We have compared the lipo-oligosaccharide (LOS) biosynthesis loci from 11 Campylobacter jejuni strains 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 allow C. 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 "allelic" 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.Many pathogenic bacteria have variable cell-surface glycoconjugates such as capsules in Streptococcus spp. and Neisseria meningitidis (1), lipopolysaccharides in Gram-negative bacteria (2), and glycosylated surface-layer proteins (3). In mucosal pathogens, the variability of cell-surface polysaccharides has been shown to play a major role in virulence (4). This variation is caused by the diversity of monosaccharide components and the linkages between them, derivatization with noncarbohydrate moieties, and in some cases, by the length and sequence of the repeating units. The variation of these glycan structures can sometimes be correlated with a specific gene complement, but it is probable that other genetic mechanisms are also employed to create variable cell-surface glycoconjugates. The DNA sequencing of the relevant genetic loci from multiple strains of a pathogen can provide insights into the genetic origins of important strain variable traits such as cell-surface glycoconjugates.The mucosal pathogen Campylobacter jejuni has been recognized as an important cause of acute gastroenteritis in humans (5) and has been shown to have variable cell-surface carbohydrates that are associated with virulence (6, 7). Epidemiological studies have shown that Campylobacter infections are more common than Salmonella infections in developed countries, and they are also an important cause of diarrheal diseases in developing countries. C. jejuni is also considered the most frequent antecedent infection to the development of GuillainBarré syndrome, a form of neuropathy that is the most common cause of generalized paralysis since the eradication of poliomyelitis in developed countries (8). The core oligosaccharides of low molecular weight lipo-oligosaccharides (LOS) 1 of many C. jejuni strains have been shown to exhibit molecular mimicry of the carbohydrate moieties of gangliosides (Fig. 1). Te...

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-Barré syndrome. We first cloned a gene encoding an ␣-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 ␤-1,4-N-acetylgalactosaminyl-transferase (cgtA), a ␤-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 ␣-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 of 3 J(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 related C. jejuni strains that express shorter ganglioside mimics in their LOS.

The genes encoding the ␣-2,3-sialyltransferases involved in lipooligosaccharide biosynthesis from Neisseria meningitidis and Neisseria gonorrhoeae have been cloned and expressed in Escherichia coli. A high sensitivity enzyme assay using a synthetic fluorescent glycosyltransferase acceptor and capillary electrophoresis was used to screen a genomic library of N. meningitidis MC58 L3 in a "divide and conquer" strategy. The gene, denoted lst, was found on a 2.0-kilobase fragment of DNA, and its sequence was determined and then used to design probes to amplify and subsequently clone the corresponding lst genes from N. meningitidis 406Y L3, N. meningitidis M982B L7, and N. gonorrhoeae F62. Functional sialyltransferase was produced from the genes derived from both L3 N. meningitidis strains and the N. gonorrhoeae F62. However, the N. meningitidis M982B L7 gene contained a frameshift mutation that renders it inactive. The expression of the lst gene was easily detected using the enzyme assay, and the protein expression could be detected when an immunodetection tag was added to the COOH-terminal end of the protein. Using the synthetic acceptor N-acetyllactosamineaminophenyl-(6-(5-(fluorescein-carboxamido)-hexanoic acid amide), the ␣-2,3 specificity of the enzyme was confirmed by NMR examination of the reaction product. The enzyme could also use synthetic acceptors with lactose or galactose as the saccharide portion. This study is the first example of the cloning, expression, and examination of ␣-2,3-sialyltransferase activity from a bacterial source. Mammalian oligosaccharides containing terminal N-acetyl-neuraminic acid (Neu5Ac) 1 residues are recognized as biologically important carbohydrates for their function as receptors for lectins involved in cellular adhesion, as receptors for toxins, and for certain viruses (1). Some pathogenic bacteria have also been shown to carry sialylated oligosaccharides in their lipooligosaccharides (LOS), which are identical in structure to those found in mammalian glycolipids. Neisseria gonorrhoeae and Neisseria meningitidis LOS contain ␣-2,3-monosialylated lacto-N-neotetraose (2, 3), and in Camplyobacter jejuni the structures are variants of a mono-, di-, or tri-sialylated ganglioside (4). The role of these sialylated oligosaccharides in the pathogenesis of N. gonorrhoeae has been clearly demonstrated (2), and although the precise role of similar sialylated LOS in the pathogenesis of N. meningitidis or C. jejuni is not known, it is presumed to be a form of molecular mimicry that aids in the evasion of the host immune response.There have been extensive studies of the sialyltransferases involved in mammalian glycoconjugate synthesis, where at least eight different enzymes with an ␣-2,3-sialyltransferase activity have been examined either through protein purification or the cloning of the genes (1). In contrast, the bacterial sialyltransferases involved in the synthesis of ␣-2,3-sialylated lacto-N-neotetraose from N. gonorrhoeae or N. meningitidis and the enzyme(s) involved in the sialylat...

The thermostability of the 20 396 Da Bacillus circulans xylanase was increased by the introduction of both intra- and intermolecular disulfide bridges by site-directed mutagenesis. Based on the 3-D structure of the enzyme, sites were chosen where favourable geometry for a bridge existed; in one case, to obtain favourable geometry additional mutations around the cysteine sites were designed by computer modelling. The disulfide bonds introduced into the xylanase were mostly buried and, in the absence of protein denaturants, relatively insensitive to reduction by dithiothreitol. The mutant proteins were examined for residual enzymatic activity after various thermal treatments, and were assayed for enzymatic activity at elevated temperatures to assess their productivity. We have examined one of these mutants by X-ray crystallography. All of the disulfide bond designs tested increased the thermostability of the B. circulans xylanase, but not all enhanced the activity of the enzyme at elevated temperatures.

The lgtB gene encoding a beta-1,4-galactosyltransferase gene and the lgtC gene encoding an alpha-1,4-galactosyltransferase from the bacterial pathogen Neisseria meningitidis were cloned into an expression vector and overexpressed in Escherichia coli. Both genes expressed very well, but problems with C-terminal proteolysis were encountered with both proteins. The lgtC protein was initially isolated from extracts of recombinant E.coli as a truncated species that retained enzymatic activity, and was subsequently shown by mass spectrometry to be 19 residues shorter than the expected protein. A specific set of engineered C-terminal deletions was constructed to investigate their effect on the expression of lgtC. As many as 28 residues could be deleted with little effect on activity, and with the concomitant improvement of the overall expression up to fivefold over the full length protein. The lgtB protein was also proteolysed in extracts of normal E.coli strains into enzymatically inactive fragments lacking 28 or 41 C-terminal residues. This degradation could be prevented by expression in an ompT protease deficient strain of E.coli. The full length lgtB protein was not stable in soluble protein extracts from all recombinant strains, however a stable enzyme preparation could be achieved with the membrane fraction from cells of the ompT deficient strain expressing lgtB. Specific deletions of lgtB were also constructed, and 15 residues could be removed without loss of enzyme activity and also with the concomitant improvement of the overall expression up to twofold over the full length protein. Longer deletions produced protein but activity could not be detected in these recombinant strains. Examination of the glycosyltransferase sequences from a wide range of bacteria showed their C-terminal segments of approximately 50 amino acids frequently contained paired basic residues. Engineering of these segments may therefore be required as a general practice to produce these enzymes for use in the large scale chemi-enzymatic synthesis of carbohydrate-based therapeutics.