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Dr Paul Williams - Calcium imaging of Cry5B action on the intestine of Ascaris suum
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Descriptif
Crystal (Cry)proteins from Bacillus thuringiensis areused as effective insecticides. These pore forming toxins act within the midgutof insects killing them. Cry5B, has been found to target and kill intestinalparasitic nematodes including Ascarissuum. Cry5B forms pores in the nematode intestines killing the parasite inless than a day. To track the time course of the action of Cry5B on theintestine beginning from application, we incubated Ascaris suum intestines in Fluo-3AM for calcium imaging. We exposedisolated intestine flaps to either 100 µg/ml or 10 µg/ml activated Cry5B andfollowed the cytosolic calcium over a6-hour period. We observed a Cry5B concentration-dependent increase andsubsequent fall in calcium that occurred after 1 hour from Cry5B application.The shortest time that we observed an increase in calcium after application ofCry5B was 10 minutes. After the initial increase in calcium, we observed smallfluctuations and oscillations in the calcium signal. We are treating sampleswith 100 mM galactose to see if there is inhibition of the effects of Cry5B andhistology to look for the microscopic structure changes. By 6 hours intestinalcells were severely damaged, with their columnar cells vacuolated and swollen.The activated Cry5B toxin can act rapidly to produce serious nematode intestinedamage. With PCR and qPCR techniques we have identified the presence of BRE-5and other Cry5B target orthologues showing intestine specific expressionpresent in Ascaris suum. The complexcalcium signal requires further study.
Financial Support: RJM is supported by NIH, the National Institute ofAllergy and Infectious Diseases grants R01AI047194-17, R21AI092185-01A1and the E. A. Benbrook Foundation for Pathology andParasitology.
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