Elongation Factor P-Dependent Translation and Metabolic Phenotypes of Salmonella

The facultative intracellular pathogen Salmonella has acquired adaptations contributing to virulence, including pathogenicity islands and regulatory alterations. Elongation factor P (EF-P) is a universally conserved translation factor, and deletion of efp in Salmonella results in pleiotropic phenotypes, including hyperactive metabolism, reduced stress resistance, and avirulence. Crystal structures demonstrated that EF-P adopts the shape of a tRNA and binds to the ribosome to stimulate peptide bond synthesis. Preliminary analyses suggested that EF-P is not a general translation factor but rather influences a specific subset of proteins. In this thesis I investigate the mechanism underlying EF-Pâ s impact on translation and how its deletion leads to the observed phenotypes. I present the Salmonella EF-P-dependent proteome, and particular proteins offer explanations for efp mutant phenotypes. Meanwhile, other groups discovered that EF-P rescues ribosomes stalled at polyproline motifs. I expand upon their work and show that polyproline motifs are not strictly necessary or solely sufficient to render a protein dependent on EF-P. I demonstrate that residues upstream of the polyproline motif significantly influence stall strength. Furthermore, translation initiation rate greatly impacts EF-P-dependence. I present a model wherein elongation stalls only reduce protein synthesis if they are more rate-limiting than translation initiation. While examining efp mutants, I discovered a novel phenotype of wild-type Salmonella. When dicarboxylates are the sole carbon source, Salmonella represses its growth using the general stress response sigma factor RpoS, the RpoS-stabilizer IraP, and repression of the dicarboxylate transporter DctA. Moreover, E. coli does not exhibit this phenotype and the E. coli dctA promoter is sufficient to induce Salmonella growth. This divergence suggests a potential role in virulence, and I examine the accumulation of succinate and itaconate in activated macrophage. I demonstrate that the dicarboxylate phenotype does not influence intracellular survival, and I propose novel hypotheses of why Salmonella has evolved this trait.Ph.D