Isolation of Sphingolipids from Sphingomonas echinoides B-3126 and their Characterization by MS/MS Analysis

EDGAR DAVID URIBE SANCHEZ: Isolation of Sphingolipids from Sphingomonas echinoides B-3126 and their Characterization by MS/MS Analysis Sphingolipids are important components in human cellular lipid membranes and are differentiated by the incorporation of serine with an amide bond between the amino acid and a lipid tail. Sphingolipids produced by bacteria differ in both headgroups attached to the serine alcohol and the architecture of the lipid backbone. While the synthesis of sphingolipids in mammalian cells is well studied, only a handful of steps in the pathway are described in bacteria.1 The observations that although sphingolipid synthesis is rare in bacteria, it is more common among gut anaerobes and that these sphingolipids are substrates for CD1 signaling in the immune system, suggest that sphingolipids are involved in immune regulation of the host by the gut microbiome. In previous research on a mouse model, Bacteroides fragilis sphingolipids showed an ability to signal the neonatal immune system, inducing smaller levels of the gut invariant natural killer T (iNKT) cells in the adult and affording protection from colitis. 2 In my project, the hypothesized role of sphingolipids participating in the immune signaling is the motivation to isolate these compounds so they can serve as substrates to synthesize new immune-stimulating/regulating compounds. When binding to CD1d, the lipid part of the sphingolipids tends to control protein binding. The headgroup however is on the surface of the protein-lipid complex, aiding in recognition of the T cell antigen receptor3 Previous research has been reported in the development of headgroups that can selectively stimulate distinct cytokine profiles from iNKT cells.4 The sphingolipids are effective substrates of human CD1d and prompt the release of cytokines from activated iNKT cells. In this project, I conducted several experiments to optimize sphingolipid production and characterize sphingolipids from the strain Sphingomonas echinoides B-3126. I compared media for growing strains based on sphingolipid yield, as well as attempting to increase yield with a salinity stressor. I worked to develop sphingolipid extraction and isolation conditions. Following sphingolipid extraction, I used LCMS screening analysis to compare the strains against known sphingolipids uploaded to the Global Natural Products Social Molecular Networking (GNPS) platform.5 The goal of this research project is to optimize the isolation of bacterial sphingolipids. In the future our lab could functionalize these molecules into novel chemical entities, aiming for a large panel of semi-synthetic sphingolipids that can be tested for triggering specific cytokine profiles or for function as vaccine adjuvants.