Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen,Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding an α-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked α-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of3J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two relatedC. jejuni strains that express shorter ganglioside mimics in their LOS

A direct NMR method for the measurement of competitive kinetic isotope effects

We present a technique that uses 13C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (kcat/Km) for enzyme-catalyzed reactions. Using only milligram quantities of isotopically labeled substrates, precise competitive KIEs can be determined while following the ongoing reaction directly in a NMR spectrometer. Our results for the Vibrio cholerae sialidase\u2013catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.Peer reviewed: YesNRC publication: Ye

Complete chemoenzymatic synthesis of the Forssman antigen using novel glycosyltransferases identified in Campylobacter jejuni and Pasteurella multocida

We have identified an \u3b11,4-galactosyltransferase (CgtD) and a \u3b21,3-N-acetylgalactosaminyltransferase (CgtE) in the lipooligosaccharide (LOS) locus of Campylobacter jejuni LIO87. Strains that carry these genes may have the capability of synthesizing mimics of the P blood group antigens of the globoseries glycolipids. We have also identified an \u3b11,3-N-acetylgalactosaminyltransferase (Pm1138) from Pasteurella multocida Pm70, which is involved in the synthesis of an LOS-bound Forssman antigen mimic and represents the only known bacterial glycosyltransferase with this specificity. The genes encoding the three enzymes were cloned and expressed in Escherichia coli as soluble recombinant proteins that can be used to chemoenzymatically synthesize the Forssman antigen, and its biosynthetic precursors, in high yields.Peer reviewed: YesNRC publication: Ye

The genetic bases for the variation in the liposaccharide of the mucosal pathogen, Campylobacter jejuni : biosynthesis of sialylated ganglioside mimics in the core oligosaccharide

We have compared the lipo-oligosaccharide (LOS) biosynthesis loci from 11 Campylobacter jejunistrains expressing a total of 8 different ganglioside mimics in their LOS outer cores. Based on the organization of the genes, the 11 corresponding loci could be classified into three classes, with one of them being clearly an intermediate evolutionary step between the other two. Comparative genomics and expression of specific glycosyltransferases combined with in vitro activity assays allowed us to identify at least five distinct mechanisms that allowC. jejuni to vary the structure of the LOS outer core as follows: 1) different gene complements; 2) phase variation because of homopolymeric tracts; 3) gene inactivation by the deletion or insertion of a single base (without phase variation); 4) single mutation leading to the inactivation of a glycosyltransferase; and 5) single or multiple mutations leading to \u201callelic\u201d glycosyltransferases with different acceptor specificities. The differences in the LOS outer core structures expressed by the 11 C. jejuni strains examined can be explained by one or more of the five mechanisms described in this work.Peer reviewed: YesNRC publication: Ye

Evidence for Acquisition of the Lipooligosaccharide Biosynthesis Locus in Campylobacter jejuni GB11, a Strain Isolated from a Patient with Guillain-Barré Syndrome, by Horizontal Exchange

Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, has been shown to be genetically closely related to the completely sequenced strain C. jejuni NCTC 11168 by various molecular typing and serotyping methods. However, we observed that the lipooligosaccharide (LOS) biosynthesis genes strongly diverged between GB11 and NCTC 11168. We sequenced the LOS biosynthesis locus of GB11 and found that it was nearly identical to the class A LOS locus from the C. jejuni HS:19 Penner serotype strain (ATCC 43446). Analysis of the DNA sequencing data showed that a horizontal exchange event involving at least 14.26 kb had occurred in the LOS biosynthesis locus of GB11 between galE (Cj1131c in NCTC 11168) and gmhA (Cj1149 in NCTC 11168). Mass spectrometry of the GB11 LOS showed that GB11 expressed an LOS outer core that mimicked the carbohydrate portion of the gangliosides GM1a and GD1a, similar to C. jejuni ATCC 43446. The serum from the GB11-infected patient was shown to react with the LOS from both GB11 and ATCC 43446 but not with that from NCTC 11168. These data indicate that the antiganglioside response in the GB11-infected patient was raised against the structures synthesized by the acquired class A LOS locus

Structural Characterization of Campylobacter jejuni Lipooligosaccharide Outer Cores Associated with Guillain-Barré and Miller Fisher Syndromes

Molecular mimicry between lipooligosaccharides (LOS) of Campylobacter jejuni and gangliosides in peripheral nerves plays a crucial role in the pathogenesis of C. jejuni-related Guillain-Barré syndrome (GBS). We have analyzed the LOS outer core structures of 26 C. jejuni strains associated with GBS and its variant, Miller Fisher syndrome (MFS), by capillary electrophoresis coupled with electrospray ionization mass spectrometry. Sixteen out of 22 (73%) GBS-associated and all 4 (100%) MFS-associated strains expressed LOS with ganglioside mimics. GM1a was the most prevalent ganglioside mimic in GBS-associated strains (10/22, 45%), and in eight of these strains, GM1a was found in combination with GD1a mimics. All seven strains isolated from patients with ophthalmoplegia (GBS or MFS) expressed disialylated (GD3 or GD1c) mimics. Three out of 22 GBS-associated strains (14%) did not express sialylated ganglioside mimics because their LOS locus lacked the genes necessary for sialylation. Three other strains (14%) did not express ganglioside mimics because of frameshift mutations in either the cstII sialyltransferase gene or the cgtB galactosyltransferase gene. It is not possible to determine if these mutations were already present during C. jejuni infection. This is the first report in which mass spectrometry combined with DNA sequence data were used to infer the LOS outer core structures of a large number of neuropathy-associated C. jejuni strains. We conclude that molecular mimicry between gangliosides and C. jejuni LOS is the presumable pathogenic mechanism in most cases of C. jejuni-related GBS. However, our findings suggest that in some cases, other mechanisms may play a role. Further examination of the disease etiology in these patients is mandatory

GM3, GM2 and GM1 mimics deigned for biosensing : Chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

In press: YesNRC publication: Ye

Lipooligosaccharide of Campylobacter jejuni: SIMILARITY WITH MULTIPLE TYPES OF MAMMALIAN GLYCANS BEYOND GANGLIOSIDES

Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering Guillain-Barré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumor-associated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and Pk antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity