The ExoY family of cytotoxins are prevalent virulent factors expressed by human pathogens of emerging clinical interest. ExoY family toxins function as nucleotidyl cyclases, enzymes that catalyze the conversion of nucleotide triphosphates into cyclic nucleotide monophosphates. Studies have shown that the enzymatic activity of ExoY family enzymes is stimulated by differing bioforms of actin, a prevalent eukaryotic cytoskeletal protein that exists in two distinct forms: monomeric, globular actin (G-actin), and polymeric actin filaments (F-actin). I have recently investigated the relationship between ExoY from Pseudomonas aeruginosa (PaExoY) and its activator, F-actin. I discovered that PaExoY can reorganize actin filaments into thick cable-like bundles. This actin bundling activity is commonly observed among eukaryotic regulatory proteins and my data suggests that it may be coupled to PaExoY catalysis. Actin bundles play key roles in numerous cellular processes, yet they are largely understudied, as they produce highly heterogeneous populations in vitro ill-suited to conventional structural analysis. I have developed an approach, outlined in this proposal, focused on reducing complexity to investigate the structural organization of the PaExoY-actin bundle. This approach integrates cutting edge structural techniques with biochemical and biophysical assays and can be applied to effectively study actin bundles of diverse origin. The results of this work will greatly expand our understanding of the molecular processes underlying actin bundle formation. I will then expand the scope of my studies to investigate the activation of a related toxin from the human pathogen Vibrio vulnificus (VvExoY), which the data suggest is activated by G-actin, not F-actin. A similar approach will be employed to characterize VvExoY-actin interactions and allow for identification of the key factors responsible for ExoY family activation and actin recognition. Such knowledge will provide a vital foundation in the design of small molecule inhibitors with the goal of generating novel therapeutic strategies against bacterial intoxication. Additionally, I will compare the results of this work with other known bacterial toxins, such as anthrax edema factor, to assess the practicality of using ExoY family toxins as a novel research tool to study actin- associated signaling processes. This work has been specifically designed to build off of my established foundation in Pseudomonas microbiology and X-ray crystallography and expand my structural biology skillset, particularly in the area of high-resolution electron microscopy. I have formed a diverse array of collaborators to assist in the proposed research and serve as an effective mentorship team throughout my postdoctoral training. Through close interactions with these experts, I will foster skills not just as an experimentalist, but as a well- rounded scientist, enhancing my skills in areas such as grantsmanship, scientific communication, and mentorship. At the conclusion of this fellowship, I will expand upon this research in the form of a K99/R00 proposal as I transition from my postdoctoral training to a position as an academic principal investigator.
The ExoY-family of bacterial toxins are prevalent virulence factors produced by human pathogens such as Pseudomonas, Vibrio, and Burkholderia, and are known to be activated by binding to different multimeric forms of actin, yet the foundational mechanisms underlying this activation are unknown. This proposal aims to explore the structural basis for ExoY-actin interactions and define the molecular mechanisms governing ExoY activation. Successful completion of the proposed aims will enhance our understanding of the basic processes governing actin bundle formation, provide foundational knowledge to guide therapeutic development, and evaluate the effectiveness of ExoY enzymes as tools to probe actin-related signaling processes.
