Synthesis and application of peptidoglycan oligomers as metabolic labeling agents for bacteria

Abstract

Peptidoglycan is the core component of every bacterial cell wall, which makes it an attractive target for the development of new bacterial targeting agents and broad-spectrum antibiotics. Although many major discoveries have been made on the bacterial cell wall in the past decade, much remains uncertain about the working mechanisms of bacterial cell wall biogenesis, as well as its dynamic transformations during a bacterium’s life cycle. One major bottleneck in these discoveries is the limited availability of tool compounds, derived from either natural or synthetic sources, to study the composition and dynamics of bacterial cell walls. The first part of this thesis describes an efficient and convenient approach to synthesize biohybrid peptidoglycan oligomers (PGOs), starting with the plentiful shrimp shell-derived biopolymer chitosan. The new method is the first that enabled top-down PGO synthesis as opposed to other bottom-up synthetic strategies reported. The whole process took thirteen steps in eight one-pot reactions and produced the final product in a practical gram scale. The highly water-soluble biohybrid PGOs were then synthetically conjugated to the fluorescent rhodamine dye and successfully incorporated into the peptidoglycan cell walls of both Gram-positive and Gram-negative bacteria strains. Using super-resolution STED confocal microscopy, PGO-rhodamine were found to be localized into the cell walls of all bacterial strains tested. Furthermore, the PGO-rhodamine was not taken up or incorporated into mammalian cells at all, thus confirming that PGOs can be a powerful tool for pan-bacteria-specific labeling and imaging. In the following part, mechanistic studies further supported the hypothesis of enzyme mediated metabolic labeling rather than non-specific binding to bacterial surface. The cell wall-deficient L-form strain of enterococcus showed a drastic reduction in PGO-rhodamine incorporation, and calorimetric studies further confirmed the strong binding between our PGOs and the penicillin-binding protein 1a (PBP1a). Moreover, the potential of PGOs was demonstrated in biosensing as a diagnostic tool based on the mechanisms explored. The agent could sensitively detect the presence of low amount of bacteria, reaching as low as ~10 CFU/mL. It was also capable of identifying antibiotic resistant strains, with the aid of respective antibiotics. Animal studies confirmed the excellent specificity and utility of PGOs for use in infection models relevant to real life situation, and no toxicity was observed in all in vivo experiments. Besides application of modified PGOs per se, the bacteria targeting capability was tested in conjugation with gold nanoparticles for colorimetric analysis in a subsequent chapter. Bacteria detection was demonstrated with high sensitivity using our design, by incubation only without the need of sample washing thereafter. Based on all the results presented in this thesis, the synthetic PGOs have been demonstrated applicable to bacterial detection and killing purposes with versatile modifications available in a feasible manner. The practical and efficient synthesis of this polymer is not an end to the project, but a starting point whereby a broad range of antibacterial applications could develop upon.