Ressource pédagogique : Dr Paul Williams - Calcium imaging of Cry5B action on the intestine of Ascaris suum

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Crystal (Cry) proteins from Bacillus thuringiensis are used as effective insecticides. These pore forming toxins act within the midgut of insects killing them. Cry5B, has been found to target and kill intestinal parasitic nematodes including Ascaris suum. Cry5B forms pores in the nematode intestines killing the parasite in less than a day. To track the time course of the action of Cry5B on the intestine beginning from application, we incubated Ascaris suum intestines in Fluo-3AM for calcium imaging. We exposed isolated intestine flaps to either 100 µg/ml or 10 µg/ml activated Cry5B and followed the cytosolic  calcium over a 6-hour period. We observed a Cry5B concentration-dependent increase and subsequent fall in calcium that occurred after 1 hour from Cry5B application. The shortest time that we observed an increase in calcium after application of Cry5B was 10 minutes. After the initial increase in calcium, we observed small fluctuations and oscillations in the calcium signal. We are treating samples with 100 mM galactose to see if there is inhibition of the effects of Cry5B and histology to look for the microscopic structure changes. By 6 hours intestinal cells were severely damaged, with their columnar cells vacuolated and swollen. The activated Cry5B toxin can act rapidly to produce serious nematode intestine damage. With PCR and qPCR techniques we have identified the presence of BRE-5 and other Cry5B target orthologues showing intestine specific expression present in Ascaris suum. The complex calcium signal requires further study.  Financial Support: RJM is supported by NIH, the National Institute of Allergy and Infectious Diseases grants R01AI047194-17, R21AI092185-01A1 and the E. A. Benbrook Foundation for Pathology and Parasitology.

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