The Bacterial Cell Envelope: Specific Labeling and Its Applications

Abstract

Although there has been significant progress in the understanding of the mechanism and regulation of bacterial cell envelope biogenesis, further exploration is required to fully understand the bacterial cell envelope and its synthesis machineries and develop new methods to kill bacteria. This thesis aims to showcase the fluorescent labeling methods for studying bacterial cell wall envelopes and their application in studying outer membrane (OM) permeability and killing antibiotic-resistant pathogens. The assays involve chemical and chemoenzymatic synthesis, advanced microscopy techniques, and microorganism assays, and expand the toolbox of labeling bacteria and make it more accessible to a wider range of researchers. Chapter 2 describes a fluorescent labeling method that can label the newly synthesized lipopolysaccharides (LPS) of Gram-negative bacteria using azido-galactose, which is a common monosaccharide in bacterial LPS. This method provides a new way to specifically label Gram-negative bacteria through a click-chemistry reaction. Chapter 3 outlines a labeling strategy for peptidoglycan (PG) by functionalizing GlcNAc. This one-step biosynthesis of UDP-GlcNAz serves as an intermediate for incorporation into PG, allowing for labeling of the glycan core of PG. This approach provides a complementary labeling strategy for bacterial labeling that targets a different component than the previous study. The study is published in iScience in 2022. Chapter 4 describes the photodynamic inactivation of antibiotic-resistant pathogens using chemical photosensitizers, including photoactive D-amino acid derivatives and antibiotic photosensitizers. The killing mechanisms involve the incorporation of D-amino acids, penicillin-binding protein (PBP) binding, and D-ala-D-ala binding, leading to membrane damage and efficient killing of MRSA and VISA. Chapter 5 investigates OM permeability through a designed approach of labeling peptidoglycan with trans-cyclooctenes (TCO) and using fluorogenic probes to indicate OM permeability through the strain-promoted inverse electron-demanding Diels-Alder cycloaddition (SPIEDAC) reaction. This provides a direct method to indicate OM permeability. In summary, this thesis showcases new and complementary fluorescent labeling methods for bacterial components and their applications in studying OM permeability and killing antibiotic-resistant pathogens. These methods expand the toolbox of labeling bacteria and provide useful tools for researchers to study bacterial cell envelope biogenesis.