Enterotoxigenic Escherichia coli (ETEC) is endemic to developing countries, in which acute infectious diarrhea is the second most common cause of childhood death, and millions of traveler's per year from the industrialized world are affected. A virulence factor produced by some strains of ETEC called the heat labile toxin (LT) can lead to these disease symptoms by detrimentally upsetting the balance of electrolytes in the intestine. The pentameric B subunit (LTB) of LT instigates this process by acting as a bacterial lectin, thereby localizing the toxin to the host cell surface through protein-carbohydrate interactions with the proper receptor(s). To date, the complexity of the number of binding partners utilized by LTB to carry out this process is likely underestimated. The primary host receptor for LTB is believed to be that of the monosialoganglioside GM1a, but its targeted binding pocket can also accommodate glycoconjugates carrying terminal N- acetyllactosamine (Gal?1-4GlcNAc?1-3-R) epitopes (albeit with significantly weaker affinity). Furthermore, there also exists a second (periphery) binding site distinct from the GM1a pocket capable of binding the blood group A and B antigens, primarily through direct contacts with these terminal trisaccharides. Currently, the role that these non-GM1a ligands play in LTB cell-surface binding/pathogenesis is still unclear, and the identity of the proteins and/or lipids that these glycan structures are conjugated to remains undetermined. This is because carbohydrate-protein interactions are notoriously difficult to study using traditional biochemical methods due to their transient/low affinity nature Therefore, the long term goals of this research program are (1) to develop methods that can be used to covalently capture cell-surface binding partners by inserting a crosslinker at specific types of monosaccharides within glycan structures and (2) to use this technology to answer the ongoing problem of which glycoprotein and glycolipid binding partners of the heat- labile toxin are responsible for its internalization during host cell infection.
Aim 1 of this proposal is to biosynthetically incorporate diazirine-modified sialic acid residues (a.k.a, SiaDAz) into GM1a and other cell- surface glycoconjugates of human intestinal epithelial cell lines (which most closely mimic the site of LTB infection). Upon photoirradiation, the diazirine is activated to form a highly reactive crosslinker that forms a covalent adduct with nearby molecules, and thus can be used to capture binding partners of LTB. The modified protein/lipid receptors within these now stable complexes will be identified by mass spectrometry, and their ability to initiate LTB internalization verified. An analogous approach will be used in Aim 2 to identify functional GlcNAc-modified LTB receptors (a.k.a., GlcNDAz), such as those possessing N- acetyllactosamine structures. These experiments will mark an important achievement in understanding the relevant proteins involved in ETEC mediated disease, and thus uncover novel therapeutic targets to complement existing treatments.
Acute infectious diarrhea due to enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children living in developing countries [1, 2], and is a major cause of traveler's diarrhea (TD) in persons visiting these regions from industrialized countries . While the heat- labile enterotoxin (LT) produced by some strains of ETEC is an attractive target for the development of preventative therapies, the events that lead to its entry into host cells are not fully understood. To address this issue, I propose to use chemical methods to identify the carbohydrate-modified receptors that are capable of interacting with this toxin.
|Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E et al. (2015) Fucosylation and protein glycosylation create functional receptors for cholera toxin. Elife 4:e09545|