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

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.