Bugging the cell wall of bacteria : novel insights into the biosynthesis of peptidoglycan and its inhibition

Abstract

The last few decades saw an alarming rise of resistance against antibiotics, including the infamous methicillin-resistant Staphylococcus aureus (MRSA) that is resistant to the large group of antibiotics. This has turned the development of new antimicrobial compounds into a crucial necessity. The bacterial cell wall forms an invaluable target for antibiotics as it is essential for the viability of the cell and its location at the cell’s exterior gives it a relatively high accessibility. Many aspects concerning the biochemical processes involving the cell wall remain unclear, which likely conceals potential targets for new antibiotics. It is therefore important to deepen our understanding of cell wall physiology, which requires the development of additional strategies for cell wall analysis. This thesis describes the development and application of novel techniques to gain more insight into peptidoglycan metabolism and the enzymes involved herein with an emphasis on the role of the cell wall precursor Lipid II in the targeting of transglycosylases. The high tolerance of the bacterial cell wall synthesis machinery is fully exploited by the use of a variety of labeled peptidoglycan derivatives. A fluorescent cell wall labeling approach is set up, which is used to study peptidoglycan metabolism in vivo. It is shown that externally supplied NBD-labeled murein tripeptide is taken up by E. coli cells and metabolically incorporated into the cell wall via the peptidoglycan recycling pathway. By analyzing the cell wall labeling patterns in wild-type cells and several division and amidase mutants, FtsZ-dependent hydrolase activity during preseptal elongation was discovered. Additionally, we could visualize the major peptidoglycan hydrolase activity of AmiC during septation. The above developed method of labeling the cell wall of E. coli with reporter groups in vivo is adapted into a proteomics format. Using a tripeptide derivative containing a photoactivatable crosslinker and an alkyne moiety, proteins interacting with the cell wall and/or its precursors were crosslinked. Using the alkyne as bait, azide derivatized tags or beads could be covalently attached to the crosslink products via click chemistry, enabling their selective detection and purification. A number of interesting proteins were identified. The focus turned to the transglycosylation process, which for long was a black box in peptidoglycan biosynthesis and a potentially interesting target for novel antibiotics. In vitro photo-crosslinking in combination with mass spectrometry techniques provided information on the substrate (Lipid II) binding site in the transglycosylase penicillin-binding protein 1b (PBP1b) of E. coli. By reacting a photoactivatable analogue of Lipid II with the enzyme in the presence of moenomycin, it is shown that the substrate is covalently captured in one specific region, possibly encompassing the binding site of Lipid II. The mechanism of action of the potential transglycosylase inhibitor compound 5b is investigated. 5b is shown to disturb the functional integrity of negatively charged membranes. Moreover, nisin-Lipid II pore formation in model membranes was inhibited by 5b, which was reduced by an excess presence of undecaprenylpyrophosphate. This points to a specific interaction of 5b with Lipid II