Final Report Summary - DIVANTI (THE DIVIDING CELL MEMBRANE: A PROMISING TARGET FOR NOVEL ANTIBIOTICS)
The worldwide rise in multidrug resistant pathogens is a major healthcare problem, and both the World Health Organization and European Union have identified antimicrobial resistance as a public health priority. The number of novel antibiotics that enter the clinic has dwindled over the last decades, and the development of new antibiotics is urgent. Cell division is a logical antimicrobial drug target, yet there are currently no clinically approved compounds that target this process. One of the main reasons is that we do not really know how bacterial cell division works. The aim of my CIG DIVANTI project was to understand the molecular mechanisms of bacterial cell division and use the generated knowledge to develop novel antimicrobial compounds.
A key discovery was the identification of the conserved cell division protein SepF. The protein makes large protein rings but it was a question whether the ring structure is relevant. We examined SepF from other species and found that they make comparable protein rings. The conserved nature of ring formation provides strong support for a functional role of this configuration. The cell division proteins FtsA and SepF are peripheral membrane proteins implying that a reversible, thus dynamic, interaction with the cell membrane is important for their functions. We have tested this and found that such dynamic interaction is not crucial for cell division. This facilitates the construction of artificial cell division systems in future synthetic cells. Most cell division genes, although conserved, are not essential in the model bacterium Bacillus subtilis. Finally, we have systematically removed all non-essential cell division genes, and it turned out that only FtsZ and SepF are required for cells to divide. This finding underlines the importance of SepF, and is also interesting from a synthetic biology point of view interesting, since it shows that division can be achieved with a minimal set of proteins.
To discover novel antibiotics active against cell division, we have built a high-throughput assay that can be used to find simultaneously compounds that inhibit either FtsA or SepF. In collaboration with the Westerdijk Fungal Biodiversity Institute (Utrecht, Netherlands), we have tested the activity of many fungal isolates for activity using a novel assay and found a compound that affects cell division. Finally, we found that antibiotic tolerant ‘persister’ cells are vulnerable for antimicrobials that target their cell membrane. Unexpectedly, this turned out to be primarily a consequence of the production of oxygen radical by the cell.
The results obtained during this period has provided some important new insights into the workings of the bacterial cell division process and opened new avenues to reconstruct a functional cell division machinery in synthetic/minimal cells. In addition, the project delivered a potent screening system for novel cell division targeting antibiotics that we will hope to explore in the near future. Another novel screening method yielded an unknown fungal compound that inhibits cell division. Finally, the project provided mechanistic insights into the killing of antibiotic tolerant persister cells that might find applications in the future.
A key discovery was the identification of the conserved cell division protein SepF. The protein makes large protein rings but it was a question whether the ring structure is relevant. We examined SepF from other species and found that they make comparable protein rings. The conserved nature of ring formation provides strong support for a functional role of this configuration. The cell division proteins FtsA and SepF are peripheral membrane proteins implying that a reversible, thus dynamic, interaction with the cell membrane is important for their functions. We have tested this and found that such dynamic interaction is not crucial for cell division. This facilitates the construction of artificial cell division systems in future synthetic cells. Most cell division genes, although conserved, are not essential in the model bacterium Bacillus subtilis. Finally, we have systematically removed all non-essential cell division genes, and it turned out that only FtsZ and SepF are required for cells to divide. This finding underlines the importance of SepF, and is also interesting from a synthetic biology point of view interesting, since it shows that division can be achieved with a minimal set of proteins.
To discover novel antibiotics active against cell division, we have built a high-throughput assay that can be used to find simultaneously compounds that inhibit either FtsA or SepF. In collaboration with the Westerdijk Fungal Biodiversity Institute (Utrecht, Netherlands), we have tested the activity of many fungal isolates for activity using a novel assay and found a compound that affects cell division. Finally, we found that antibiotic tolerant ‘persister’ cells are vulnerable for antimicrobials that target their cell membrane. Unexpectedly, this turned out to be primarily a consequence of the production of oxygen radical by the cell.
The results obtained during this period has provided some important new insights into the workings of the bacterial cell division process and opened new avenues to reconstruct a functional cell division machinery in synthetic/minimal cells. In addition, the project delivered a potent screening system for novel cell division targeting antibiotics that we will hope to explore in the near future. Another novel screening method yielded an unknown fungal compound that inhibits cell division. Finally, the project provided mechanistic insights into the killing of antibiotic tolerant persister cells that might find applications in the future.