Characterization of the importance of post-translational protein glycosylation in the pathogenesis of Staphylococcus aureus infections

• Applicant: Privatdozentin Dr. Christine Heilmann
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 377557971

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains largely unknown. We found that the plasmin-sensitive surface protein Pls is a post-translationally modified glycoprotein and identified the glycosyltransferases (Gtfs) involved in the sugar transfer. Previously, the pls gene has been demonstrated to be a virulence factor in mouse septic arthritis. Glycosylation occurs at serine residues in the Pls serine-aspartate (SD)-repeat region. Functional characterization revealed that Pls glycosyl residues are directly involved in biofilm formation via stimulation of intercellular adherence. Moreover recently, we found that another S. aureus SD-repeat surface glycoprotein, SdrE, also promotes biofilm formation, when glycosylated. In this project, we intend to exactly define the molecular mechanisms underlying increased biofilm formation mediated by the sugar modifications. For this, S. aureus strains will be generated that express glycosylated or non-glycosylated versions of the surface glycoproteins Pls, SdrE, SdrC, and SdrD and characterized using confocal laser scanning microscopy. To identify the respective glycosyl residue-specific interaction partner(s) on the neighbouring staphylococcal cell, pull-down assays and ELISAs will be performed. To produce the bait in the pull-down assays, the pls, sdrE, sdrC, and sdrD genes will be cloned in Escherichia coli, the respective proteins will be purified and in-vitro glycosylated. We are also interested to elucidate the glycosylation machinery, therefore different combinations of Gtfs and activated sugar precursors will be used in the in-vitro glycosylation assays and subsequently, glycosylated proteins will be analysed by mass spectrometry. This will define the positions and compositions of the modifying sugars, which may have an important impact on the function and antigenicity of surface glycoproteins. Additionally, different assays are planned to characterize a potential involvement of the modifying glycosyl residues of Pls, SdrE, SdrC, and SdrD in the adaptive and innate immune functions. X-ray crystallographie of glycosylated surface proteins Pls and SdrE is expected to clarify the mechanisms involved in intercellular adherence and biofilm formation mediated by the carbohydrate modifications. We also intend to analyse the role of the modifying glycosyl residues in host tissue colonization and internalization by human host cells. When the carbohydrates are involved in the adherence to human host cells, colonization, and/or internalization, we will perform pull-down assays to identify the putative host cell receptor(s). Finally, the in vivo importance of the modifying glycosyl residues of surface glycoproteins will be characterized in a mouse model of biofilm formation recently developed at our institute.

• DFG Programme: Research Grants