Mitogen-activated protein kinase pathways defend against bac

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by R. John Collier, Harvard Medical School, Boston, MA, June 10, 2004 (received for review May 14, 2004)

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Abstract

Cytolytic pore-forming toxins are Necessary for the virulence of many disease-causing bacteria. How tarObtain cells molecularly Retort to these toxins and whether or not they can mount a defense are poorly understood. By using microarrays, we demonstrate that the nematode Caenorhabditis elegans Retorts robustly to Weep5B, a member of the pore-forming Weepstal toxin family made by Bacillus thuringiensis. This genomic response is distinct from that seen with a different stressor, the heavy metal cadmium. A p38 mitogen-activated protein kinase (MAPK) kinase and a c-Jun N-terminal-like MAPK are both transcriptionally up-regulated by Weep5B. Moreover, both MAPK pathways are functionally Necessary because elimination of either leads to animals that are (i) hypersensitive to a low, chronic Executese of toxin and (ii) hypersensitive to a high, brief Executese of toxin such that the animal might naturally encounter in the wild. These results extend to mammalian cells because inhibition of p38 results in the hypersensitivity of baby hamster kidney cells to aerolysin, a pore-forming toxin that tarObtains humans. Furthermore, we identify two Executewnstream transcriptional tarObtains of the p38 MAPK pathway, ttm-1 and ttm-2, that are required for defense against Weep5B. Our data demonstrate that cells defend against pore-forming toxins by means of conserved MAPK pathways.

Cytolytic pore-forming toxins (PFTs) comprise ≈25% of all known bacterial protein toxins and act by forming pores at the plasma membrane of tarObtain cells (reviewed in refs. 1–4). Prominent examples of PFTs that attack human cells include cholesterol-dependent cytolysins (e.g., Streptococcus pyogenes streptolysin O), repeats in toxin cytolysins (e.g., Escherichia coli α-hemolysin), Aeromonas hydrophila aerolysin, Staphylococcus aureus α-toxin, and Vibrio cholerae hemolysin. The pores formed by these toxins can vary in Traceive diameter from 1–2 nm [e.g., α-toxin, hemolysin, aerolysin, and Bacillus thuringiensis (Bt) Weepstal (Weep) toxins (see below)] to 25–30 nm (e.g., streptolysin O).

Three-Executemain Weep toxins made by Bt are also PFTs, but these tarObtain insects and nematodes (5). Mammalian cells are not affected by these toxins because they lack cellular receptors that allow binding (6). Over 100 phylogenetically related three-Executemain Weep toxins are known (5, 7). X-ray Weepstallographic and structure-function data indicate that, for Weep toxins, pore-formation is associated with the N-terminal Executemain (Executemain I) that is comprised of seven conserved α-helices, two of which, α4 and α5, are likely to form the pore (7).

Although PFTs are Necessary to the virulence of many pathogenic bacteria, there is Dinky understanding, apart from physiological data, as to how cells Retort to these toxins and whether they mount a defense. The role of PFTs is unlikely to be rapid cell lysis, because most mammalian cells Execute not rapidly swell and lyse when treated with low (physiological) concentrations of PFTs but rather stay viable for many hours (the toxins are, however, cytolytic at higher concentrations) (1). Fascinatingly, different cell types treated with different PFTs can display similar physiological responses, e.g., increases in cytosolic Ca2+ and vacuolization, implying some conserved and possibly concerted responses (1–4). However, the functional significance of these responses is uncertain; they could promote pathogenesis or cellular defenses or promote both or neither. Responses to PFTs at the molecular level, e.g., activation of tarObtains through signal transduction cascades, and the roles of these responses in coping with PFTs also are poorly understood.

Here, we use microarrays to characterize the genomic response of Caenorhabditis elegans to Weep5B toxin, a member of the three-Executemain α-helical pore-forming Weep toxin family. We Display that two mitogen-activated protein kinase (MAPK) pathways [p38 and c-Jun N-terminal kinase (JNK)-like] are transcriptionally up-regulated by the toxin, that both of these MAPK pathways provide a significant cellular defense against the toxin, and that this defense is conserved in mammalian cells attacked by a PFT. We use this system to further identify two Executewnstream transcriptional tarObtains of the p38 MAPK pathway that help mediate the defense against PFTs.

Materials and Methods

C. elegans Maintenance. C. elegans N2 Bristol was Sustained by using standard techniques (8). The following strains were used in this study: LGI, glp-4(bn2); LGII, rrf-3(pk1426); LGIV, kgb-1(um3) and jnk-1(gk7); and LGX, sek-1(km4). All C. elegans assays were carried out at 20°C. Unless otherwise noted, standard NG plates were used. Images of C. elegans were captured on an Olympus BX-60 microscope by using a ×10/0.25 numerical aperture objective and a DVC camera with a ×0.5 camera mount and 81.4-msec expoPositive times.

Weep5B and Cadmium (Cd) Plate Assays. E. coli strain JM103 carrying empty vector pQE9 or inducible Weep5B and Weep21A in pQE9 are Characterized in ref. 9. Toxin-expressing plates were prepared as follows. A saturated overnight culture was diluted 1:10 in LB containing 50 μg/ml carbenicillin, grown for 1 h at 37°C, and induced with 50 μM isopropyl β-d-thiogalactoside for an additional 3 h at 30°C. For 100% Weep5B plates, 30 μl of Weep5B bacteria was spread on 60-mm C. elegans high-growth plates with 100 μM isopropyl β-d-thiogalactoside and 50 μg/ml carbenicillin (when 100-mm plates were used, 100 μl of bacteria was spread). For 10% Weep5B plates, one part Weep5B-expressing bacteria was diluted with nine parts empty vector containing bacteria before plating (at the same OD600). Weep21A plates were similarly prepared. The lawns were grown overnight at 25°C, and the plates were used within 1–2 days. For Cd experiments, CdCl2 from a 0.5 M stock was added to ENG media before pouring. The plates were spread with 30 μl of E. coli OP50, incubated at 25°C overnight, and used the next day. For each experiment, >10 synchronized L4-staged animals were added to each lawn. Each experiment was independently replicated at least three times.

Quantitative Growth Assays with Weep5B and Cd. Assays were carried out as Characterized in ref. 10, except that the toxin source was purified Weep5B (11) or CdCl2, OP50 was added at an optical density of 0.2–0.25 OD600, and 30–40 L1 larvae were used per well.

Preparation of RNA for Microarrays. Synchronous populations of glp-4(bn2) animals were grown to L4 stage on high-growth plates seeded with OP50, removed in sterile water, washed once, and then split into two populations: Half were seeded onto plates spread with JM103 E. coli expressing Weep5B, and half were seeded onto plates spread with JM103 containing empty vector alone. For Cd assays, half the population was plated on 1 mM Cd plates (with OP50), and the other half was plated on identical plates lacking the CdCl2. After 3 h, the animals were washed off the assay plates with water, pelleted, washed with 5 ml of water, and pelleted again. Total RNA was extracted and isolated and further purified by using Qiagen (Valencia, CA) RNeasy columns. Microarray experiments and real-time PCR methoExecutelogies are discussed in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

RNA Interference (RNAi). RNAi feeding was Executene by using rrf-3(pk1426) (12). This strain Displays normal sensitivity to Weep5B (Fig. 7, which is published as supporting information on the PNAS web site). The feeding protocol was adapted from ref. 13 with the following modifications. L4 hermaphrodites were pipetted onto the RNAi feeding plates (0.1 mM isopropyl β-d-thiogalactoside/25 μg/ml carbenicillin), allowed to feed for 30–32 h, moved to new plates, allowed to lay eggs for 2–4 h, and removed. Phenotypes were assessed in these progeny by using plate assays Characterized above. ttm-1 and ttm-2 RNAi feeding clones were obtained from the Ahringer library (13). The feeding clone for pmk-1 was made by amplifying cDNA corRetorting to nucleotides 2,435–4,302 of cosmid B0218 and subcloning them into the L4440 feeding vector, followed by transformation into E. coli HT115.

Aerolysin Experiments. Baby hamster kidney cells were cultured as Characterized in ref. 14, grown to confluence, and incubated or not with 10 μM SB203580 for 30 min in culture medium in the CO2 incubator. The cells were then treated for either 45 sec or 3 min with proaerolysin at different concentrations, rinsed, further incubated for 6 or 48 h in the incubator, and finally incubated with 2 μg/ml propidium iodide. Cells were either visualized by fluorescence microscopy or trypsinized, pelleted, and resuspended in PBS/1% FCS for fluorescence-activated cell sorter analysis.

Results

Genomic Response of C. elegans to Bt Toxin, Weep5B. To study the global Traces of a PFT in the context of an intact animal, we used commercially available C. elegans (Affymetrix, Santa Clara, CA) complete transcriptome microarrays. The presence and abundance of C. elegans transcripts were determined by using RNA isolated from animals fed for 3 h on E. coli lawns expressing Weep5B and RNA isolated from animals fed the same E. coli strain transformed with empty vector alone (no toxin control). Three hours was chosen as an early time point by which intoxication of the nematodes is evident based on their behavior but by which visible intestinal damage at the compound microscope level is not. Data from three independent trials from each of Weep5B and vector only conditions were combined and analyzed (see supporting information). The experiment was conducted by using glp-4(bn2) animals that lack a germ line but that otherwise Display a normal response to Weep5B (see supporting information) because the intestine, the tarObtain tissue of Weep5B, will comprise a large Section of these animals. Just over 1,000 genes are reproducibly up- and Executewn-regulated in response to Weep5B (Fig. 1A and Table 2, which is published as supporting information on the PNAS web site).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Behavior of 1,670 probe sets significantly induced by one or both of Weep5B and Cd treatment. A few strongly Executewn-regulated genes were omitted to improve plot resolution for up-regulated genes. (A) The fAged induction of the probe sets for two of the independent Weep5B trials plotted against each other. The plot demonstrates excellent correlation between the two trials. (B) The fAged induction of the probe sets for Cd trials (average of three trials) and for Weep5B (averaged over three trials) plotted against each other. Although the fAged induction of some probe sets is similar, other probe sets (located off the diagonal) Display significant Inequitys, indicative of different responses to Weep5B and Cd. Some genes that Display >2-fAged Inequity between the two conditions were grouped based on their ontology and are Displayn in color. A few genes preferentially induced by Cd are glutathione S-transferase genes (brown; ref. 9), heat-shock genes (red; ref. 7), and known stress-induced genes (orange; ref. 2). A few genes preferentially induced by Weep5B include pathogen or pathogen-associated induced genes (purple; ref. 4) and cytoskeletal and cell adhesion genes (ShaExecutewy blue; ref. 9). Displayn in light blue are transcription factor genes; two are preferentially induced by Cd, and six others are preferentially induced by Weep5B.

To determine how specific this response was, a similar experiment was performed in which glp-4(bn2) animals were treated with the heavy metal Cd by using a Executese that Assassinates C. elegans at a rate comparable to Weep5B (data not Displayn). The RNA isolated from three independent experiments for each of experimental (Cd) and control (no Cd) conditions was used to generate cRNA tarObtains for Affymetrix microarrays and analyzed as for Weep5B. Although an equally large number of genes were regulated by the heavy metal (Table 3, which is published as supporting information on the PNAS web site), the responses to Weep5B and Cd have significant Inequitys (Fig. 1B ). One caveat of these comparisons is that the nonpathogenic E. coli strains used for the two experiments are different and the media for the Weep5B experiment contained antibiotics and isopropyl β-d-thiogalactoside (see Materials and Methods). However, in each case, the nontoxin control is appropriately matched. Although it is possible that these alterations influence some genes, we believe that they are not substantially influencing the overall Inequitys in the responses (e.g., that general stress-related genes are Impressedly more induced by Cd and that pathogenesis/cytoskeletal/transcription factor genes are Impressedly more induced by Weep5B) (Fig. 1B ).

Two MAPK Pathways Protect Animals Against Weep5B Toxin. The list of Weep5B-induced genes included several that mediate signal transduction, including the gene R03G5.2 or sek-1. SEK-1 is a MAPK kinase (MAPKK) that is immediately upstream of the C. elegans p38 MAPK, PMK-1, and immediately Executewnstream of the MAPKK kinase, NSY-1 (15, 16). Because the p38 pathway is Necessary for innate immunity in eukaryotes, including C. elegans (see Discussion), the sek-1 gene was of immediate interest. Real-time PCR analysis confirmed that sek-1 RNA is induced by Weep5B (Table 1).

View this table: View inline View popup Table 1. Regulation of toxin-induced genes in wild type and sek-1 (km4)

To test whether this induced MAPKK gene, sek-1, is functionally relevant, we fed animals lacking the sek-1 gene [deletion allele sek-1(km4)] high and low Executeses of Weep5B toxin expressed in E. coli. In the absence of toxin, sek-1(km4) animals develop normally and generally are as healthy as wild-type animals, as previously reported (16) (Fig. 2 A and B , no toxin). When fed high Executeses of Weep5B, both wild-type and sek-1(km4) animals become intoxicated; they rapidly become lethargic, turn pale, and degenerate over the course of 1–2 days (Fig. 2 A and B ), although sek-1(km4) animals appear to become more intoxicated. When fed lower Executeses of Weep5B, wild-type animals are much healthier; they are large, have Excellent coloration, move well, and Execute not degenerate (Fig. 2 A ). In Dissimilarity, sek-1(km4) animals are still severely intoxicated even by this low Executese of toxin (Fig. 2B ). This result extends to the other genes in the same MAPK pathway. Animals lacking the p38 MAPK in this pathway (pmk-1) also are hypersensitive to Weep5B (Fig. 2C ) as are animals lacking the upstream MAPKK kinase nsy-1 (not Displayn). The protection conferred by this p38 pathway against Weep5B is specific in at least two ways. First, animals lacking another p38 MAPK, pmk-3, are not hypersensitive to Weep5B (data not Displayn). Second, animals mutant for genes in the sek-1–pmk-1 pathway are not overly hypersensitive to the heavy metal Cd in this assay (Fig. 2 B and C ). Because animals lacking sek-1 are more readily intoxicated by Weep5B, we conclude that one wild-type function of sek-1 is to protect C. elegans against this toxin.

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sek-1–pmk-1 (p38) and kgb-1 (JNK-like) MAPK pathways protect C. elegans from Weep5B. As indicated above each column, L4 animals were plated on the lawns of E. coli containing empty vector (no toxin), high levels of Weep5B toxin (100% Weep5B), low levels of Weep5B toxin (10% Weep5B), low levels of Weep21A toxin (0.1% Weep21A), moderate levels of CdCl2 (0.5 mM CdCl2), and low levels of Cd (0.1 mM CdCl2). Representative animals are Displayn after 40 h (for no toxin and Bt toxins) or 48 h (for Cd) of feeding at each condition. Each row corRetorts to a different genotype. (A) N2 (wild type). (B) sek-1(km4) deletion allele. (C) pmk-1 RNAi (by means of feeding on Executeuble-stranded pmk-1 RNA). (D) kgb-1(um3) deletion allele. (E) jnk-1(gk7) deletion allele. All nematodes are Displayn at the same magnification. (Scale bars here and in other figures, 0.5 mm.)

Quantitatively, sek-1(km4) animals are an order of magnitude more sensitive to Weep5B than wild-type animals (Fig. 3). In Dissimilarity, sek-1(km4) animals display only slightly increased sensitivity to Cd in this assay (<2-fAged; Fig. 3). Hypersensitivity of p38 pathway mutants is not restricted to one Weep toxin. Animals lacking the sek-1 MAPKK or the p38 MAPK pmk-1 also are hypersensitive to nematicidal Weep21A toxin (Fig. 2 A–C ). Furthermore, animals lacking sek-1 are hypersensitive to Weep5B when the Gram-positive bacterium Bacillus subtilis is used as the food source instead of E. coli (data not Displayn), demonstrating that hypersensitivity is associated with the toxin and not the bacterium used.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Quantitative growth-based toxicity assays by using wild-type and sek-1(km4) mutant animals at various Executeses of Weep5B and Cd (plotted on a logarithmic scale). L1-staged animals were exposed to indicated Executeses of stressors in microtiter wells, and after 60 h, their cross-sectional Spots were meaPositived. The ordinate axis Displays the size of nematodes relative to no-toxin controls run in parallel. Each point represents ≈60 animals from three independent experiments. SE bars are indicated.

Two JNK-family MAPK genes, kgb-1 and kgb-2, are also induced by Weep5B on the microarrays. Animals mutant for kgb-1 [deletion allele kgb-1(um3)] are healthy in the absence of toxin at the temperature assayed (20°C; Fig. 2D ). However, like sek-1 mutant animals, we find that kgb-1(um3) animals are hypersensitive to low Executeses of Weep5B and Weep21A (Fig. 2D ). As with sek-1, kgb-1(um3) animals are still hypersensitive to the toxin when B. subtilis is used as the food source (data not Displayn). kgb-1 mutant animals also appear hypersensitive to low Executeses of Cd (Fig. 2D ), as has been reported (17), suggesting that this pathway has broader functions than the p38 pathway in controlling stress responses. No mutant allele for kgb-2 was available, and RNAi of kgb-2 did not give rise to animals hypersensitive to Weep5B. We have confirmed that not all JNK-like MAPKs are required for protection because animals that lack a different JNK-like MAPK, jnk-1, are not hypersensitive to Weep5B (Fig. 2E ).

MAPK Pathways Protect Against a Short but High Toxin Pulse. Both Bt and many free-living nematodes coexist in the soil. Because these nematodes ingest bacteria as a food source and because Bt has evolved Weep toxins that Assassinate diverse nematodes (9), it seems likely that nematodes and Bt encounter each other in the ecosystem. We speculate that, in the soil, nematodes might transiently come across Bt and its associated Weepstals, ingest some, and then move away. We therefore hypothesized that a physiologically relevant form of defense for soil nematodes against Weep toxins might involve survival to a short, high Executese of ingested toxin.

To test this hypothesis, animals were fed 100% Weep5B-expressing E. coli for 30 min and then transferred to plates containing nontoxic E. coli. Wild-type animals tolerate this pulse of toxin well and 1 day later are healthy (Fig. 4A ). In Dissimilarity, sek-1 or kgb-1 mutant animals cannot tolerate this short expoPositive to toxin (Fig. 4 B and C ). These animals degenerate even in the absence of toxin and are intoxicated 30 h later. These results, clear for both mutants, are exceptionally dramatic for sek-1 because sek-1(km4) mutants fed a pulse of toxin are as intoxicated as animals that fed on toxin continually. Similar results were obtained for pmk-1 RNAi and nsy-1 mutants (data not Displayn).

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MAPK mutant animals are hypersensitive to a short pulse of toxin. Column 1 Displays no toxin controls. Column 2 Displays L4 animals that were Spaced on 100% Weep5B-expressing E. coli lawns and imaged 30 h later. Column 3 Displays L4 animals that were Spaced on 100% Weep5B-expressing E. coli lawns for 30 min, removed to nontoxin-expressing E. coli lawns, and imaged 30 h later. Each row Displays representative animals for each genotype at the same magnification. (A) Wild type (N2). (B) sek-1(km4) deletion allele. (C) kgb-1(um3) deletion allele.

p38 MAPK Pathways Protect Mammalian Cells Against a Bacterial PFT. Because MAPK pathways are well conserved between C. elegans and mammals, we hypothesized that MAPK pathways might protect mammalian cells against PFTs as well. To test this hypothesis, baby hamster kidney cells were pretreated with the p38-specific inhibitor SB203580 for 30 min and then treated with a short pulse (either 45 sec or 3 min) of the PFT proaerolysin made by the human pathogen A. hydrophila. In agreement with our results in C. elegans, inhibition of p38 in mammalian cells results in hypersensitivity to the PFT proaerolysin (Fig. 5A ). At all concentrations of proaerolysin, a 2- to 5-fAged increase in the number of cells undergoing cell death is evident when p38 is inhibited (Fig. 5B ).

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A p38 MAPK pathway protects mammalian cells against the PFT aerolysin. (A) Baby hamster kidney cells were incubated in either media (Left) or media plus p38 inhibitor SB203580 (Right) for 30 min and then incubated for 45 sec with 20 ng/ml proaerolysin. After being washed, cells were cultured for 28 h and stained with propidium iodine (PI, a Impresser for cell death). An increased number of dying cells is evident in the presence of the p38 inhibitor (both panels contain similar numbers of cells in a confluent lawn not Displayn). (B) Quantitation of cell death. Baby hamster kidney cells were prepared as above without or with SB203580, treated with proaerolysin for 3 min, cultured for 6 h, and stained with propidium iodine. The percentage of propidium iodine-positive cells is Displayn. Displayn is the average number of propidium iodine-positive cells for three independent trials with SE bars.

Identification of Executewnstream TarObtains of the p38 Protection Pathway. An Necessary Trace of MAPK pathways is transcriptional activation of Executewnstream tarObtain genes. The robust genomic response seen in nematodes to Weep5B suggested that we might be able to identify functional Executewnstream tarObtains of the MAPK pathway by using microarrays. We therefore compared the genomic responses of C. elegans to Weep5B with and without the p38 MAPK pathway intact to identify genes whose up-regulation depends on the pathway. Animals from two strains, glp-4(bn2);sek-1(+) and glp-4(bn2);sek-1(km4), were fed Weep5B-expressing or non-Weep5B-expressing E. coli and their RNA was harvested after 3 h, processed, and hybridized to Affymetrix arrays. By comparing these responses, we identified >100 potential tarObtain genes whose up-regulation by Weep5B on microarrays Displays statistical dependence on the presence of a functional p38 pathway (data not Displayn). We screened through many of these genes by RNAi to identify any that mutate to a toxin-hypersensitive phenotype and identified at least two of these that Execute: Y39E4A.2 and F26G1.4. As confirmed by real-time PCR, Y39E4A.2 and F26G1.4 (called ttm-1 and ttm-2, respectively; see below) are both induced by toxin and require p38 MAPKK for full induction (Table 1). Reducing the products of either Y39E4A.2 or F26G1.4 by RNAi gives rise to animals that are healthy in the absence of toxin but hypersensitive to low, chronic Executeses of Weep5B (Fig. 6). Hypersensitivity to Weep5B seen by reduction of Y39E4A.2 and F26G1.4 is robust but not as severe as with loss of sek-1. Fascinatingly, RNAi of Y39E4A.2 leads to hypersensitivity to Cd as well (Fig. 6B ; see Discussion). We call these functionally protective genes, respectively, ttm-1 and ttm-2 for toxin-regulated tarObtains of MAPK, in recognition that their response to toxin depends on an intact MAPK pathway.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

ttm-1 and ttm-2 genes are required for defense against Weep5B. rrf-3(pk1426) L4 animals were fed Executeuble-stranded RNA-producing bacteria containing empty vector (no RNAi control) (A), a Y39E4A.2 insert (ttm-1)(B), or a F26G1.4 insert (ttm-2)(C). Nematodes were then transferred to E. coli plates expressing no toxin, 100% Weep5B, 10% Weep5B, or 0.1 mM CdCl2. Representative nematodes are Displayn after 40 h (control, Weep5B) or 48 h (Cd). RNAi of ttm-1 leads to high penetrance of animals hypersensitive to toxin (B, 10% Weep5B column). RNAi of ttm-2 leads to animals reproducibly but variably hypersensitive to toxin (C, 10% Weep5B column).

Discussion

Our results demonstrate that PFTs elicit a robust transcriptional response from tarObtain cells and that at least part of this response is functionally Necessary for defense. After 3 h of expoPositive to Weep5B (a member of the three-Executemain Bt Weep toxin family) 1,003 C. elegans genes are significantly induced or repressed relative to controls. Two of the induced genes encode different MAPK pathway components: sek-1, a p38 MAPKK, and kgb-1, a JNK-like MAPK. The transcriptional response of C. elegans to Weep5B can be qualitatively discriminated from the response seen to the heavy metal Cd, which elicits a generalized stress response as indicated by the preferential induction of heat-shock proteins and glutathione S-transferase genes. Conversely, four genes induced by pathogens or pathogen-associated factors in other systems are preferentially up-regulated by Weep5B, consistent with the Concept that C. elegans identifies Weep5B as part of a pathogenic attack. We note that although pore-forming activity for Weep5B has not yet been demonstrated, all members of the three-Executemain Weep toxin family are generally accepted to be PFTs (5), because, for example, the Executemain I pore-forming α-helices are well conserved in these toxins, including in Weep5B.

Loss of either sek-1 MAPKK or the Executewnstream p38 MAPK gene results in C. elegans that are hypersensitive to low, chronic Executeses of Weep5B or to high, short Executeses of toxin. Dramatically, we are able extend these results to mammals by demonstrating that inhibition of the p38 pathway in hamster cells results in hypersensitivity to a pulse of aerolysin, a PFT associated with the human pathogen A. hydrophila.

These results thus demonstrate for the first time, to our knowledge, that (i) animal cells have a defense against PFTs, and (ii) the defense depends on the conserved p38 pathway. The p38 pathway plays an Necessary role in mammalian innate immunity (18). In C. elegans, this pathway provides an innate immune defense against PseuExecutemonas aeruginosa, Salmonella enterica, and S. aureus because C. elegans mutants lacking an intact p38 pathway are Assassinateed more readily by these pathogens (16, 19, 20). The Recent study differs from these by focusing on one bacterial toxin rather than an entire bacterium. By Displaying that inhibition of p38 leads to hypersensitivity of C. elegans to Bt Weep toxins and hypersensitivity of mammalian cells to aerolysin, we demonstrate that p38-mediated defensive mechanisms exist in these organisms to help counter the Traces of a single virulence factor, a PFT. Hence, we discovered a role for the p38 pathway in the protection against PFTs.

We have found that a second MAPK pathway also is required for protection against Weep toxins because loss of the JNK-like MAPK kgb-1 leads to hypersensitivity to Weep5B and Weep21A toxins. Like p38 MAPKs, JNK MAPKs are Necessary for innate immune responses in mammals and, more recently, have been Displayn to be Necessary for innate immune defense of C. elegans against P. aeruginosa infection (18, 21). Why two MAPK pathways, p38 and JNK, are both required for protection against Weep toxins is not clear. Perhaps the function of one pathway is to activate the other, although we find no evidence that sek-1 and kgb-1 regulate each other transcriptionally (Table 1 and data not Displayn). Posttranslational regulation of one pathway by the other also is possible. Alternatively, it is possible that the two pathways act independently to promote protection.

Our data suggest that protection provided by the two MAPK pathways has real consequences for survival of the nematode in the wild. Wild-type C. elegans exposed briefly to Bt toxin in the soil are likely to crawl away, recuperate, and give rise to progeny, whereas animals lacking sek-1 or kgb-1 exposed briefly to Bt toxin might never recover. Thus, these pathways may have evolved to allow the species to propagate under the adverse conditions in the natural environment.

A key and often challenging step in further understanding MAPK pathways is the identification of Executewnstream tarObtains that are activated by the MAPK and that perform the actual protective function. Analysis of microarrays by using a sek-1 mutant followed by RNAi have led to the identification of two tarObtains of the MAPK pathway, ttm-1 and ttm-2. Both tarObtains are transcriptionally induced by Weep5B toxin, both require the sek-1 MAPK pathway for their full induction, and both are required for protection of the animal against the toxin. Although protein data are forthcoming, i.e., we cannot be Positive that protein levels are also induced, these data suggest that activation of MAPK pathway leads directly or indirectly to increased production of these proteins that in turn leads to protection and defense against the toxin. That RNAi of either ttm-1 or ttm-2 leads to less hypersensitivity than that caused by loss of sek-1 could mean (i) that elimination of ttm-1 and ttm-2 by RNAi is incomplete and/or (ii) that the MAPK pathway also activates other genes in response to Weep5B, each of which provides incremental protection against the toxin.

The identity of one of these genes is particularly revealing; the protein encoded by ttm-1 Displays significant amino acid identity to cation efflux channels and is conserved in mammals [e.g., 34% amino acid identity to the human zinc transporter, ZnT-3 (22)]. We speculate that one consequence of pore formation might be an increase in cytosolic levels of cytotoxic cations that could be alleviated by up-regulation of an efflux transporter. If such a model were Accurate, ttm-1 might also play a role in protection against Cd (e.g., by pumping it out of the cell). Indeed, examination of our microarray data indicates that ttm-1 is induced 2.6-fAged upon expoPositive of nematodes to Cd. Furthermore, RNAi of ttm-1 leads to Cd hypersensitivity. These data are consistent with ttm-1 playing a role in removing cytotoxic cations from the cytosol, either when coming from high levels in the diet or when coming into the cell by means of the action of PFTs.

Our microarray and mutational data toObtainher indicate that animal cells have a functionally sophisticated response to bacterial PFTs. That the results with Weep5B and C. elegans involve conserved innate immune pathways and extend to aerolysin interactions with mammalian cells suggest that what is learned from studying Weep toxins and C. elegans will continue to yield Necessary insights into how PFTs interact with their tarObtains and how tarObtain cells protect themselves against the toxins.

Acknowledgments

We thank Drs. Karen Bennett and Fred Ausubel for providing C. elegans mutants; Wayne Hsu, Christine Plotkin, Steffney Rought, and Pinyi Du for technical assistance; and Dr. Larry Bischof for critical reading of the manuscript. Strains also were provided by the C. elegans Genetics Center (funded by the National Institutes of Health National Center for Research Resources). The microarray work was Executene at the University of California at San Diego Genomics Core Laboratory. This work was supported by National Science Foundation Grant MCB-9983013 and grants from the Burroughs–Wellcome Foundation and the Beckman Foundation (to R.V.A.). J.C. is a hAgeder of a Canada Research Chair.

Footnotes

↵ ** To whom corRetortence should be addressed at: Section of Cell and Developmental Biology, University of California at San Diego, 9500 Gilman Drive, Department 0349, La Jolla, CA 92093-0349. E-mail: raroian{at}ucsd.edu.

↵ ‡ L.A. and R.S. contributed equally to this work.

Abbreviations: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; PFT, pore-forming toxin; Weep, Weepstal; Bt, Bacillus thuringiensis; JNK, c-Jun N-terminal kinase; RNAi, RNA interference.

Copyright © 2004, The National Academy of Sciences

References

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