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

The bacterial cell cycle relies on the coordinated and dynamic interactions between division proteins and those involved in peptidoglycan (PG) synthesis. However, visualizing these interactions in vivo remains technically challenging. Here, we established fluorescence-lifetime imaging microscopy combined with Forster resonance energy transfer (FLIM-FRET) as a robust, spatially resolved technique to visualize protein interactions in living Staphylococcus aureus using fluorescent proteins. After systematically optimizing growth conditions and the analysis pipeline, we validated the method with cytosolic and membrane-anchored control proteins, achieving FRET efficiencies of up to 40%. Using FLIM-FRET, we mapped the protein interactions of the critical glycosyltransferase FtsW within the septal PG synthesizing complex. We confirmed its interaction with the cognate transpeptidase PBP1 and the regulatory protein DivIB. Notably, we found that FtsW also self-interacts, an observation corroborated by an alternative FLIM-FRET method employing Halo-Tag labelled with Janelia Fluor dyes. These findings support the hypothesis that septal PG synthesis may be carried out by a complex of multimers, capable of simultaneously synthesizing more than one glycan strand. Inhibition of PG synthesis by directly targeting PBP1 with the beta-lactam antibiotic imipenem weakened the interaction between PBP1 and FtsW, whereas the FtsW self-interaction was enhanced in a dose-dependent manner. In contrast, inhibition of PG synthesis by targeting the lipid II flippase, therefore depleting the FtsW-PBP1 substrate from the outer surface of the cell membrane, had little effect on these interactions. This suggests that alterations in FtsW interactions result primarily from antibiotic-induced conformational changes or from uncoupling the activities of FtsW and PBP1, resulting in the presence of uncrosslinked glycans, rather than merely from a loss of PG synthesis activity.