Unravelling the cellular mechanisms that provide specificity of bacterial pesticidal proteins against invertebrates and some cancer cells

Heath, Emily 2023. Unravelling the cellular mechanisms that provide specificity of bacterial pesticidal proteins against invertebrates and some cancer cells. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Background The entomopathogenic bacteria Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) produce numerous proteins packed in natural crystals that are stable in the environment and, once ingested, are active against a range of invertebrate pests. These proteins are diverse in terms of their structure, invertebrate targets and mechanisms of action. In addition to toxicity against their invertebrate targets, some of these bacterial proteins have reported activity against cancer cell lines, namely parasporins (Bt) and Tpp1/Tpp2 (Ls). The mechanisms that provide specificity of these proteins towards cancer cells are currently unknown. Methods Through cell viability assays, utilising sugar competition techniques and treatment with glycosylation inhibitors, in addition to sugar/lipid dot blots and lipid thin layer chromatography, this thesis explores the potential role of lipids and glycans in mediating specificity of proteins produced by Bt and Ls (namely Cry41Aa1 and Tpp1/Tpp2) against target insects and HepG2 human cancer cells. Additionally, the structure of App6 from natural Bt crystals was solved at multiple pH values, from data generated by serial femtosecond crystallography and this protein was also tested against HepG2 cells. Results/ Conclusions There appear to be complex requirements for toxicity of Tpp1/Tpp2 against HepG2 cancer cells, which from this work remain to be elucidated. However, both Tpp1/Tpp2 were shown to interact with lipids from target insect cells and human cancer cells, which may indicate a role in specificity. The results presented in this thesis provide evidence for a role of glycoconjugates in Cry41 toxicity towards HepG2 cancer cells, with both glycosphingolipids and N-linked glycans seemingly required for toxicity and interactions specifically with GlcNAc and GalNAc shown. The natural structure of App6 showed high similarity to published recrystalised App6 structures. Although the natural structure provided insights into how App6 is packed within the natural crystals, the pH mixing studies at present provided little insight into crystal dissolution in vivo

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Funders:	South West Biosciences DTP BBSRC
Date of First Compliant Deposit:	24 May 2024
Last Modified:	24 May 2024 13:23
URI:	https://orca.cardiff.ac.uk/id/eprint/169160

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