Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Amongst these parasporins, parasporin-3 most closely resembles the commercially used insecticidal toxins and presents the narrowest activity spectrum, showing moderate cytotoxicity against only two cancer cell lines, HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells). Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards these two cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made at the N- or C-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60 respectively) which we hypothesize occlude a binding motif. Preliminary work carried out on binding showed a lack of correlation between binding and toxicity since toxin binds to both susceptible and non-susceptible cancer cell lines. In an attempt to better understand the mechanism of action of Cry41Aa against these cells, we evolved resistance in HepG2 through repeated exposure to increasing doses of the toxin. Morphological, physiological and genetic characteristics of the resistant cell line were compared with susceptible cells. Toxin was shown to bind to resistant HepG2 similarly to the susceptible line. RNA sequencing identified AQP9 as a potential mediator of resistance but extensive investigations failed to show a direct link. The involvement of certain intracellular signalling pathways were also investigated in order to understand cell responses to the toxin and showed that in response to the toxin p38, but not ERK1/2, is activated and in a dose dependent manner.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:759607 |
| Date | January 2018 |
| Creators | Souissi, Wided |
| Publisher | University of Sussex |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://sro.sussex.ac.uk/id/eprint/80446/ |
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