Characterisation of VapBC Toxin-Antitoxins from Mycobacterium tuberculosis

Toxin-antitoxin (TA) systems were identified more than 20 years ago on the mini F plasmid of Escherichia coli as plasmid stability elements; components responsible for purging bacterial cells that lack the plasmid from the population. More recent discovery of TA systems spanning a wide diversity of prokaryotic chromosomes, including that of Mycobacterium tuberculosis (M. tb), suggests a broader biochemical role. TA systems can be classified into a number of families, with the vapBC systems being by far the largest. The biochemical role of vapBC systems in M. tb remains unclear despite their abundance within the genome. This thesis describes the biochemical and functional characterisation of two vapBC systems encoded by operons Rv0065a/c and Rv0617a/c in the M. tb genome. VapCRv0617 overexpression had a bacteriostatic effect on the growth of Mycobacterium smegmatis cultures. Therefore, the biochemistry underlying this phenotype was investigated, along with that of the Rv0065a/c system. VapCRv0065 and VapCRv0617 are Mg2+-dependent, sequence-specific ribonucleases targeting GC rich 4-mers. Ribonuclease assays with in vitro synthesised RNA transcripts suggested an additional layer of target recognition resides in RNA secondary structure, and revealed that both VapC proteins exhibit high activity against isolated M. smegmatis 16S and 23S rRNA. Electrophoretic mobility shift assays with purified VapBC protein and labelled DNA demonstrated an autoregulatory function for the VapBCRv0617a/c complex and not the VapBCRv0065a/c complex. VapBCRv0617a/c bound specifically to a near-perfect inverted repeat in the Rv0617a/c promoter region that overlaps an annotated -10 M. tb promoter element. In contrast, the VapBCRv0065a/c complex exhibited no DNA binding activity against a putative Rv0065a/c promoter region. Individualised vapBC transcriptional regulation mechanisms may help explain the persistence of such an expanded number of these systems in the genome. Knowledge of the physiological role of vapBC systems in M. tb will enable a better understanding of how they contribute to the pathogenicity of this bacterium. This would serve as the basis for the design of drugs which interfere with vapBC system functioning and in turn the ability of M. tb cells to enter the persistent state.

Engineering bacterial nitroreductases for anticancer gene therapy and targeted cell ablation

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Engineering bacterial nitroreductases for anticancer gene therapy and targeted cell ablation

Bacterial nitroreductases are members of a diverse family of oxidoreductase enzymes that can catalyse the bioreductive activation of nitroaromatic compounds, including anti-cancer and antibiotic prodrugs. Nitroreductases have diverse applications in medicine and research, including anti-cancer gene therapy and targeted ablation of nitroreductase‑expressing cells in transgenic zebrafish to model degenerative diseases. Research in these fields to date has focused almost exclusively on the canonical nitroreductase NfsB from Escherichia coli (NfsB_Ec), which is a relatively inefficient choice for most applications. By exploring alternative nitroreductase candidates from a variety of bacterial species, in concert with enzyme engineering to fine-tune specific activities, we have generated improved prodrug-activating enzymes. The nitroreductase NfsB from Vibrio vulnificus (NfsB_Vv) has been central to our efforts, and following solution of its crystal structure, was selected as a scaffold for directed evolution via site-saturation mutagenesis. By applying library screening strategies that involved rounds of both positive and negative selection, several mutants that displayed improved activity with a promising next-generation cancer prodrug were identified. In parallel work, an engineered NfsB_Vv variant from the same library was found to be substantially improved in activation of the antibiotic prodrug metronidazole, which is widely used for targeted cell ablation in transgenic zebrafish. Current methods of ablation employing NfsB_Ec require high, near lethal concentrations of metronidazole to achieve total ablation of nitroreductase-expressing cells. A transgenic zebrafish line expressing a lead NfsB_Vv variant was generated and we found we could achieve robust ablation of nitroreductase-expressing cells at a 100-fold reduced metronidazole concentration compared to the NfsB_Ec line (0.1 mM challenge for 24 hours vs 10 mM challenge for 48 hours respectively). The identification of these superior nitroreductase variants offers improved tools for researchers aiming to achieve targeted cell ablation in either a cancer therapy or degenerative disease-modelling context

Discovering and engineering novel prodrug activating and detoxifying enzymes to improve targeted cell ablation

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Discovery and evolution of primordial antibiotic resistance genes from soil microbes

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Metagenomic discovery and directed evolution of genes that defend against chemotherapeutics

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Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall

Accessing bacterial dark matter for improved enzyme discovery and engineering

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