Date of Award
Microbial infection involves complex and dynamic interplay of host defense and pathogen virulence pathways that requires new methods for mechanistic investigation. Towards this goal, I have developed chemical reporters of nucleic acid and protein synthesis and posttranslational modifications to explore their functions in bacterial pathogenesis as well as basic biological pathways. Initially, I developed amino acid chemical reporters that allow the cell-selective labeling of bacterial proteomes to facilitate the analysis of bacterial protein expression during infection of host cells. These reporters are based on unnatural amino acids that are incorporated into the bacterial proteome instead of methionine or phenylalanine when used in combination with mutant aminoacyl-tRNA-synthetases. This technology allows the visualization of bacterial protein synthesis during infection as well as the enrichment, identification and proteomic analysis of bacterial proteins from infected host cells. Additionally, I developed a chemical reporter for the study of protein AMPylation, a key posttranslational modification used by various bacterial pathogens to rewire host-signaling pathways. This reporter is used by all of the known AMPylation enzyme classes and allows the proteomic identification of enzyme-specific substrates in cell lysates. This project also stimulated the development of a chemical reporter for RNA synthesis, which in contrast to previous RNA reporters allows labeling of mRNA polyadenylation tails. In conclusion, I have developed chemical reporters for the cell selective labeling of bacteria in the presence of mammalian cells, a chemical reporter for protein AMPylation, and a chemical reporter for RNA synthesis and mRNA polyadenylation. These reagents should help to identify bacterial proteins that are differentially expressed during infection, new AMPylation substrates and the analysis of mRNA polyadenylation dynamics in basic cell biology and microbial pathogenesis studies in the future.
Grammel, Markus, "Chemical Reporters for Bacterial Pathogenesis and Beyond" (2012). Student Theses and Dissertations. 160.