Activation of the Unfolded Protein Response Is Required for Defenses against Bacterial Pore-Forming Toxin In Vivo
Figure 5
Relationship of the p38 MAPK and UPR pathways in response to PFT and unfolded proteins.
(A) The xbp-1 pathway is not required for phosphorylation of p38 MAPK by Cry5B. Wild-type N2 and xbp-1(zc12) were exposed to either control buffer or purified Cry5B toxin for one hour. Worm lysates were analyzed by immunoblotting for phospho P38 MAPK along with α-tubulin as a loading comparison. Positions of molecular weight markers in kilodaltons are shown on left side of gel. Data are representative of three independent experiments. (B) Cry5B induced splicing of xbp-1 requires sek-1 (MAPKK). Splicing of xbp-1 mRNA was compared in glp-4(bn2) and glp-4(bn2);sek-1(km4) after 3 hours of exposure to either control E. coli or E. coli expressing Cry5B. Size markers in nucleotides are indicated on the left. This is a representative experiment of three independent experiments. (C) Tunicamycin induced splicing of xbp-1 does not require sek-1 (MAPKK). Splicing of xbp-1 mRNA was compared in glp-4(bn2) and glp-4(bn2);sek-1(km4) after 3 hours of exposure to either control (DMSO) or tunicamycin (2 µg/mL). This is a representative experiment of three independent experiments. (D) In vivo induction of hsp-4::GFP by Cry5B requires pmk-1 (p38 MAPK). The strains hsp-4::GFP and hsp-4::GFP;pmk-1(km25) were fed either control E. coli or E. coli expressing Cry5B for 8 hours and the expression of GFP was then analyzed. Cry5B induces GFP within the intestinal cells of the strain hsp-4::GFP but not in the strain containing the pmk-1(km25) mutant. The experiment was performed three times and representative worms are shown. Scale bar is 0.2 mm. (E) In vivo induction of hsp-4::GFP by tunicamycin does not require pmk-1 (p38 MAPK). The strains hsp-4::GFP and hsp-4::GFP;pmk-1(km25) were exposed to either control (DMSO) or tunicamycin (2 µg/mL) for 8 hours and the expression of GFP was then analyzed. Tunicamycin induces GFP throughout both the strains hsp-4::GFP and hsp-4::GFP;pmk-1(km25), including within the intestinal cells. The experiment was performed three times and representative worms are shown. Scale bar is 0.2 mm. (F) Downstream targets of the UPR require the p38 MAPK pathway for induction by PFT but not unfolded proteins. The fold change in the levels of hsp-4 and Y41C4A.11 mRNA transcripts by Cry5B and tunicamycin were determined for glp-4(bn2), glp-4(bn2);xbp-1(zc12) and glp-4(bn2);sek-1(km4) using real-time PCR. In addition, the fold change in ttm-2 transcripts was determined in response to Cry5B. Data are mean and standard deviation of three independent experiments. (G) Animals lacking sek-1 MAPKK are more sensitive to Cry5B than animals lacking xbp-1. Wild-type N2, sek-1(km4), and xbp-1(zc12) animals were placed on plates spread with E. coli transformed with empty vector (0%) or spread with empty vector E. coli diluted 9∶1 (10%) or 3∶1 (25%) with Cry5B-expressing E. coli (% thus gives toxin dose on a plate relative to undiluted Cry5B-expressing E. coli). The assay was initiated with L4 stage worms and photographs were taken 48 hours later. In the absence of Cry5B, the worms developed into dark, gravid, active, healthy adults. On 10% Cry5B-expressing E. coli, xbp-1(zc12) were slightly smaller than N2 but healthier than sek-1(km4), which were as small, pale, inactive, and severely intoxicated. On 25% Cry5B-expressing E. coli, xbp-1(zc12) was more intoxicated than N2 but not as intoxicated as sek-1(km4) animals. Scale bar is 0.2 mm.