Subcellular protein localization in B. subtilis

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

In the last year, we have continued our studies that investigate how cells differentiate in an effort to understand how these processes may fail during diseases like cancer. In this report, we outline progress that we have made in the past year that extends our studies on how proteins localize and assemble during growth and development of the model organism Bacillus subtilis. Our lab has extended our analysis on the assembly of the bacterial spore coat as a model for understanding cellular morphogenesis. Previously, we had discovered a novel cytoskeletal protein, SpoIVA, that utilizes ATP hydrolysis to drive its static polymerization around the assembling bacterial spore. Curiously, SpoIVA is descended from a family of GTPases, but the selective pressure that drove this switch from GTP binding to ATP binding was unknown. We approached this problem by employing a computational approach to identify residues that were likely involved in ATP binding specificity, and used site-directed mutagenesis to restore the ancestral GTPase activity of SpoIVA. We discovered that, en route to polymerization, SpoIVA forms a stable ADP-bound intermediate that is not formed during GTP hydrolysis. Further, by measuring ATP and GTP levels during spore formation, we discovered that intracellular GTP levels remain very low, whereas ATP levels are relatively higher. We proposed that this paucity of GTP is the selective pressure that drove the switch in SpoIVA from utilizing to GTP to utilizing ATP. A manuscript describing these results were published in eLife in 2021. Previously, we have used our basic science discoveries to develop artificial bacterial spore-like particles that we proposed can be used as novel drug delivery vehicles. In the last year, we have engineered these particles to contain a sample drug cargo and have modified the surface of these particles to directly bind to certain epitopes that are overrepresented on cancer cells. We have demonstrated, in a mouse model, that these particles are safe when administered and can prevent the growth of tumors. Further, we demonstrated that drug delivery using this platform resulted in fewer side effects than a leading nanoparticle-mediated method. A manuscript describing these results has been posted to bioRxiv and is currently under revision at a peer-reviewed journal. Finally, we are investigating the efficacy of artificial spore-like particles as a vaccine display platform that displays peptide antigens. Although peptide antigens may provide high specificity and are relatively easy to generate, they are usually largely ineffective because of their short biological half-life. We are currently testing peptide antigens from various bacterial and viral pathogens, including Staphylococcus aureus and SARS-CoV2.