Exploration of the bacterial polyadenylase PAP I as an antimicrobial drug target

The purpose of this project is to understand how polyadenylation of RNA transcripts contributes to antimicrobial resistance and bacterial pathogenesis. Polyadenylation in bacteria represents a critically understudied process that accelerates decay of target transcripts. The major bacterial polyadenylase PAP I is widely conserved among b- and g- proteobacteria but has never been studied in mammalian pathogens. In non-pathogenic E. coli, PAP I promotes plasmid replication and plasmid-encoded antimicrobial resistance. However, the role of PAP I in maintaining clinically-significant plasmids in bacterial pathogens is unknown. Our preliminary data suggest that PAP I promotes the maintenance of both native virulence plasmids and engineered antimicrobial resistance plasmids in the human pathogens Yersinia pseudotuberculosis and Shigella flexneri. The Yersinia and Shigella native virulence plasmids each encode a type III secretion system (T3SS) used to subvert host defenses. Yersinia and Shigella require PAP I for sufficient T3SS expression as well as for plasmid-encoded antimicrobial resistance. This underscores PAP I as a promising target for antimicrobial drug development. However, the polyadenylation landscape outside of lab-strain E. coli is completely unexplored, despite preliminary data suggesting species-specific roles for PAP I in resistance to cellular stress. We hypothesize that PAP I stabilizes diverse plasmids in pathogenic Enterobacteriaceae by polyadenylating transcripts that regulate plasmid replication and stability, promoting plasmid-mediated virulence and antimicrobial resistance. To address these gaps in knowledge and to test this hypothesis, we will carry out the following three aims. In Aim 1, we will assess the role of PAP I in antimicrobial resistance and cellular stress resistance in Klebsiella pneumoniae and Salmonella enterica, clinically relevant pathogens often associated with antimicrobial resistance plasmids in humans. We will extend our study to include a collection of antibiotic resistance plasmids isolated from human blood samples. In Aim 2, we will identify transcripts polyadenylated by PAP I in Y. pseudotuberculosis, K. pneumoniae, and S. enterica as well as those encoded by clinically-isolated antimicrobial resistance plasmids, using parallel transcriptomic approaches. We will then validate polyadenylation of prioritized PAP I targets predicted to be involved in plasmid maintenance or stress resistance. In Aim 3, we will determine how PAP I inactivation impacts bacterial pathogenicity when it depends on plasmid replication, using Y. pseudotuberculosis mouse infection as a model. This exploratory investigation sets the stage for comprehensive mechanistic studies of polyadenylation in pathogenic bacteria and lays a foundation for a drug discovery initiative targeting PAP I.