Bacterial protein toxins : tools to study mammalian molecular cell biology

Bacterial protein toxins are genetically encoded proteinaceous macromolecules that upon exposure causes perturbation of cellular metabolism in a susceptible host. A bacterial toxin can work at a distance from the site of infection, and has direct and quantifiable actions. Bacterial protein toxins can target and interfere with almost any aspect of cellular metabolism. They are among the most potent, per unit weight, of all toxic substances, and are effective even in minute concentrations. The ability to harness those very attributes can turn bacterial protein toxins into invaluable tools. Bacterial protein toxins have been instrumental in exploring host cell biology, and we owe many of our insights into cellular physiology to their use. The application range of a native bacterial toxin as a tool however will eventually be limited by its biology. By combining new possibilities of protein engineering with technological advances, and thus enhancing the tool itself, bacterial protein toxins can be increasingly precisely tailored to address specific questions. The bacterial protein toxin cytolethal distending toxin (CDT) is the only known bacterial protein toxin that acts as a DNase in the nucleus. Chapter 2 and Appendix 1 describe the discovery of host proteins that are essential for bringing the enzymatically active CDT subunit CDTB to its target location. With a newly developed haploid genetic screen, we identify for the first time cellular host factors that are essential for intracellular CDT trafficking, and reveal that CDTs from different bacterial species exploit different subsets of the human proteome to achieve intoxication. Chaper 3 comprises the development of CDT as a tool via sortase-mediated trans-peptidation, to further explore CDT biology and the accompanying host cell biology. TMEM181, not only an essential factor for E. coli CDT intoxication, but a highly conserved yet previously completely uncharacterized cellular protein, is explored in some detail. In addition, we identify GRB2 as a novel CDT interactor, not uncovered in the haploid genetic screens possibly because of its essential nature for eukaryotic cell survival. In Chapter 4, bacterial protein toxin engineering using sortase is expanded to aerolysin, a model for pore-forming toxins. This is the first description of chemo-enzymatic engineering of aerolysin. This enzymatic means to install labels at precise predefined locations and the generation of single and double-labeled aerolysin monomers enables investigation of the fate of individual N and C-terminal domains, while preserving the functionality of the toxin. Chapter 5 describes the generation of mouse monoclonal antibodies against the fluorophore Alexa Fluor 647 (AF647) by microengraving screening. As an accessory tool, it complements the bacterial protein toxin toolkit as it transforms the single purpose AF647 into a multi-functional handle that can be used not only in fluorescence detection applications, but also in biochemical approaches such as immunoprecipitation and immunoblotting. Bacterial protein toxins are very precise modifiers of specific host factors, they are exact instruments to dissect basic host cell physiology. Recent advances in molecular engineering allow us now to tweak, calibrate and target those tools better. In a virtuous cycle, this drives knowledge accumulation of both toxin and host cell biology. Moreover, we are now able to tailor bacterial toxins to a variety of biomedical and molecular applications as diverse as immunotoxins, vaccines and next generation sequencing. We can expect many more exciting and surprising insights and biomedical applications to emerge from the field of bacterial protein toxin research.