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. 2016 Jan 21;12(1):e1005389.
doi: 10.1371/journal.ppat.1005389. eCollection 2016 Jan.

Bacillus Thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis Elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

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Bacillus Thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis Elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

Fengjuan Zhang et al. PLoS Pathog. .
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Abstract

Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The specific interactions between Cry6Aa and ASP-1.
(A) The expression and purification of ASP1-GST fusion protein in E. coli. Lane 1, E. coli BL21 containing pGEX-6p-1 vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet; lane 2, E. coli BL21 containing pASP1-GSTvector without IPTG induction, cell pellet. lane 3, E. coli BL21 containing pASP1-GST vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet. lane 4, purified ASP1-GST fusion protein eluted from the glutathione Sepharose bulk; lane 5, purified GST tag eluted from the glutathione Sepharose bulk. (B) The binding of Cry6Aa to ASP1-GST fusion proteins by ligand blotting. The purified ASP1-GST fusion proteins (lane 6) or GST (lane 7) were separated by SDS-PAGE gels, and were transferred to a PVDF membrane. Filters were blocked overnight, and then probed with biotinylated Cry6Aa, Unbound toxin was removed by washing. The bound protein was detected streptavidin-horseradish peroxidase (HRP) conjugate. And finally the membrane was visualized. GST was the control. (C) ASP-1 proteins were dotted on a NC membrane directly and were probed with biotin labeled Cry6Aa or with biotin labeled Cry6Aa plus unlabeled Cry6Aa (1000-fold). GST was the control. (D) Binding affinity of Cry6Aa to ASP-1 was determined by ELISA. Ninety-six-well microtiter plates coated with ASP-1 were incubated with increasing concentrations of biotinylated Cry6Aa alone or with 1000-fold molar excess of unlabeled Cry6Aa to determine specific binding. Each point represents mean amounts of protein specifically bound. Specific binding was determined by subtracting nonspecific binding (with 1000-fold molar excess of unlabeled Cry6Aa) from total binding (without excess unlabeled Cry6Aa). (E) Isothermal titration calorimetric analysis of Cry6Aa binding to ASP-1. Titration of ASP-1 (2.20 μM) with 21.16 μM Cry6Aa. The top panel show the raw data of the heat released, and the bottom panel show the binding isotherm fitted using nonlinear binding models. Data were analyzed in Origin 8.6 software after subtracting the heat released from titrating Cry6Aa alone into buffer. One of three representative experiments is shown.
Fig 2
Fig 2. The susceptibility of mutant asp-1(tm666) to Cry6Aa.
(A) Cry6Aa quantitative growth assay with wild type N2 and mutant asp-1(tm666). (B) Cry6Aa lethality assay with N2 and asp-1(tm666). The strains (as shown) were exposed to five doses of Cry6Aa. (C) Survival of wild type N2 and mutant asp-1(tm666) exposed to Cry6Aa. (D) Growth assay of different concentrations of Cry6Aa against L1 larvae of N2 (top panels), asp-1(tm666) (middle panels), and asp-1 (tm666) rescued with the whole asp-1 gene (bottom panels) observed under a light microscope. (E) Cry6Aa quantitative growth assay with N2, asp-1(tm666) and asp-1(tm666) transformed with the whole asp-1 gene. Data were showed as mean ± SD (n = 3).
Fig 3
Fig 3. Intestinal cell plasma membrane integrity was lost in wild type nematode N2 after exposure to Cry6Aa or heat stroke.
Heat stroke was a positive control. Fluorescence microscopy was used to monitor propidium iodide uptake. Arrows indicate intestinal cells stained with propidium iodide due to loss of membrane integrity. Two of the fluorescent images were magnified (boxed inset). The numbers of cell corpses per nematode were counted (Right). These results are the mean ± SD of three independent experiments. Double asterisks indicate p < 0.01. The bar denotes 20 μm.
Fig 4
Fig 4. Cry6Aa-induced intestinal cell lysosomal rupture in C. elegans.
(A) DIC and fluorescence microscopy of nematodes labeled with the intestinal lysosomal marker Lysotracker. (B) DIC and fluorescence microscopy of nematode RT258 (LMP-1::GFP strain). The intestinal lysosomal marker LMP-1::GFP was used to examine lysosomal integrity. The controls without Cry6A show significant staining, indicating that fluorescent gut lysosomes remained intact. In the Cry6A treated nematode, the labeled lysosome was found to be diffused (Fig 4A), indicating that Cry6Aa induces intestinal cell lysosomal rupture.
Fig 5
Fig 5. Heat stroke induced intestinal cell lysosomal rupture in C. elegans.
(A) DIC and fluorescence microscopy of nematodes labeled with the intestinal lysosomal marker Lysotracker. (B) DIC and fluorescence microscopy of nematode RT258 (LMP-1::GFP strain). The intestinal lysosomal marker LMP-1::GFP was used to examine lysosomal integrity. In the nematode without heat stroke, Fluorescent gut lysosomes remained intact. In the heat stroke treated nematode, the loss of lysosomal membrane integrity was accompanied by diffused fluorescence.
Fig 6
Fig 6. Cry6Aa-induced an increase in cytoplasm calcium concentration in C. elegans.
(A) The rise in [Ca2+]i in the intestine of wild type nematode N2 induced by 63 μg/mL Cry6Aa for 6 days. Fluorescence microscopy was used to monitor calcium concentration by measuring cytoplasmic fluorescence using the calcium indicator Fluo-4 AM. The right part shows the quantification of the fluorescence levels. These results are the mean ± SD of three independent experiments. A single asterisk indicates p < 0.05. The bar denotes 20 μm. (B) Representative CFP, FRET and Ratio images in transgenic nematode KWN190 upon exposure to 63 μg/mL Cry6Aa for 6 days. Calcium levels were visualized using the calcium indicator d3cpv expressed from the intestine-limited promoter in KWN190. Right shows that the FRET ratio increased upon exposure to Cry6Aa. The image is representative of three independent experiments. A single asterisk indicates p < 0.05. The bar denotes 20 μm.
Fig 7
Fig 7. The susceptibility of necrosis mutants to Cry6Aa.
(A) Cry6Aa lethality assay with itr-1(sa73), tra-3(e1107), and vha-12(ok821) necrosis mutants. The strains (as shown) were exposed to five doses of Cry6Aa. (B) Survival of itr-1(sa73), tra-3(e1107), vha-12(ok821), asp-3(tm4559), and asp-4(ok2693) necrosis mutants exposed to Cry6Aa. Mutations in itr-1(sa73), tra-3(e1107), and vha-12(ok821) reduced the sensitivity of nematodes to Cry6Aa. (C) Cry6Aa lethality assay with asp-3(tm4559) and asp-4(ok2693). The strains (as shown) were exposed to five doses of Cry6Aa. (D) Survival of itr-1(sa73), tra-3(e1107), vha-12(ok821), asp-3(tm4559), and asp-4(ok2693) necrosis mutants exposed to heat stroke. Mutations in itr-1(sa73), tra-3(e1107), and vha-12(ok821) reduced the sensitivity of nematodes to heat stroke. A single asterisk indicates p < 0.05. Data were showed as mean ± SD (n = 3).
Fig 8
Fig 8. The protein expression of asp-1 was up regulated in C. elegans upon exposure to Cry6Aa.
Western blot shows the increased ASP-1 protein levels in C. elegans upon exposure to Cry6Aa. The blot is one of the three independent experiments.
Fig 9
Fig 9. The susceptibility of necrosis mutants to Cry5Ba.
(A) Cry5Ba quantitative growth assay with wild type N2 and mutant asp-1(tm666). (B) Cry5Ba lethality assay with N2 and asp-1(tm666). The strains (as shown) were exposed to five doses of Cry6Aa. (C) Lethality of necrosis mutants exposed to Cry5Ba. Data were showed as mean ± SD (n = 3).
Fig 10
Fig 10. Cry5Ba is not able to bind to ASP-1.
(A) Purified Cry5Ba and Cry6Aa were dotted on a NC membrane directly and were probed with biotin labeled ASP-1. Cry6Aa was the positive control. (B) Binding affinity of Cry5Ba to ASP-1 was determined by ELISA. Ninety-six-well microtiter plates coated with ASP-1 were incubated with increasing concentrations of biotinylated Cry5Ba.
Fig 11
Fig 11. The working model of crystal protein Cry6Aa.
Cry6Aa toxin triggers the Ca2+-dependent calpain–cathepsin necrosis pathway using Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of calcium, lysosomal lyses, the killer cathepsin proteases mediated by ASP-1, uptake of propidium iodide, and burst of death fluorescence. Abbreviations: PI, propidium iodide; DF, death fluorescence.

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Grant support

MS was supported by the National High Technology Research and Development Program (863) of China (2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), and the National Natural Science Foundation of China (31170047). LR was supported by the National Natural Science Foundation of China (31171901). FZ was supported by State Key Laboratory of Agricultural Microbiology Program (AMLKF201306). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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