Research Article: Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

Date Published: January 21, 2016

Publisher: Public Library of Science

Author(s): Fengjuan Zhang, Donghai Peng, Chunsheng Cheng, Wei Zhou, Shouyong Ju, Danfeng Wan, Ziquan Yu, Jianwei Shi, Yaoyao Deng, Fenshan Wang, Xiaobo Ye, Zhenfei Hu, Jian Lin, Lifang Ruan, Ming Sun, David S. Schneider.


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.

Partial Text

Cell death plays critical roles in development and in pathological conditions. Apoptosis and necrosis are the two major modes of cell death [1]. Apoptosis, the most well- known mode of the cell death, plays a significant role in development, tissue homeostasis, and host defense [2,3]. Unlike apoptosis, necrosis is characterized by loss of plasma membrane integrity [4,5]. Necrotic cell death can contribute to many pathological conditions, such as inflammation [6], human neurodegenerative and aging-associated diseases [5,7]. Moreover, Necrosis plays an important role in microbial pathogenesis. In some cases, necrosis plays a significant role in antiviral/antibacterial host defense [2,8]; in others, necrosis is utilized as pathogen survival strategy to aid its spread [2].

The classical, pore-forming model is the widely accepted model for describing the mode of action of 3d-Cry. This model elucidates crystal protein action at the biochemical level, which includes crystal protein activation, receptor binding, pore formation, and cell lysis [10–13]. The identified receptors in the pore-forming model, such as cadherin, aminopeptidase (APN), and alkaline phosphatase (ALP), mediate insect resistance to 3d-Cry [45]. However, these receptors were not identified in C. elegans by the Cry6Aa-ligand blotting assay. These results indicate that the mode of action of non-3d crystal protein Cry6Aa against C. elegans may be different from the classical pore-forming model. The signal transduction model proposed that crystal proteins activate a Mg2+-dependent adenylyl cyclase (AC)/protein kinase A (PKA) signaling pathway [14,15]. This work show that crystal protein Cry6Aa triggers the Ca2+-dependent calpain–cathepsin necrosis pathway in C. elegans. Thus, in contrast to the classical pore-forming model, which elucidated at the biochemical level, the present findings reveal that crystal toxin triggers cell death by necrosis signaling pathway. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein.




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