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.

Objective Aim of this study was to investigate the impact of human PDL-derived fibroblasts (HPDF) and human alveolar bonederived osteoblasts (HABO) co-culture on the expression of cytokines involved in tissue remodeling using an in vitro compressive force (CF) model. Materials and methods Static compressive force (CF) of 47.4 g/cm 2 was applied on mono-and co-cultured HPDFs and HABOs for 1, 2, or 4 h at 30°C. TNFA, PTGS2, and IL6 gene expressions were determined by quantitative real-time polymerase chain reaction. TNF, PGE 2 , and IL6 concentrations were measured using enzyme-linked immunosorbent assay. Results In mono-culture, TNFA, PTGS2, and IL6 gene expressions were upregulated under CF as compared to controls for each time period in both cell types. PGE 2 increased at 1 and 2 h in both cell types, and IL6 increased only at 2 and 4 h in HPDFs. Coculture alleviated the force-induced increase of the expression of TNFA, PTGS2, IL6, PGE2, and IL6 in HPDFs at any time point. In HABOs, co-cultivation decreased the expression of PGE 2 after 1 h and 4 h, and that of IL6 after 1 h compared to mono-culture. Conclusions CF application on co-cultures of HPDFs and HABOs causes significant changes of TNFA, PTGS2, and IL6 gene expressions and PGE 2 and IL6 production in comparison to mono-culture indicating intercellular communication. Clinical relevance Mechanical stimulation of HPDFs and HABOs in co-culture induces a different gene expression pattern than stimulation of individual cell types alone. Co-culture might therefore be a relevant method to elucidate periodontal regeneration during orthodontic therapy.

Plant-parasitic nematodes cause huge agricultural economic losses. Two major families of Bacillus thuringiensis crystal proteins, Cry5 and Cry6, show nematicidal activity. Previous work showed that binding to midgut receptors is a limiting step in Cry toxin mode of action. In the case of Cry5Ba, certain Caenorhabditis elegans glycolipids were identified as receptors of this toxin. However, the receptors for Cry6 toxin remain unknown. In this study, the C. elegans CUB-like-domain containing protein RBT-1, released by phosphatidylinositol-specific phospholipase C (PI-PLC), was identified as a Cry6Aa binding protein by affinity chromatography. RBT-1 contained a predicted glycosylphosphatidylinositol (GPI) anchor site and was shown to locate in lipid rafts in the surface of the midgut cells. Western ligand blot assays and ELISA binding analysis confirmed the binding interaction between Cry6Aa and RBT-1 showing high affinity and specificity. In addition, the mutation of rbt-1 gene decreased the susceptibility of C. elegans to Cry6Aa but not that of Cry5Ba. Furthermore, RBT-1 mediated the uptake of Cry6Aa into C. elegans gut cells, and was shown to be involved in triggering pore-formation activity, indicating that RBT-1 is required for the interaction of Cry6Aa with the nematode midgut cells. These results support that RBT-1 is a functional receptor for Cry6Aa.

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Pore-forming toxins (PFTs) are important weapons of multiple bacterial pathogens to establish their infections. PFTs generally form pores in the plasma membrane of target cells; however, the intracellular pathogenic processes triggered after pore-formation remain poorly understood. Using Caenorhabditis elegans as a model and Bacillus thuringiensis nematicidal Cry PFTs, we show here that the localized PFT attack causes a systemic mitochondrial damage, important for the PFT toxicity. We find that PFTs punch pores only in gut cells of nematodes, but unexpectedly mitochondrial disruption is able to occur in distal unperforated regions, such as the head and muscle tissues. We demonstrate that PFTs affect the activity of the mitochondrial respiratory chain (MRC) complex I resulting in the loss of mitochondrial membrane potential (ΔΨ m ), which causes further mitochondrial fragmentation and the reduction of total mitochondrial content. Worms with decreased ΔΨ m or inhibited MRC activity show higher sensitivity to PFTs. The inhibition of mitochondrial fission or the increase of mitochondrial content markedly improves the survival of animals treated with PFTs. These findings suggest that mitochondrial changes underpin PFT-mediated toxicity against nematodes and that systemic mitochondrial disruption caused by localized pore-formation represents a conserved key intracellular event in the mode of action of PFTs.