coli, S. marcescens, and P. aeruginosa) on nematodes harboring mutations in either nsy-1, dbl-1, or daf-16. For this analysis, we used real-time RT-PCR to monitor expression of the three duf-141 genes that were most strongly induced by gram-negative pathogens; we also examined pathogen-induced expression of the two genes that were regulated by all three immune signaling pathways in the presence of E. coli: clec-85 and lys-8. Interestingly, the effects of daf-16 and dbl-1 were more moderate in the presence of E. coli when the experiment was carried out on solid media (Fig. 6A and B) compared to liquid media (Fig. 4C and D), but all three pathways were required for full pathogen-mediated induction of clec-85 and lys-8 expression (Fig. 6A and B). Thus, all three pathways modulate clec-85 and lys-8 expression, regardless of the bacterial inducer. The duf-141 gene dod-22 was strongly regulated by nsy-1 and daf-16 but not dbl-1 when exposed to any of the three gram-negative bacteria (Fig. 6C). Similarly, all three pathways exerted differing effects on the regulation of F55G11.7 and K08D8.5 expression in the presence of different bacteria (Fig. 6D and E). Thus, these three signaling pathways affect not only the "basal" antimicrobial expression in the presence of nonpathogenic E. coli but also the "induced" antimicrobial expression when the nematodes are exposed to either S. marcescens or P. aeruginosa.
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DISCUSSION |
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Interestingly, all of the antimicrobial::gfp fusions were expressed in the presence of E. coli, which is usually not pathogenic to C. elegans (although E. coli can be toxic to C. elegans under certain special conditions or late in life) (12, 13, 15). Previous C. elegans studies used DNA microarrays to investigate gene regulation in response to infection and compared nematodes exposed to pathogens with nematodes exposed to nonpathogenic E. coli (7, 32, 41, 47, 56). However, because C. elegans does not usually encounter E. coli, its food source in the laboratory, in its native soil environment, it is possible that E. coli exposure itself could be inducing immune response genes. Consistent with this hypothesis, inhibition of the immune response pathways affected the expression of numerous antimicrobial genes. We also note that inhibition of tir-1 inhibits not only nlp-29 expression in the presence of pathogens but also the "basal" level of expression in the presence of E. coli (7). Moreover, we found that although some antimicrobial genes were induced when exposed to pathogens, some actually exhibited stronger expression in the presence of the nonpathogenic E. coli or B. subtilis control strains. Therefore, it is reasonable to think of E. coli as just one more bacteria to which C. elegans can sense and respond.
Several immune response signaling pathways have been identified in C. elegans. The best characterized pathway is the MAPK signaling pathway, which affects survival when nematodes are exposed to any of numerous pathogens. Previous data suggested that tir-1 functions through the nsy-1 MAPK pathway (5, 29). Moreover, tir-1 was shown to regulate two antimicrobial peptides, including nlp-29 (7). Here we show that many candidate antimicrobial genes are regulated by tir-1 and nsy-1 (Fig. 7). Notably, this pathway does not regulate all antimicrobial genes. Instead, the two genes in this pathway regulate an identical subset of the antimicrobial genes tested. The unique set of antimicrobial genes regulated by this pathway, or the tir-1 nsy-1 pathway "antimicrobial fingerprint," is distinct from that regulated by the other immune response pathways tested and may be useful in characterizing novel innate immunity genes and assigning them to known pathways. Consistent with this hypothesis, we have identified several novel genes in a genomic screen for antimicrobial gene regulators that have an "antimicrobial fingerprint" identical to tir-1 and nsy-1 (S. Alper, J. H. Freedman, and D. A. Schwartz, unpublished data).
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dbl-1 mutant animals are more susceptible to some pathogens, and several antimicrobial genes are reported to have altered expression in dbl-1 mutant animals, as determined using microarrays (32, 36). The set of genes regulated by dbl-1 in our study overlaps with but is distinct from those regulated by the tir-1 nsy-1 pathway (Fig. 7).
The daf-2 pathway regulates the rate of aging in C. elegans, with daf-2 mutant animals living longer than wild-type animals (26). The long life span of these daf-2 mutant animals depends on the activity of the downstream transcription factor daf-16 (26). daf-2 mutant animals are resistant to a wide variety of stresses, including pathogens, and this stress/pathogen resistance is also dependent on daf-16 (14). We identified several antimicrobial genes that were regulated by daf-16 (Fig. 7). Again, although there is overlap in the genes regulated by this signaling pathway and the two other pathways, the pattern of antimicrobial genes regulated by the insulin signaling pathway is still unique.
We find that a distinct set of genes is also induced by different pathogenic bacteria. The two gram-negative pathogens induced a similar but not identical set of antimicrobial genes. In contrast, the gram-positive pathogen tested induced a completely different set of genes. This pathogen-mediated gene induction required the same signaling pathways that were required for the "basal" antimicrobial gene expression present when nematodes were grown on E. coli. Not only were different genes induced by all three pathogens, but the genes induced cannot be assigned to any one genetic signaling pathway. Because no single pathway is activated by any given pathogen, this suggests that C. elegans can distinguish between different pathogens and activate multiple pathways that interact at some level to turn on the appropriate set of antimicrobial genes. In higher organisms, pathogen recognition receptors (PRRs) are thought to recognize a unique set of pathogenic compounds (termed pathogen-associated molecular patterns, or PAMPs) (34). The identities of the specific PAMPs recognized by C. elegans remain unclear, although Salmonella lipopolysaccharide has been identified as one potential PAMP (1). The identity of the C. elegans PRRs is still unclear, although our data as well as previously published microarray data (7, 21, 32, 41, 47, 56) suggest the existence of numerous PRRs that can uniquely identify different pathogens to induce a differential response. The most obvious candidate, tol-1, the only Toll-like receptor in C. elegans, plays a role in a pathogen avoidance response, but it does not affect pathogen susceptibility (43). Although the identity of the PRRs in C. elegans remains elusive, the complicated, highly specific response that we observed suggests that several exist. It remains to be seen whether these PRRs are similar to those used in higher organisms. However, the downstream immune signaling components identified in C. elegans so far are homologous to immune genes that function in higher organisms. It therefore seems likely that novel genes identified in C. elegans will play a role in immunity in higher organisms as well.
Although we have only examined several members of selected antimicrobial gene classes, we can draw some general conclusions based on our results and others reported in the literature. Different antimicrobial gene families exhibit distinct patterns of regulation in response to different pathogens. For example, we showed that different members of the duf-141 gene family are induced by each of the three pathogens and that this pattern is distinct from those family members induced by Microbacterium nematophilum (7, 32, 41, 47, 56). Likewise, different pathogens induce different members of the lysozyme family: lys-7 is induced by S. aureus, lys-1 and lys-8 are very weakly induced by S. marcescens (32) and more strongly induced by P. aeruginosa (as is lys-2) (47), and lys-3, lys-7, and lys-8 are induced by M. nematophilum (41). Different members of the C-type lectin family and the nlp-29 family are also induced by different pathogens (7, 32, 41, 47, 56). In contrast, the two defensin-like genes and the two saposin genes that we studied exhibited less change in response to pathogens and were not strongly regulated by any of the three immune signaling pathways, suggesting that their expression could be constitutive. As observed in higher organisms, we see some antimicrobial genes with inducible expression and some without.
We have identified many novel targets in each of the three signaling pathways tested. We note that two genes, clec-85 and lys-8, are regulated by all three signaling pathways. Therefore, clec-85::gfp and lys-8::gfp will be useful screening tools to identify novel innate immunity signaling genes or PRRs in C. elegans.
Innate immunity has traditionally been described as a relatively nonspecific response (in contrast to the highly specific adaptive immune response). More recently, a large body of work has demonstrated that far more specificity exists in the innate response than originally believed, and this response is critical to many aspects of host defense (58). Strikingly, even in the "more modest" C. elegans innate immunity model system, which lacks many of the cellular mechanisms of defense present in higher organisms, we can observe this complexity and specificity in response at the level of antimicrobial gene regulation.
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ACKNOWLEDGMENTS |
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Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources.
We thank Joy Alcedo, Cynthia Kenyon, Adam Driks, Emily Troemel, Fred Ausubel, and the NARSA repository for nematode and bacterial strains. Thanks to Julie Rice, Dan Snyder, and Windy Boyd for assistance with the COPAS Biosort, Brooke Baker for assistance with the confocal microscope, Dave Sherwood for use of his microscope, and Javier Apfeld for critical reading of the manuscript.
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FOOTNOTES |
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Published ahead of print on 25 May 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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