FtsW protein-protein interactions visualized in live Staphylococcus aureus cells by FLIM-FRET

Abstract

The bacterial cell cycle relies on coordinated and dynamic interactions between division and peptidoglycan synthesis proteins. However, visualizing these interactions in vivo remains technically challenging. Here, we established fluorescence-lifetime imaging microscopy combined with Förster resonance energy transfer (FLIM-FRET) as a robust, spatially resolved technique to visualize protein interactions in living Staphylococcus aureus. We set up and validated the method using cytosolic and membrane-anchored control proteins, achieving FRET efficiencies of up to 40%. Using FLIM-FRET, we mapped protein interactions of the glycosyltransferase FtsW within the septal peptidoglycan-synthesizing complex. We confirmed its interaction with the cognate transpeptidase PBP1 and the regulatory protein DivIB. Notably, we found that FtsW also self-interacts, suggesting that septal peptidoglycan synthesis is performed by a complex of multimers, able to synthesize more than one glycan strand. Inhibition of peptidoglycan synthesis by directly targeting PBP1 with the beta-lactam antibiotic imipenem, but not by targeting the lipid II flippase, therefore depleting the FtsW-PBP1 substrate from the outer surface of the cell membrane, weakened FtsW-PBP1 interaction. This suggests that alterations in FtsW interactions result primarily from antibiotic-induced conformational changes or from uncoupling FtsW-PBP1 activities, resulting in the presence of uncrosslinked glycans, rather than merely from a loss of peptidoglycan synthesis activity.