A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis

Abstract

Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg(2+)-dependent cytotoxic event upon binding of the toxin to its receptor BT-R(1). Here we show that binding of Cry toxin to BT-R(1) provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (G(alphas)) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifested by sequential cytological changes that include membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis. The discovery of a toxin-induced cell death pathway specifically linked to BT-R(1) in insect cells should provide insights into how insects evolve resistance to Bt and into the development of new, safer insecticides.

Fig. 1.

Cytological changes associated with the progression of Cry1Ab toxin-induced cell death. (A) S5 cells exposed to toxin in the presence of EDTA (Center Lower), EDTA + Ca²⁺ (Right Lower), EGTA (Center Upper), and EDTA + Mg²⁺ (Right Upper). S5 cells treated with Cry1Ab toxin (Left Upper) and untreated S5 cells (Left Lower). (B) Sequence of cytological changes during the course of toxin-induced cell death as viewed by phase-contrast microscopy. The long arrow beneath the photographs indicates the relative time for each stage of cell death, i.e., toxin binding, membrane blebbing, and cellular swelling. The symbols below the time line show the points at which cell death can be blocked by EDTA and cell swelling, but not cell death, can be blocked by raffinose (Raf). A time-lapse movie is provided as Movie 1. (C) Effect of osmotic protectants raffinose (Left), sucrose (Suc, Center), and glucose (Glu, Right) on membrane blebbing and cellular swelling. (Scale bars: 15 μm.)

Fig. 2.

Involvement of AC/PKA pathway in Cry1Ab toxin-induced cytotoxicity. (A) Stimulation of cAMP production by Cry1Ab toxin. Cells were incubated with isobutyl-1-methylxanthine (0.5 mM) before the addition of Cry1Ab, and cAMP was measured by using the Bridge-It cAMP Assay (Mediomics) in S5 and H5 cells (3 × 10⁴). (B) Effect of Cry1Ab, FSK, and EDTA on cAMP production. cAMP levels were measured in S5 cells after 10 min of treatment with either Cry1Ab (Cry, 180 nM) or FSK in the absence or presence of EDTA. (C) Percent relative cytotoxicity of Cry1Ab in the presence of NF023 (1 μM) or NF449 (1 μM). (D) Effect of ddADP on Cry1Ab cytotoxicity. S5 cells were incubated for 30 min with ddADP (0–40 μM), a cell-permeable inhibitor of AC, before the addition of Cry1Ab toxin (60 nM). Cell death was determined by trypan blue exclusion analysis (14). (E) Percent relative cytotoxicity of Cry1Ab in the presence of cell-permeable inhibitors of PKA (H-89, ≈0–50 μM, and PKAI 14–22-amide, ≈0–8 μM) for 30 min at the specified concentration before addition of Cry1Ab toxin. (F) Synergetic effect of pCPT-cAMP (200 μM) and FSK (0.2 μM) on Cry1Ab toxin-induced cell death. The concentration of ddADP was 40 μM. Cry1Ab toxin was administered at 60 nM, the concentration required to kill 50% of S5 cells (14). Data are presented as the mean ± SD of six experiments.

Fig. 3.

Proposed model for the action of Cry toxin. Cry toxin binds to BT-R and stimulates G protein and AC, which promotes production of intracellular cAMP. In turn, PKA activation destabilizes the cytoskeleton and ion channels, leading to cell death.