Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

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Dr. Jacques-Philippe Colletier Jan 23, 2020 Scanning electron micrographs (SEM) were acquired using a Zeiss LEO 1530 microscope and the associated commercial software. Atomic force microscopy (AFM) images were acquired using the NanoScope software. Protein gels were digitalized using a ChemiDoc XRS+ imaging system piloted by the Image Lab software version 6.0.0. FACS raw data were collected, treated and processed using the MACSQuantify software v2.11. For exposure of cells to FITC-dextran beads, cell images were acquired using the commercial software associated with the laser scanning confocal microscope (LSM), Zeiss LSM 510. Black lipid membrane (BLM) data were recorded using an Axon Digidata 1440A digitizer and associated commercial software. Transmission electron micrographs (TEM) were acquired using the commercial software associated with the FEI Tecnai T12 and FEI F20 microscopes.
All collected diffraction pattern images were indexed with DIALS (ref. 37), using the cctbx.xfel graphical user interface. Data were merged using cxi.merge. pH7 dataset was phased by molecular replacement with Phaser. Further refinement in real space was performed using Coot, and refinement in the reciprocal space using Refmac. Fo-Fo maps were q-weighted and produced using a CNS custom-written script (all scripts and methods available in refs 43 to 45). ImageJ v1.51k was used to determine crystal sizes (SEM), diameters and surfaces of pores (AFM), and to evaluate the size of proteins present in SDS-PAGE bands. AFM Images were processed with Gwyddion 35 and, when needed, stripe noise was removed using DeStripe. MALDI data were processed with Flexanalysis 3.0 software. BLM data analysis was performed with clampfit. The software R 3.5.2 was used to calculate all statistical indicators. pH solubility and mortalities were analyzed using a modified code from Savi et al. 2018 (binomial model selection and SP and LC measurements).

October 2018
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