How to find new antibiotics

The story of a protein complex that turns out to be an essential on/off switch for bacterial cell division

17 October 2018

Scientific discoveries seldom are the result of a Eureka!-moment of a single person. They usually take years of hard labour by multiple people and lots of collaboration. This is the story of how we are starting to unravel a protein complex that is essential for bacterial cell division, as told by Tanneke den Blaauwen from the Swammerdam Institute for Life Sciences at the University of Amsterdam (UvA).

Life as we know it would not be possible without bacteria. Bacteria have a bad name, because some of them make us very ill. But there are also many beneficial bacteria, for example the ones in our guts. Without them, we would not be able to properly digest our food. 

New antibiotics

Still, the ‘bad’ bacteria are a problem, and increasingly so as pathogenic bacteria that are resistant to multiple antibiotics are on the rise. There is a constant arms race between these bacteria and scientists worldwide who are trying to find new antibiotics to stop them. But, where to start when you want to develop new antibiotics?

A good starting point is trying to unravel how bacteria multiply. Because if you can stop this, you can stop them in their tracks. We know bacteria multiply by cell division, so the trick is to find proteins or other molecules that are essential for this process. At the UvA’s Swammerdam Institute, the Bacterial Cell Biology & Physiology research group focuses on a protein called FtsQ.

Initial discovery

The story of this protein starts in 1994, when professor Nanne Naninga started to investigate this mysterious but seemingly essential protein. His PhD student Nienke Buddelmeijer discovered that FtsQ is localized in the middle of the bacterial cell when it is about to divide. So, exactly at the spot where the division will take place. Making this discovery was really not an easy job, since Buddelmeijer had to do it without the help of the fluorescent dyes we can now use to label proteins and follow them in the cell.

Picking up the thread

Next up, den Blaauwen’s PhD student Thesse Vinkenvleugel managed to determine which amino acids within FtsQ are important for its localization in the cell, using the newly discovered technique of using fluorescent protein fusions to tag proteins and thus make their localization visible. In collaboration with Jan Löwe of the Cambridge University – the star of bacterial cytoskeleton crystallization – the first crystal structure of FtsQ was produced in 2008. In the meantime, Buddelmeijer had discovered that another protein called FtsB is FtsQ’s major partner in crime during bacterial cell division. Joen Lurink of the VU University picked up the thread. After studying the mechanism of protein translocation in the bacterial cell membrane using FtsQ, he realized that the FtsQLB protein complex is essential for cell division in most bacterial species and started to study the interaction between these three proteins.

Potential target

The discovery that the FtsQLB protein complex is essential for cell division in so many bacteria means it is a very interesting potential target for new broad spectrum antibiotics. Basically, this protein complex is the general on/off switch for bacterial cell division. And it is absent in human cells; so antimicrobial drugs that interact with this protein complex will not hurt us.

The current state of affairs

In order to develop such drugs, we need to understand the protein complex in greater detail. A collaboration of the Molecular Microbiology group of Joen Luirink, three Chemistry groups from the Amsterdam Institute for Medicines and Systems (AIMMS), the Bacterial Cell Biology and Physiology group of the Swammerdam Institute for Life Sciences and the MRC laboratory of Molecular Biology of Jan Löwe allowed the precise analysis of the interaction between FtsQ and FtsB. Using methods such as site directed mutagenesis, localization studies, in vitro surface plasmon resonance protein interaction analysis, peptide synthesis and crystallization, this collaboration culminated in a publication in mBio this year (2018), where the exact interaction of the amino acids 64-87 of FtsB with the very conserved binding site of FtsQ was determined. This study provided us with atomic details of the interaction interface. Knowing these details makes  it possible to  design inhibitory molecules, that have a potential to serve as novel antimicrobial drugs.

Divisome

Figure: Schematic representation showing the complexity of the division machinery (“divisome”) of bacteria. The proteins shown are involved in gram negative bacterial cell envelope synthesis. The envelope consists of an outer membrane and a cytoplasmic membrane with in between the periplasm with the peptidoglycan layer. The peptidoglycan (PG) layer is a very strong network of sugar strands that are crosslinked by peptide side bridges. It is a unique bacterial structure and essential for their survival. Therefore, many of the proteins involved in its synthesis are suitable antibiotic targets (i.e. the well-known penicillin binding proteins). The PG building unit is synthesized in the cytoplasm by Mur proteins. After synthesis, it is coupled to a lipid linker and flip-lopped across the cytoplasmic membrane by MurJ. It was shown only this year that MurJ is part of the divisome (Liu et al. Mol Microbiology 2018). Subsequently, the PG precursors are inserted into the existing layer by FtsW, PBP3 and PBP1B. This is a tricky business, because the layer has to be opened by hydrolases such as PBP4 to allow insertion of new material. If this is not coordinated well, the cells will explode due to a high internal pressure. This illustrates that also the coordination and regulation process would be a good target to kill the cells. FtsQLB (purple complex) are the on/off switch for cell division. The proteins on the left of FtsQLB are involved in positioning the new septum at mid cell.

And now…

Presently, this research is being followed up in many research groups. The FtsQB interaction site is being analysed by Daan Geerke (Computational Chemistry, VU), while a collaboration between Tom Grossmann, Joen Luirink (both VU) and Stanislav Gobec (University of Ljubljana) is ongoing to design small molecule and peptidomimetic inhibitors against the division complex. Moreover, at the Swammerdam Institute for Life Sciences, Nils Meiresonne from the Bacterial Cell Biology and Physiology group is collaborating with Joachim Goedhart from the Molecular Cytology group to use FRET, a fluorescence based interaction assay (bioRxiv 2018).

The ultimate message: collaborate

The story of all the work on this protein complex illustrates that new antibiotics do not come with express service. But they will be ultimately delivered when collaborations are supported, without first and leading author issues.

 

Publications

Buddelmeijer, N., Aarsman, M. E., Kolk, A. H., Vicente, M., & Nanninga, N. (1998). Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli. Journal of Bacteriology, 180(23), 6107–6116.

Buddelmeijer, N., & Beckwith, J. (2004). A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region. Molecular Microbiology, 52(5), 1315–1327. doi: 10.1111/j.1365-2958.2004.04044.x

 Van den Ent, F., Vinkenvleugel, T. M. F., Ind, A., West, P., Veprintsev, D., Nanninga, N., et al. (2008). Structural and mutational analysis of the cell division protein FtsQ. Molecular Microbiology, 68(1), 110–123. doi: 10.1111/j.1365-2958.2008.06141.x

Alexeeva, S., Gadella, T. W. J., Verheul, J., Verhoeven, G. S., & Blaauwen, den, T. (2010). Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET. Molecular Microbiology, 77(2), 384–398. doi: 10.1111/j.1365-2958.2010.07211.x 

van den Berg van Saparoea, H. B., Glas, M., Vernooij, I. G. W. H., Bitter, W., Blaauwen, den, T., & Luirink, J. (2013). Fine-mapping the contact sites of the Escherichia coli cell division proteins FtsB and FtsL on the FtsQ protein. The Journal of Biological Chemistry, 288(34), 24340–24350. doi: 10.1074/jbc.M113.485888 

Glas, M., van den Berg van Saparoea, H. B., McLaughlin, S. H., Roseboom, W., Liu, F., Koningstein, G. M., et al. (2015). The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity. The Journal of Biological Chemistry, 290(35), 21498–21509. doi: 10.1074/jbc.M115.654756

Kureisaite-Ciziene, D., Varadajan, A., McLaughlin, S. H., Glas, M., Montón Silva, A., Luirink, R., et al. (2018). Structural Analysis of the Interaction between the Bacterial Cell Division Proteins FtsQ and FtsB. mBio, 9(5), 321. doi: 10.1128/mBio.01346-18

Meiresonne, N. Y., Consoli, E., Mertens, L. M., Chertkova, A., Goedhart, J., & Blaauwen, den, T. (2018). Superfolder mTurquoise2ox optimized for the bacterial periplasm allows high efficiency in vivo FRET of cell division antibiotic targets. bioRxiv 415174; doi: 10.1101/415174 

(from figure) Liu, X., Meiresonne, N. Y., Bouhss, A., & Blaauwen, den, T. (2018). FtsW activity and lipid II synthesis are required for recruitment of MurJ to midcell during cell division in Escherichia coli. Molecular Microbiology, 109(6), 855–884. doi: 10.1111/mmi.14104

Published by  Swammerdam Institute