Biosynthesis of Ganglioside Mimics in Campylobacter jejuni OH4384

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 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. 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 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. lipooligosaccharide cytidine monophosphate-N-acetylneuraminic acid correlated spectroscopy 6-(5-fluorescein-carboxamido)-hexanoic acid succimidyl ester heteronuclear multiple bond coherence heteronuclear single quantum coherence lipopolysaccharide nuclear Overhauser effect NOE spectroscopy total correlation spectroscopy amino acid(s) kilobase pair(s) open reading frame polymerase chain reaction 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid isopropyl-1-thio-β-d-galactopyranoside matrix-assisted laser desorption ionization/time of flight Since the late 1970s, Campylobacter jejuni has been recognized as an important cause of acute gastroenteritis in humans (1.Skirrow M.B. Br. Med. J. 1977; 2: 9-11Crossref PubMed Scopus (902) Google Scholar). Epidemiological studies have shown that Campylobacterinfections are more common in developed countries thanSalmonella infections, and they are also an important cause of diarrheal diseases in developing countries (2.Ketley J.M. Microbiology. 1997; 143: 5-21Crossref PubMed Scopus (309) Google Scholar). In addition to causing acute gastroenteritis, C. jejuni infection has been implicated as a frequent antecedent to the development of Guillain-Barré syndrome, a form of neuropathy that is the most common cause of generalized paralysis (3.Ropper A.H. N. Engl. J. Med. 1992; 326: 1130-1136Crossref PubMed Scopus (638) Google Scholar). One of the most commonC. jejuni serotypes associated with Guillain-Barrésyndrome is O:19 (4.Kuroki S. Saida T. Nukina M. Haruta T. Yoshioka M. Kobayashi Y. Nakanishi H. Ann. Neurol. 1993; 33: 243-247Crossref PubMed Scopus (223) Google Scholar), and this prompted detailed study of the LOS structure of strains belonging to this serotype, including strains OH4382 and OH4384, which were isolated from two siblings who developed the Guillain-Barré syndrome (5.Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Mills S.D. Kurjanczyk L.A. Penner J.L. J. Bacteriol. 1992; 174: 1324-1332Crossref PubMed Scopus (77) Google G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. 33: PubMed Scopus Google G.O. McDonald A.G. Pang H. 33: PubMed Scopus Google G.O. S. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. PubMed Google Scholar). The oligosaccharides of O:19 strains were shown to of to of and GT1a of are to J. PubMed Scopus Google Scholar). have been in O:19 strains. The most a ganglioside mimic of has been in the OH4384. of by the of LOS is to be a of which could this to the Med. PubMed Google G.O. J. Scopus Google Scholar). the of the genes involved in LOS synthesis and the study of their is of for a of the used by these The cloning and of a gene involved in the synthesis of the LOS has been PubMed Scopus Google Scholar), while two Microbiology. PubMed Scopus Google J.M. Microbiology. PubMed Scopus Google have the cloning of biosynthesis of these genes are to bacterial have been linked to the synthesis of the LOS The genes by Microbiology. PubMed Scopus Google and J.M. Microbiology. PubMed Scopus Google could be involved in the synthesis of the or in the synthesis of the sequence of the C. NCTC 11168 has been completed by the The of this is structure is sequence a of information for the of the genes involved in the synthesis of the of the LOS. In addition to their for bacterial have been shown to be for the of oligosaccharides with activity M. J. 1997; PubMed Scopus Google M. S. Y. PubMed Scopus Google Scholar). Since bacterial the of oligosaccharides to and they are to in M. S. Y. PubMed Scopus Google PubMed Scopus Google Scholar), they are to the The ganglioside mimics synthesized by C. jejuni O:19 strains α-2,3- and sialic this is then a of α-2,3- and In this the of a locus involved in the biosynthesis of the LOS and the cloning and of four which for the biosynthesis of ganglioside mimics by C. jejuni OH4384, which has been implicated in Guillain-Barré syndrome. One of these is a bifunctional which and acid The NMR analysis was on nanomole amounts of using and heteronuclear the and acid were confirmed by using a gradient inverse broadband nano-NMR to correlation across the glycosidic The C. were used in this O:19 strains OH4382 and OH4384 were obtained from the for and Escherichia was used for the while was used to express the different cloned from jejuni strains was using of for and were as by the or the of the used for the were using the as by the to amplify open reading were using polymerase as by the and were from was using an model and the The was using a partial of the of C. jejuni OH4384. The partial was on a and was with the and the were on with and were in of and were in of with of each were used to of with of at was to and the were for The were by in of and for The were assayed for sialyltransferase activity as that the and were and The were for single and were and for activity in of the of the were individually, which to the of a two and Using and the of the two were on The with were also for sialyltransferase and the of the cloned in was to using in to the in the with to the The used to amplify the LOS biosynthesis locus of C. jejuni OH4384 were on from the complete of the NCTC 11168 the from the The and were used to amplify an locus using the The was on a and on using a of and of were using the The were using the and were cloned in the J. M. PubMed Scopus Google Scholar). was using the acid of the of activity was as the of that of oligosaccharides were as J. PubMed Scopus Google Scholar). The screening for α-2,3-sialyltransferase activity in of and in a of The were for the of glycosyltransferase a of the with were by and the were at The β-1,3-galactosyltransferase was assayed using of and The was assayed using and The α-2,3-sialyltransferase was assayed using and The was assayed using and The reaction were with and by using the and detection as M. J. PubMed Scopus Google Scholar). The from the were using with the detection of from the reaction were by on as M. J. PubMed Scopus Google Scholar). NMR were on a 600 NMR were using a gradient NMR were from to of The compounds were in and the was to with the were in 600 of NMR were as J. 1993; PubMed Google S. M. 1997; PubMed Scopus Google using as and the the of was at the of was at to at were on the of The for with and a of was for a of each and with a of the of the at were The were used to the of in and of and in of the were using The in was by the The total was to the of was that the was for The was obtained to were applied in The were at a of in the and in the the of the the was at a of using and a the NMR were using with the or The was used for gradient inverse broadband nano-NMR was used to the gradient J. Scopus Google T. J. Scopus Google for the The nano-NMR probe, which is a probe, of in J. PubMed Scopus Google Scholar). The was by the and in of for a of The of the could be The gradient was at a of of of and for a of were obtained using a of each was with a a of and were using the the cloning of the C. jejuni OH4384 and NCTC 11168 for an activity was then the to The was and detection of activity in the M. J. PubMed Scopus Google Scholar). We the NCTC 11168 and the Guillain-Barré syndrome OH4384 for the for the GT1a ganglioside mimic OH4384 the for the synthesis of this and The NCTC the β-1,3-galactosyltransferase and the Since the LOS structure for NCTC 11168 has been the or of a activity in this with the structure of LOS from an partial of from C. jejuni OH4384 which were to form of We used a and screening from which two were obtained and and analysis and with C. jejuni OH4384 that that were in the The sequence of nucleotide in was while the first were to be the sequence of The analysis and with that of the were in C. jejuni OH4384 two partial and two were in the sequence of The first a that is with the amino acid of the chain from The of the of the chain is also the first of the of an open reading frame that spans in was sialyltransferase and encodes a that is with a The H. encodes a that is to the region of the The sequence of an and a partial that that are with the two and of In to that the encodes sialyltransferase and The expressed was used to sialic acid to was by NMR to the linkage specificity of and and NMR for the fluorescent of the ganglioside mimics synthesized using the cloned NMR are in from obtained at 600 for for for for and The of is at The is for and for the The is for the of and to NMR are in from obtained at 600 for for for for and The of is at The is for and for the The is for the of and to in a of the from a of the of The two of the and are for which is with is for each The two for the common in the two compounds are by the two or The linkage is identified from the The is and the is are using a for for for for for and for and by the as in and of the sequence at the of the C. jejuni NCTC 11168 that the two involved in the synthesis of the of the were by sequence with bacterial The region between the two spans 13.49 in NCTC 11168 and at on in Since structure is for the LOS of NCTC was to for the glycosyltransferase genes in that on in the and to amplify the region between We obtained a of 13.49 using jejuni NCTC 11168 and a of 11.47 using from C. jejuni OH4384. The of the from NCTC 11168 was with the The of the from OH4384 between the strains in the region between the two genes and that the genes for of the to OH4384 could be in that We the using a of and of The of the was of from of the sequence the of complete in addition to the two partial encoding the two comparing the amino acid that the two strains genes that are and four genes that are between and genes are to C. while gene is to C. jejuni OH4384 genes that are as and in C. jejuni OH4384 are in an in C. jejuni NCTC and of the of the LOS biosynthesis locus from C. jejuni in sequence of the C. jejuni NCTC 11168 be obtained from the in the in in the that were are in are on homologues from from H. from H. biosynthesis from N. first of from H. from from H. sialyltransferase from N. acid from N. acid of from acid from H. from H. The sequence of the C. jejuni NCTC 11168 be obtained from the The that were are in are on homologues from in a were to express each of the glycosyltransferase genes between the two from C. jejuni OH4384. The the and a of this activity assayed using as The was for a was a of the activity with The reaction obtained from the as by and an in the to that for the the structure of the of C. jejuni OH4384, this for glycosyltransferase has a specificity to the of The linkage specificity of was confirmed by the NMR analysis of and and The in role of cgtA in the synthesis of a mimic is confirmed by the by C. jejuni OH4382 of the from C. jejuni OH4382 a of of which in the of a cgtA of The LOS structure jejuni OH4382 is with the of as the galactose is with sialic acid G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. 33: PubMed Scopus Google Scholar). The the and an of this activity using as an was to and was to have the by the structure of the LOS of C. jejuni OH4384, that this for glycosyltransferase has specificity to the of The linkage specificity of was confirmed by the NMR analysis of and and which was synthesized by using and The and an sialyltransferase activity using and as acceptors. sialyltransferase from OH4384 was was shown to be as could sialic acid α-2,3- to the of and also to the sialic acid of NMR analysis of a reaction with confirmed the of the first sialic acid on the and the of the sialic acid and The in of cst-II from C. jejuni OH4384 in the synthesis of a GT1a ganglioside mimic is by with the cst-II from C. jejuni O:19 that the ganglioside are nucleotide that amino acid between these two expressed coli, the cst-II from C. jejuni O:19 has α-2,3-sialyltransferase activity activity which is with the of sialic acid in the LOS G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. 33: PubMed Scopus Google of C. jejuni O:19 from C. jejuni NCTC 11168 expressed α-2,3-sialyltransferase activity the homologues from O:19 or OH4384 and We could an on a cst-II from NCTC 11168 was expressed in The from NCTC 11168 with the homologues from O:19 or OH4384. We could the sequence could be responsible for the activity expressed in of the activity of the from C. activity is expressed in of of total in the of the activity on by the activity on were expressed in as with the in the J. M. PubMed Scopus Google Scholar). were assayed using or The activity is expressed in of of total in the of the activity on by the activity on in a The were expressed in as with the in the J. M. PubMed Scopus Google Scholar). were assayed using or the LOS biosynthesis is to cst-II first with from C. jejuni OH4384 or C. jejuni NCTC 11168 The two homologues between and are the amino of of amino at the was to be which that the of is for sialyltransferase the at the are for in they with for or for The of between the C. jejuni is different from was for the and the are more at the between the two and between different of the M. J. PubMed Scopus Google Scholar). In to the linkage specificity of an identified was by NMR In to the for the of the NMR analysis was on nanomole In the NMR of compounds are compounds are and with of a the are The is for which has to the of the in the nano-NMR the of the were on the of to the were also to of the with were also to a in the from to were at in to of the with the be from the compounds were and or that were with the NMR which was as J. 1993; PubMed Google S. M. 1997; PubMed Scopus Google Scholar). the of S. J. 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Google Scholar), and PubMed Scopus Google and have been and have been and have been and have been and have been and have been at the are NMR are in from the obtained at 600 for for for for and The of is at to at The is for and for the The is for to of was to the of the compounds S. J. Scopus Google S. J. Google to to at of between the of the and the corresponding are in and have been and have been in a The at the are NMR are in from the obtained at 600 for for for for and The of is at to at The is for and for the The is for to of was to the of the compounds S. J. Scopus Google S. J. Google to to at of between the of the and the corresponding are in The linkage on the was from a of the of the with of the to as for S. J. Scopus Google Scholar). of comparing are more be to a the from of the are with of the the at the linkage while of the are more to and The of the be identified from a of with of or the of the S. J. Scopus Google Scholar). obtained from or also are used to the of the NOE are to sequence the by the of between the for sialic and the The NOE is on the linkage also on that are to the linkage at 600 the of and are or these compounds with a of to the of the the synthetic the for the of were confirmed by the the of the were from a on the of with a of for was used to the to of the The and of the were then from the for the were in with PubMed Scopus Google Scholar). The for the have been M. J. PubMed Scopus Google Scholar). of the of from was with the addition of sialic acid to the The was identified as the and of the of the were to for the or (1.Skirrow M.B. Br. Med. J. 1977; 2: 9-11Crossref PubMed Scopus (902) Google J.M. Microbiology. 1997; 143: 5-21Crossref PubMed Scopus (309) Google A.H. N. Engl. J. Med. 1992; 326: 1130-1136Crossref PubMed Scopus (638) Google S. Saida T. Nukina M. Haruta T. Yoshioka M. Kobayashi Y. Nakanishi H. Ann. 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Google Scholar). was with the of the and in and which are from the S. J. Google Scholar). the were on of with the for of was confirmed by in the with in the The is to the of in galactose S. J. Scopus Google Scholar). In the and of of the sialic in to be PubMed Scopus Google Scholar), as confirmed from the of and shown in C. The of the obtained from were in with of the oligosaccharides shown in of were for and these are to different at the these the of the in between the and their were of and with and of or were used to the linkage specificity for The of using is that the be to the of the of the at the linkage In of the are the most by and then Using a nano-NMR of a NMR on the of the total the of an In to the LOS from C. the activity screening that used to the α-2,3-sialyltransferase from N. M. J. PubMed Scopus Google Scholar). The activity screening two which two of the α-2,3-sialyltransferase gene analysis that a is responsible for the α-2,3-sialyltransferase The activity screening to the that are for the synthesis of the GT1a have been to a of to the the of an LOS biosynthesis locus in the complete sequence jejuni NCTC 11168 to to in the corresponding locus from C. in of to a (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase The in synthesis of fluorescent of nanomole amounts of ganglioside mimics and their NMR analysis the linkage specificity of the four cloned on these that the in is used by C. jejuni OH4384 to a GT1a The role for cgtA is by the that C. jejuni which an of this have in LOS The cst-II gene jejuni OH4384 α-2,3- and in an in from C. jejuni O:19 α-2,3-sialyltransferase activity is with the role for cst-II in the addition of a sialic acid in C. jejuni OH4382 and OH4384, of which have cst-II in C. are amino acid between the homologues from C. jejuni O:19 and and the of which amino are responsible for the bifunctional activity is Since to a locus the LOS biosynthesis be that by gene and and that the to sialic acid to form the or that the cst-II gene from C. jejuni OH4384 this the gene The have an on the of the C. jejuni infection has been that the of the be involved in the development of as the Guillain-Barré syndrome S. L.A. M. G.O. Kurjanczyk L.A. Penner J.L. PubMed Google Scholar). is also that bifunctional activity is the a bifunctional glycosyltransferase activity has been for the acid from J. 1992; PubMed Google Scholar). The activity of cst-II and the of cgtA to be two of that C. jejuni to different that are to the In addition to gene that are the O:19 strains and are the are between C. and NCTC 11168 for the the locus is in C. jejuni OH4384. are in the organization of the LOS biosynthesis locus between strains OH4384 and NCTC of the genes are and of are while are to or the genes that are as and in OH4384 are as an in NCTC 11168 We have this for activity in this which that at the cgtA of the be The in gene and organization between the from C. jejuni OH4384 and NCTC 11168 that and have to the of LOS The of the LOS biosynthesis in serotypes to the and the of the that are used by C. jejuni to LOS We for synthesis and for We also and for the analysis by and for

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