Characterization and exploration of the rationale for sphingolipid metabolism in host-associated Bacteroides

172 pagesSphingolipids, lipids based on aliphatic amino alcohols, historically derived their name from their mysterious ”sphinx-like” nature and continued to be described as enigmatic for their diverse and once-mysterious functions in cell structure and signaling. Yet, it could be argued that the now greater enigma is their presence in limited bacterial phyla, in which their roles and metabolic pathway are only beginning to be understood. The mystery grows when one considers the association of sphingolipid-producing bacteria with eukaryotic hosts (e.g., Bacteroidetes spp. abundant in the human gut) as the mechanisms underlying the maintenance and functional role of microbiome composition are likewise not yet fully uncovered. Bacterial sphingolipids, with recently described roles in host immune function and metabolism, serve as a possible trans-kingdom signaling mechanism linking these worlds of lipid and microbiome study. In the following chapters, I characterize sphingolipid and inositol lipid production in the model commensal bacterium Bacteroides thetaiotaomicron using a strain with tunable, inducible sphingolipid synthesis, as well as the first knockout strains of genes with predicted involvement in inositol lipid synthesis (myo-inositol-phosphate synthase and phosphoinositol-dihydroceramide synthase) in the Bacteroidetes. In Chapter 3, I use these strains in a transcriptomic analysis to identify how sphingolipid- or inositol lipid-deficient bacteria compensate for the loss of these lipids, tying changes in membrane lipid composition to altered carbohydrate metabolism, capsule synthesis, and transmembrane proteins (among others) that together form many of the adaptive mechanisms enabling bacterial life in an ever-changing mammalian gut. Then, in Chapter 4, I analyze two knockout strains of novel extracytoplasmic function sigma factors that are differentially expressed with the induction of sphingolipid synthesis by RNA-seq, likewise identifying roles in capsule synthesis and cell stress response. Finally, Chapter 5 outlines a role for bacterial sphingolipid-dependent interaction in a mammalian host, showing exchange of bacterial lipids with host cells and characterizing the importance of bacterial sphingolipids in the regulation of host liver ceramide levels. Sphingolipid and inositol lipid synthesis are phylogenetically restricted traits in Bacteria, yet common among host-associated species, with sphingolipid-producing Bacteroidetes spp. comprising 30-50% of the Western human gut microbiome. Together, these chapters strive to answer how the unique array of lipids in the Bacteroides spp. influence diverse aspects of bacterial physiology. In future work, these findings can be expanded to identify how these lipids may help facilitate a life in the mammalian gut, which may happen through (i) influence on membrane-mediated nutrient sensing, uptake, and antigen presentation mechanisms directly within the bacterial cell, and (ii) signaling or lipid provision to their mammalian host, in which sphingolipid signaling is widespread and fundamental