UC Berkeley

The complexity of the bacterial cell envelope along with the clinical implications arising from biological discoveries have produced steady research over the last century. Orchestration of cell envelope biosynthesis requires the coordination of proteins, carbohydrates, glycolipids and lipids alike. The majority of these biomolecules are not encoded genetically and their dynamics can be challenging to interpret from standard genetic and biochemical approaches. Throughout my doctoral studies, I have endeavored into developing chemical reporters to visualize individual components of the bacterial cell envelope in live cells. This chemical biology strategy allowed the discovery of front-line tuberculosis drug effects on cell envelope dynamics, mapping of the cell envelope assembly during cell growth and division, as well as tracking individual glycolipids during in vivo mycobacterial infection. These investigations are the subject matter of this dissertation.

In the first chapter, I discuss a suite of chemical approaches to label the non-protein components of the bacterial surface. In particular, covalent and non-covalent modifications are described based on specificity towards labeling individual components from the cell envelope as well as examples of species-specific strategies. This overview of the literature sets the stage for improving metabolic reporters that can reveal underlying molecular processes concerning bacterial cell envelope structure and dynamics. The lessons learned from this survey are utilized to provide the framework for rational design of metabolic reports outlined in the following chapters.

Chapter 2 describes the visualization of mycomembrane dynamics in real time in live bacteria. Metabolic labeling of trehalose mycolates associated with the cell envelope directly reports on mycomembrane fluidity. This strategy revealed that mycomembrane fluidity correlates with mycolic acid structure in Actinobacteria, and the glycolipids in Mycobacteria are nearly immobile. Our platform sets the stage for interrogation of mycomembrane fluidity as a proxy for increased susceptibility in mycobacteria during drug treatment.

The work presented in Chapter 3 relates to the study of cell wall dynamics during cell growth in live mycobacteria visualized by metabolic labeling. Development of molecular tools revealed the subcellular organization trehalose monomycolates with super resolution microscopy. Pulse-chase experiments revealed that mycobacteria significantly remodel their cell envelope during front-line tuberculosis drug treatment. This work reveals the underlying dynamics in cell wall biogenesis of pathogenic Mycobacterium marinum and provides insight into immediate stress responses. These findings also enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of new drug cocktails.

In Chapter 4, the investigation of cell envelope assembly during actinobacterial growth and division is described. Actinobacteria divide by a process called "V-snapping", where daughter cells remained joined until rapid snapping to form the characteristic V-shape. However, the exact cell wall layer that undergoes rupture during V-snapping and the temporal landscape for this process remains unclear. Unnatural cell wall reporters amenable to ligation of fluorophores by bioorthogonal chemistry were used to visualize how cell envelope organization is coordinated in actinobacteria. Dynamics of the cell envelope revealed that new peptidoglycan and arabinogalactan are actively biosynthesized during septation, but non-covalently membrane- bound glycolipids are only mobilized to the septum right before V-snapping occurs. We propose that peptidoglycan undergoes mechanical rupture to facilitate millisecond V-snapping.

Finally, Chapter 5 describes the investigation of trehalose glycolipids during infection in the M. marinum-zebrafish model. In efforts to characterize in vitro dynamics of these glycolipids, fast turnover was observed and this phenomenon was correlated to the release of outer membrane vesicles. Super resolution microscopy shed light on the ultrastucture of glycolipids within the cell envelope. Metabolic labeling of trehalose mycolates revealed dynamic trafficking of such species during infection in cellulo and in vivo, where outer membrane vesicles could play an important role during early host-pathogen interactions.