Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins

Abstract

Cytolytic pore-forming toxins are important for the virulence of many disease-causing bacteria. How target cells molecularly respond 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 responds robustly to Cry5B, a member of the pore-forming Crystal 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 Cry5B. Moreover, both MAPK pathways are functionally important because elimination of either leads to animals that are (i) hypersensitive to a low, chronic dose of toxin and (ii) hypersensitive to a high, brief dose 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 targets humans. Furthermore, we identify two downstream transcriptional targets of the p38 MAPK pathway, ttm-1 and ttm-2, that are required for defense against Cry5B. Our data demonstrate that cells defend against pore-forming toxins by means of conserved MAPK pathways.

Fig. 1.

Behavior of 1,670 probe sets significantly induced by one or both of Cry5B and Cd treatment. A few strongly down-regulated genes were omitted to improve plot resolution for up-regulated genes. (A) The fold induction of the probe sets for two of the independent Cry5B trials plotted against each other. The plot demonstrates excellent correlation between the two trials. (B) The fold induction of the probe sets for Cd trials (average of three trials) and for Cry5B (averaged over three trials) plotted against each other. Although the fold induction of some probe sets is similar, other probe sets (located off the diagonal) show significant differences, indicative of different responses to Cry5B and Cd. Some genes that show >2-fold difference between the two conditions were grouped based on their ontology and are shown 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 Cry5B include pathogen or pathogen-associated induced genes (purple; ref. 4) and cytoskeletal and cell adhesion genes (dark blue; ref. 9). Shown in light blue are transcription factor genes; two are preferentially induced by Cd, and six others are preferentially induced by Cry5B.

Fig. 2.

sek-1–pmk-1 (p38) and kgb-1 (JNK-like) MAPK pathways protect C. elegans from Cry5B. As indicated above each column, L4 animals were plated on the lawns of E. coli containing empty vector (no toxin), high levels of Cry5B toxin (100% Cry5B), low levels of Cry5B toxin (10% Cry5B), low levels of Cry21A toxin (0.1% Cry21A), moderate levels of CdCl₂ (0.5 mM CdCl₂), and low levels of Cd (0.1 mM CdCl₂). Representative animals are shown after 40 h (for no toxin and Bt toxins) or 48 h (for Cd) of feeding at each condition. Each row corresponds to a different genotype. (A) N2 (wild type). (B) sek-1(km4) deletion allele. (C) pmk-1 RNAi (by means of feeding on double-stranded pmk-1 RNA). (D) kgb-1(um3) deletion allele. (E) jnk-1(gk7) deletion allele. All nematodes are shown at the same magnification. (Scale bars here and in other figures, 0.5 mm.)

Fig. 3.

Quantitative growth-based toxicity assays by using wild-type and sek-1(km4) mutant animals at various doses of Cry5B and Cd (plotted on a logarithmic scale). L1-staged animals were exposed to indicated doses of stressors in microtiter wells, and after 60 h, their cross-sectional areas were measured. The ordinate axis shows 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.

Fig. 4.

MAPK mutant animals are hypersensitive to a short pulse of toxin. Column 1 shows no toxin controls. Column 2 shows L4 animals that were placed on 100% Cry5B-expressing E. coli lawns and imaged 30 h later. Column 3 shows L4 animals that were placed on 100% Cry5B-expressing E. coli lawns for 30 min, removed to nontoxin-expressing E. coli lawns, and imaged 30 h later. Each row shows 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.

Fig. 5.

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 marker 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 shown). (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 shown. Shown is the average number of propidium iodine-positive cells for three independent trials with SE bars.

Fig. 6.

ttm-1 and ttm-2 genes are required for defense against Cry5B. rrf-3(pk1426) L4 animals were fed double-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% Cry5B, 10% Cry5B, or 0.1 mM CdCl₂. Representative nematodes are shown after 40 h (control, Cry5B) or 48 h (Cd). RNAi of ttm-1 leads to high penetrance of animals hypersensitive to toxin (B, 10% Cry5B column). RNAi of ttm-2 leads to animals reproducibly but variably hypersensitive to toxin (C, 10% Cry5B column).