Exploring the Therapeutic Potential of Pseudomonas aeruginosa Exotoxin A and Identifying Eukaryotic Factors Involved in its Intracellular Routing Pathway

Pseudomonas aeruginosa Exotoxin A (ETA) is a 66.6 kDa bacterial toxin comprised of a single polypeptide chain that folds into three distinct structural and functional domains. Domain I serves as a ligand for binding susceptible eukaryotic cells. Domain II facilitates the transport of ETA to the cytosol. Domain III contains the active site by which ETA inhibits protein synthesis and initiates cell death. Although extensive research has gone into characterizing each domain and manipulating ETA to create targeted therapies, the intracellular routing of ETA and the role of domain II in this pathway remains uncertain. We hypothesized that ETA domain II interacts with intracellular components in the human cell to facilitate transport of the toxin from the endosome to the cytosol. This thesis investigates the use of ETA domain II as a strategy to increase the intracellular delivery of protein therapeutics, and explores cellular components that may be involved in the routing pathway of ETA in human cells. The first study demonstrated that fusing ETA domain II to cell penetrating peptides (CPPs) and their cargos enhanced the delivery of these recombinant proteins to the cytosol through retrograde transport. The second study identified 33 putative eukaryotic genes required for ETA cytotoxicity using a high-throughput lentiviral screen. Additionally, a number of proteins involved in endosome ←→ Golgi apparatus ←→ endoplasmic reticulum ←→ cytosol trafficking were identified from human cell lysate pull-down assays using ETA domain II and full-length ETA, supporting the retrograde transport hypothesis. Overall, the results of these studies confirm that ETA follows a retrograde transport pathway in human cells and that domain II plays a role in this routing. ETA can be manipulated in creative ways for both investigative and therapeutic purposes and, as such, a further understanding of ETA and other such toxins is beneficial for converting them into useful tools.