Project Summary:
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.