Who interacts with whom inside living bacteria?
Oeiras, 20th May 2026
To divide, bacterial cells depend on a highly coordinated multiprotein taskforce, but understanding which components interact closely inside these crowded cells has remained a major challenge. As a result, this key cell-division machinery remained an underexplored target for antibiotic development, particularly in pathogens like Staphylococcus aureus.
Researchers at ITQB NOVA's Bacterial Cell Biology Lab, led by Mariana Pinho, improved and implemented a method to detect who is meeting whom inside living bacteria. They used an advanced microscopy approach called FLIM-FRET, which allows them to detect when members of this taskforce interact inside the cell. The results are now published in Nature Communications.
At the heart of this “surveillance system” is FRET (Förster Resonance Energy Transfer). Researchers label two proteins of interest with different fluorescent tags: a donor and an acceptor. If the proteins come extremely close, energy can transfer from the donor to the acceptor. When that happens, the donor’s signal weakens while the acceptor’s signal grows stronger, revealing that the proteins were in close proximity.
The second part, FLIM (Fluorescence Lifetime Imaging Microscopy), adds a crucial advantage. Instead of measuring how bright the fluorescence is, FLIM measures how long the donor fluorophore “glows” after being excited. If FRET occurs, the donor has an extra way to release energy, so its lifetime becomes shorter.
Together, FLIM-FRET provides information not only on whether proteins are interacting, but also where. However, using this method in bacteria is challenging. First, the research team had to optimize the growth conditions of Staphylococcus aureus and correct for the light scattering resulting from bacteria sitting near the microscope coverslip during imaging, improving the reliability of measurements.
Only then were they able to apply this approach to living cells, focusing on FtsW, a protein essential for bacterial cell division and cell wall construction. The researchers revealed that FtsW interacts with its known partner PBP1 and its regulatory protein DivIB. But the team also discovered that FtsW interacts with itself. This suggests that cell wall synthesis is carried out by clusters of this protein working together as a larger assembly.
But there is more: “These interactions are not static. When cells are treated with a beta-lactam antibiotic, the interaction between FtsW and one of its partners weakens, while self-interaction increases,” explains Nils Meiresonne, first author of the paper and a postdoc at ITQB NOVA. “This indicates that antibiotics can reshape the architecture and dynamics of the cell wall synthesis machinery, not simply shut it down.” These insights, together with those that may emerge from applying this method more broadly, could help guide future antibiotic development.
Image caption: Fluorescence lifetime image of Staphylococcus aureus cells producing the acceptor fluorescent protein (mNeonGreen) in the cytosol and the donor fluorescent protein (superfolder mTurquoise2 ox) attached to the membrane. The acceptor was photobleached in regions shaped “FLIM,” where the membrane-bound donor can be observed. Fluorescence lifetime provides an additional source of contrast (see color scale). The image was acquired using the ZEISS LSM880 confocal system combined with the PicoQuant FLIM upgrade kit available through the ITQB bacterial imaging cluster.
Original Paper:
Nature Communications | https://doi.org/10.1038/s41467-026-72752-7
FtsW protein-protein interactions visualized in live Staphylococcus aureus cells by FLIM-FRET
Authors: Nils Y. Meiresonne, Sara F. Costa, Simon Schäper, Mário J. Ferreira,
Patricia Reed, Zach Hensel, Fábio Fernandes & Mariana G. Pinho
