Cloning of the Lipooligosaccharide α-2,3-Sialyltransferase from the Bacterial Pathogens Neisseria meningitidis and Neisseria gonorrhoeae

Abstract

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...

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure

“…LOS sialylation is catalyzed by an ␣-2,3-sialyltransferase (Lst) (38). We inactivated lst (38) and lgtB (28) in 7946 by insertion of antibiotic resistance cassettes to generate mutant strains, 7946⌬lst and 7946⌬lgtB. lst and lgtB were amplified by PCR with the use of primers in Table 3.…”

Biosynthesis of Ganglioside Mimics in Campylobacter jejuni OH4384

“…The ␣-2,8-sialyltransferase was assayed using 0.5 mM GM3-FCHASE, 0.2 mM CMP-Neu5Ac, 50 mM Hepes, pH 7, and 10 mM MnCl 2 . The reaction mixes were diluted appropriately with 10 mM NaOH and analyzed by capillary electrophoresis performed using the separation and detection conditions as described previously (19). The peaks from the electropherograms were analyzed using manual peak integration with the P/ACE Station software.…”

“…The peaks from the electropherograms were analyzed using manual peak integration with the P/ACE Station software. For rapid detection of enzyme activity, samples from the transferase reaction mixtures were examined by thin layer chromatography on Silica-60 TLC plates (Merck) as described previously (19).…”

“…When an enzyme activity was detected, we then optimized the assay conditions (described under "Experimental Procedures") to ensure maximal activity. The capillary electrophoresis assay we employed was extremely sensitive and allowed detection of enzyme activity in the microunits/ml range (19). We examined both the sequenced strain NCTC 11168 and the Guillain-Barré syndrome åssoci-ated strain OH4384 for the enzymes required for the GT1a ganglioside mimic synthesis.…”

Purification, crystallization and preliminary crystallographic characterization of the α2,6-sialyltransferase fromPhotobacteriumsp. JT-ISH-224

“…In general, bacterial STases are also important for the enzymatic modification of glycoconjugates and the enzymatic synthesis of glycans because they are more stable and productive than mammalian enzymes. Since 2,3-STase was first cloned from Neisseria meningitidis and N. gonorrhoeae (Gilbert et al, 1996), bacterial STases have been cloned from several microorganisms (Gilbert et al, 2000;Yamamoto et al, 1998;Bozue et al, 1999;Shen et al, 1999). To date, the crystal structures of three bacterial STases have been reported: the bifunctional STase CstII from Campylobacter jejuni (Chiu et al, 2004), the 2,3-STase CstI from C. jejuni (Chiu et al, 2007) and the multifunctional STase Á24PmST1 from Pasteurella multocida (Ni et al, 2006(Ni et al, , 2007.…”