Towards novel antibacterial development: from peptidoglycan to lipoprotein biogenesis

Abstract

Antibiotic resistance has been an emerging threat to the public health. Since the discovery of penicillin reported in 1929, continuous effort has been made in the past decades to discover more effective antibacterial molecules through drug-target interactions and molecule modifications . Antibiotic-mediated cell death starts with physical interaction between the drug molecule and its specific target that, depending on its mode of action, induces DNA damage, protein misfolding and mistranslation, cell envelope damage, and loss of structural integrity of the membrane(s). In addition, it was reported that major classes of bactericidal antibiotics induce cell death through the production of highly toxic hydroxyl radicals in both Gram-negative and Gram-positive bacteria, regardless of drug-target interaction. These results point out that the mechanism by which the current antibiotics kill bacteria is multilayered and complex. Bacterial cell envelope provides a rich source of drug targets and at the mean time also a barrier for certain antibacterial treatment. The major part of this PhD thesis describes the characterization of the phospho-MurNAc-pentapeptide translocase MraY, an enzyme that catalyzes the first membrane step of peptidoglycan synthesis. Being an alpha-helical membrane protein, MraY had been challenging to produce and purify with high yield. Characterization of MraY was also hampered due to lack of a crystal structure until very recently. Kinetics studies reported in the literature presented controversial evidences what the catalytic mechanism is. Furthermore, the development of MraY inhibitor was not very successful because the enzyme has two substrates, one being membrane embedded and not well documented, while the other resides in the cytoplasm, which is difficult to reach. In this PhD thesis, thorough kinetics experiments by varying the concentration of both substrates were conducted. The true kinetics values of this two-substrates enzyme were obtained. It is found that MraY must bind concomitantly to both substrates before the release of either of its products. Docking experiments gave insights to the binding model of MraY to its lipid substrate. The role of a catalytically important histidine residue was given. Furthermore, by using a novel styrene maleic acid copolymer system instead of the conventional detergent system to produce and characterize MraY, it was demonstrated that the accessibility of the embedded MraY to the lipid substrate can largely influence the starting rate of the MraY-catalyzed reaction. In the second part of the PhD thesis, a split-SNAP fluorescent reporter system for the localization of E. coli outer membrane lipoprotein is described. It was demonstrated that it is possible to detect defects along the lipoprotein biogenesis pathway using such a system. By modifying the constructs, the exploitation of such a reporter system can be expanded to outer membrane protein biogenesis as well. Finally in the last part, the overview of a promising alternative approach, antibacterial photodynamic therapy, to combat resistant bacterial strains was given. In this context, the bacterial cell envelope not only provides targets but also barriers for the delivery of the photosensitizers.