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Bacterial cell wall synthesis
The bacterial cell wall synthesis pathway is the most accessible essential pathway of bacteria and hence a very important target for antibiotic development. The pathway starts in the cytosol with the synthesis of two UDP-activated precursors UDP-GlcNAc and UDP-MurNAc-pentapeptide (see figure). This is followed by the assembly of the complete peptidoglycan subunit on a polyisoprenoid carrier, undecaprenyl phosphate, resulting in a product called Lipid II, the ultimate peptidoglycan precursor. Lipid II is then transported to the exterior side of the plasma membrane and used by bi-functional penicillin binding proteins (PBPs) for the synthesis of the cell wall. The resulting undecaprenyl pyrophosphate is then recycled back to the cytosol, de-phosphorylated after which it is ready to be used again. Our research mainly focuses on the membrane events of this cycle and the different projects are outlined below.
Characterization of the transport process of peptidoglycan subunits
Our group has identified the proteins that are involved in the transport of peptidoglycan subunits in 2011 that had so far remained elusive. These proteins FtsW, RodA and SpoVE were known to be essential during cell division, elongation and spore-formation respectively, and their essential roles can now be attributed to the transport of cell wall precursors. We now are characterizing the transport process by using our transport assays in combination with FtsW mutants and Lipid II variants. We also aim to determine the structure of this transport protein up to the atomic detail and are searching for novel antibiotics that target this essential step.
Mode of action of (l)antibiotics
With the ever-increasing prevalence of antibiotic resistance and the almost empty antibiotic pipelines of the pharmaceutical industry, there is a great need for the development of new antibiotics. In designing such new antibiotics, it is generally viewed optimal to look at the antibiotics that bacteria themselves use in their fight for survival. Evolutionary forces have shaped and continue to shape these antibiotics into excellent weapons. By learning how they work, new targets can be identified and we might be able to design better versions or even completely new antibiotics and so keep ahead in the arms race against resistant bacteria. As such, detailed investigation into the working mechanisms of lantibiotics has been lagging behind over the past years. We aim to reveal novel lantibiotic’ (validated) targets, which then may be used for the development of novel antibiotics. Most likely, these targets represent essential proteins or molecules that play a vital role in important cellular processes. Alternatively, we will reveal novel ways to inhibit known targets, from which, again, novel antibiotics may be designed.
Finding novel antibiotics targeting Gram-negative bacteria
Gram-negative bacteria are difficult to target due to the major distinguishable feature with their Gram-positive counterparts: the (extra) outer membrane (OM) within their cell envelope structure. The Gram-negative outer membrane acts as a formidable permeability barrier for antibiotic compounds and is the main reason why infections caused by Gram-negative bacteria are much more difficult to treat than those caused by Gram-positive bacteria. The outer membrane directly interacts with the external environment, and is essential for bacterial viability, thus the outer membrane itself and its biogenesis are interesting targets for antibiotic development.
In order to identify novel antibiotics we are developing HT-capable whole-cell based screening assays that are designed to identify antibacterial compounds that act specifically on the outer membrane and/or the biogenesis pathways of its essential and characteristic constituents, i.e. the lipoproteins, the outer membrane proteins (OMPs) and lipopolysaccharides (LPS).
Lipid II targeting antibiotics
The essential cell wall precursor lipid II is a very interesting target for antibiotic development. In collaboration with the Fungal Biodiversity Centre (CBS) of the KNAW, we are screening fungal extracts for novel antibiotics that act on Lipid II.