In silico and empirical analyses of the evolution and activity of diverse AB5 toxins found within the Salmonella genus
Creators: Ojiakor, Adaobi
Publisher: name: University of Alberta Library
Year: 2023

<pre><code>  Bacterial AB5 toxins are secreted protein complexes composed of an enzymatic A subunit that disrupts host cell functions and a pentameric B subunit that facilitates cellular entry of the toxin by binding to specific cell surface receptors. AB5 toxins are widely recognized for their roles in the pathogenesis of several bacterial pathogens frequently associated with human disease. In Salmonella, two distinct AB5 toxins, ArtAB and typhoid toxin appear to contribute to the virulence and disease properties of highly pathogenic strains and serovars like Salmonella enterica serovar Typhimurium definitive phage type (DT)104 and Salmonella enterica serovar Typhi, both of which cause severe infections in humans. However, the distribution and potential roles of AB5 toxins in other virulent Salmonella serovars that are often implicated in human disease is not well understood.      Using an array of in silico methods and molecular and cell biology techniques, this thesis explores the broader arsenal of AB5 toxins found within the Salmonella genus. We provide evidence that many Salmonella serovars harbour genes encoding the ArtAB and typhoid toxins and that there is substantial variation among these toxins. The B subunit sequences of these toxins are particularly variable, which is likely indicative of different glycan binding preferences, indicating that the toxins produced by different lineages likely exhibit differences in the nature of the cell type(s) they target. We also identify two novel “hybrid” AB5 toxins, RIP-TT and RIP-HLT within the Salmonella genus that combine subunits from well-established and distinct toxin families. Phylogenetic and sequence analyses showed that the A subunit sequences of RIP-TT and RIP-HLT exhibit similarity to the A subunits of Shiga family toxins, but their B subunit sequences exhibit sequence similarity to the B subunits of the unrelated ArtAB (RIP-TT) and type II heat- labile toxins (RIP-HLT), respectively. Examination of the genomic loci where these toxins are encoded revealed a likely role for prophages and transposases in the evolution of both toxins. For further analyses, we assessed and confirmed A-B interactions in both toxins using molecular cloning and protein purification systems and subsequently investigated the cellular effects of purified RIP-TT and RIP-HLT in HeLa cells using an MTT cytotoxicity assay. Here, we showed that both toxins induce significant levels of cytotoxicity in a dose dependent manner. Importantly, mutations to amino acid residues postulated to be essential for their enzymatic and binding activities abolish toxin activity, indicating that these toxins enter and intoxicate cells using the anticipated pathway. Interestingly, we observed that the Stx2a A subunit can form synthetic hybrid toxins with the B pentamers of RIP-TT and RIP-HLT, and that both of these synthetic toxins are capable of causing cytotoxicity in HeLa cells. Our collective findings reveal a remarkably diverse arsenal of AB5 toxins is encoded by various Salmonella serovars and provide fundamental insights into the evolution of AB5 toxins.
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