Elucidating the roles of protein glycosylation in prokaryotes through functional glycoproteomics - Project Summary Protein glycosylation, the post-translational attachment of carbohydrates to proteins, is crucial for a wide array of cellular processes spanning all domains of life. Yet for most prokaryotes, little is known about which proteins undergo glycosylation, and how this modification affects fundamental processes such as the formation of biofilms (microbial communities encased within an extracellular matrix) or resistance to antibiotics. While the complexity and diversity of protein glycosylation in prokaryotes have so far posed significant challenges to system-wide analyses, recent technological advances have opened new avenues to decipher the molecular mechanisms through which glycoproteins impact prokaryotic cell biology. Thus, this research program uses an interdisciplinary approach that combines proteomics, advanced bioinformatics, and cell biological assays to gain a more comprehensive, functional view of protein glycosylation in prokaryotes. The opportunistic bacterial pathogen Pseudomonas aeruginosa will be used to perform quantitative glycoproteomic analyses of different stages of biofilm formation, as well as different time points before and after exposure to an antibiotic. In addition, we will establish the Prokaryotic Proteome Project (ProkPP) for the large- scale, combined analysis of public proteomic datasets to identify and quantify glycoproteins across a broad range of experimental conditions and strains. We propose that the results from both approaches will allow us to elucidate which glycoproteins are involved in biofilm formation, and which ones are important for the resistance to antibiotics. Subsequently, we will generate knockout and glycosite-directed mutants for selected glycoproteins, and will characterize them through cell biological assays. The observed phenotypes for these mutants, in comparison to the wild type, will not only serve as a validation of the proteomic analyses, but will also allow us to gain further insights into the specific functions of these glycoproteins. Student researchers will be involved in all steps of this project, thereby obtaining valuable research experience, as well as cross-disciplinary training in proteomic techniques and data analysis. Importantly, the combined workflow of this project will serve as a blueprint that can also be used for functional glycoproteomic analyses of various other biological processes, such as host-pathogen interactions or intercellular communication (e.g. quorum sensing). The universal applicability of the employed bioinformatic tools will further allow us to expand the ProkPP to virtually any bacterial species. Altogether, this research program matches the complexity and ubiquity of glycoproteins with an interdisciplinary and generalizable approach to understand the fundamental roles of glycoproteins in bacterial pathogens.
