Abstract
Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, produces a series of simple glycolipids where diacylglycerol and cholesterol serve as the precursor. The cholesterol-based glycolipids, cholesteryl 6-O-acyl-β-D-galactopyranoside (ACGal) and cholesteryl-β-D-galactopyranoside (CGal) are immunogenic and proposed to contribute to the pathogenesis of Lyme disease. Detailed studies of CGal and ACGal in Bb have been hampered by a lack of knowledge of their underlying biosynthetic processes. The genome of Bb encodes four putative glycosyltransferases, and only one of these, BB0572, was predicted to be an inverting family 2 glycosyltransferase (GT2 enzyme) capable of using UDP-galactose as a substrate and forming a β-glycosidic bond. Comparison of the 42 kDa BB0572 amino acid sequence from Bb with other Borrelia spp demonstrates that this protein is highly conserved. To establish BB0572 as the galactosyltransferase capable of cholesterol glycolipid formation in Bb, the protein was produced as a recombinant product in Escherichia coli and tested in a cell-free assay with 14C-cholesterol and UDP-galactose as the substrates. This experiment resulted in a radiolabeled lipid that migrated with the cholesterol glycolipid standard of CGal when evaluated by thin layer chromatography. Additionally, mutation in the predicted active site of BB0572 resulted in a recombinant protein that was unable to catalyze the formation of the cholesterol glycolipid. These data characterize BB0572 as a putative cholesterol galactosyltransferase. This provides the first step in understanding how Bb cholesterol glycolipids are formed and will allow investigations into their involvement in pathogen transmission and disease development.
Highlights
Borrelia burgdorferi (Bb) is the causative agent of Lyme disease (LD)
A galactosyltransferase capable of cholesterol glycolipid formation in Borrelia burgdorferi induced by Bb can result in a multisystem disease characterized by damage in various organs including the brain, central nervous system (CNS), heart, eyes, skeleton and joints [1]
A galactosyltransferase capable of cholesterol glycolipid formation in Borrelia burgdorferi catalyze the transfer of glycosyl residues from nucleotide-sugar or polyprenyl-phospho-sugar donors onto a variety of acceptors comprise multiple glycosyltransferase families [38]
Summary
Borrelia burgdorferi (Bb) is the causative agent of Lyme disease (LD). This disease is transmitted when a mammalian host is fed upon by an Ixodes tick infected with Bb and is the most common tick-borne disease in North America [1,2]. The outer membrane structure of Bb is atypical of most Gram-negative bacteria as it is composed of phosphatidylcholine, phosphatidylglycerol and multiple lipoproteins, but lacks the classical Gram-negative lipopolysaccharide or lipooligosaccharide [4] This bacterium, produces several glycolipids composed of a single galactose residue that modifies either diacylglycerol or cholesterol [5] to form monogalactosyl diacylglycerol (MGal) [6], cholesteryl β-D-galactopyranoside (CGal) and cholesteryl 6-O-acyl-β-D-galactopyranoside (ACGal) respectively. The cholesterol-based glycolipids are formed by the use of host-derived cholesterol and have been shown to be immunogenic, as well as possibly contribute to Lyme disease pathogenesis [7,8] These cholesterol-glycolipids constitute a significant portion, 45% [9], of the total lipid from in vitro grown spirochetes suggesting that they are important in the physiology of Bb
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