Membrane Protein Comparison Between Cell Membranes and ExtracellularVvesicle Membranes of S. pneumoniaeProvide Insights into Extracellular Vesicle Formation and Shedding.

Extracellular vesicles (EVs) are complex, cell-derived nanoparticles generated by all cell types. EVs are composed of lipid bilayer membranes and their associated membrane proteins, nucleic acids, and luminal proteins. The mechanism by which Gram-positive bacteria shed EVs is still unknown. EVs from the Gram-positive human pathogen S. pneumoniae, which is a major cause of otitis medi and pneumonia, are of particular interest because of how they EVs modulate the host immune response. To uncover possible mechanisms for EV production and shedding in S. pneumoniae, we have performed a comparative proteomics analysis of EV membrane proteins versus whole-cell membrane proteins. Membrane proteins were enriched from intact S. pneumoniae cells or their EVs using a ProMTag labeling and capture workflow. ProMTag is a bifunctional protein tag where one moiety of the tag is able to form a reversible, covalent link to primary amines on proteins. The other moiety is methyltetrazine, which can form an irreversible, covalent bond with trans-Cyclooctene (TCO) on the surface of beads to capture ProMTagged proteins for cleanup and elution. Using this workflow plasma membrane proteins can be tagged, captured, washed to remove non-plasma membrane proteins, and then eluted in their original, unmodified state. In this study, intact cells and EVs from S. pneumoniae cultures were separated and the extracellular domains of membrane proteins in these two fractions were labeled with ProMTag. The membrane proteins were then enriched, washed, and eluted using the ProMTag workflow. These membrane protein populations were then TMT labeled and analyzed using mass spectrometry. Comparative analysis revealed membrane proteins that are concentrated or absent in EV membranes relative to bulk plasma membrane from whole cells, indicating a selective process for EV formation in S. pneumoniae. With this information, we present a new model for EV formation and shedding in S. pneumoniae.