Quantitative study of microphage cellular proteome and phagosomal membrane proteome in response to mycobacterial lipids.

摘要

The recent advances in technology for mass spectrometry-based protein identification and quantification have opened new avenues for a broad range of proteomic applications in biological research. In this thesis, quantitative proteomic techniques have been applied to the study of macrophage responses to mycobacterial lipids at both a cellular level and an organellar level. The overall research goal is to expand the knowledge of how lipid factors produced by Mycobacterium tuberculosis (M. tb) modulate host protein expression patterns, particularly in the phagosome, which is a critical organelle for host defense against invading pathogens.;Chapter 1 provides an overview of the contribution of organellar proteomics to our understanding of phagosome biogenesis, maturation, and activation by certain cytokines. The latest technical platforms for quantitative proteomics are also discussed, with special attention given to stable isotope labeling techniques. Chapter 2 details our study of the global cellular proteome of macrophages upon exposure to M. tb lipid and mutual validation of protein quantification results. Chapter 3 describes our effort in analyzing the membrane proteome of latex bead-containing phagosomes isolated from macrophage cells. The extensive proteome profile suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling and autophagy. Finally, Chapter 4 describes a new workflow that incorporates several techniques for phagosome purification, membrane fractionation, and proteomic quantification in order to probe the effects of certain mycobacterial lipoglycans in mediating phagosome composition. Enlightened by our proteomic findings, we further describe biochemical and imaging experiments to uncover the role of a particular M. tb lipid toxin in undermining host autophagy activity.