Complete Structures of Bordetella bronchiseptica and Bordetella parapertussis Lipopolysaccharides

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-α-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria. The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-α-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria. The genus Bordetella currently comprises nine species of Gram-negative bacteria. The most extensively studied of these are the respiratory pathogens B. pertussis, B. parapertussis, and B. bronchiseptica. B. pertussis infects only humans and is the causative agent of whooping cough in infants and persistent respiratory infections in adults (1Stevenson A. Roberts M. FEMS Immunol. Med. Microbiol. 2003; 37: 121-128Crossref PubMed Scopus (28) Google Scholar). B. parapertussis exists as two separate lineages. One is adapted to the human host and causes whooping cough; the other is adapted to the ovine host in which it can cause chronic pneumonia (2Mattoo S. Cherry J.D. Clin. Microbiol. Rev. 2005; 18: 326-382Crossref PubMed Scopus (863) Google Scholar). In contrast, B. bronchiseptica colonizes the respiratory tract of a large number of animals, and although it causes respiratory infections in some farm, companion, and wild animals, most B. bronchiseptica infections are asymptomatic and chronic. B. bronchiseptica is occasionally isolated from the respiratory tract of humans and is likely acquired through contact with infected animals (3Woolfrey B.F. Moody J.A. Clin. Microbiol. Rev. 1991; 4: 243-255Crossref PubMed Scopus (221) Google Scholar, 4Gueirard P. Weber C. Le C.A. Guiso N. J. Clin. Microbiol. 1995; 33: 2002-2006Crossref PubMed Google Scholar). Although these three pathogens are very closely related genetically (5Parkhill J. Sebaihia M. Preston A. Murphy L.D. Thomson N. Harris D.E. Holden M.T. Churcher C.M. Bentley S.D. Mungall K.L. Cerdeno-Tarraga A.M. Temple L. James K. Harris B. Quail M.A. Achtman M. Atkin R. Baker S. Basham D. Bason N. Cherevach I. Chillingworth T. Collins M. Cronin A. Davis P. Doggett J. Feltwell T. Goble A. Hamlin N. Hauser H. Holroyd S. Jagels K. Leather S. Moule S. Norberczak H. O'Neil S. Ormond D. Price C. Rabbinowitsch E. Rutter S. Sanders M. Saunders D. Seeger K. Sharp S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Unwin L. Whitehead S. Barrell B.G. Maskell D.J. Nat. Genet. 2003; 35: 32-40Crossref PubMed Scopus (758) Google Scholar), they synthesize different lipopolysaccharide (LPS) 2The abbreviations used are: LPS, lipopolysaccharide; HMBC, heteronuclear multiple bond connectivity; MS, mass spectrometry; HF, hydrogen fluoride; HPLC, high pressure liquid chromatography; FucNAc4NMe, 2-acetamido-4-methylamino-2,4,6-trideoxy-galactose; FucNAc4N, 2-acetamido-4-amino-2,4,6-trideoxy-galactose; GalNA, galactosaminuronic acid; GlcN, glucosamine; Hep, l-glycero-d-manno-heptose; Kdo, 3-deoxy-d-manno-octulosonic acid; ManNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-mannuronic acid; GalNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-galacturonic acid; GlcNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-glucuronic acid. molecules. All three LPS share similar lipid A and core structures (6Caroff M. Karibian D. Cavaillon J.M. Haeffner-Cavaillon N. Microbes. Infect. 2002; 4: 915-926Crossref PubMed Scopus (160) Google Scholar), yet only B. parapertussis and B. bronchiseptica synthesize O antigens. Initially, the O antigens of both species were reported to be identical and composed of linear polymers of 1,4-linked 2,3-diacetamido-2,3-dideoxy-α-galacturonic acid (7Di Fabio J.L. Caroff M. Karibian D. Richards J.C. Perry M.B. FEMS Microbiol. Lett. 1992; 76: 275-281Crossref PubMed Scopus (75) Google Scholar), but later differences between the end groups on B. bronchiseptica O antigens were described (8Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google Scholar). The core oligosaccharides of B. pertussis and B. bronchiseptica possess an almost identical structure of a branched nonasaccharide with several free amino and carboxyl groups linked to a distal trisaccharide, called band A trisaccharide, whereas the B. parapertussis core comprises a heptasaccharide that lacks band A trisaccharide and two other monosaccharides (9Caroff M. Aussel L. Zarrouk H. Martin A. Richards J.C. Therisod H. Perry M.B. Karibian D. J. Endotoxin. Res. 2001; 7: 63-68Crossref PubMed Scopus (52) Google Scholar). However, until now, the question of how the O antigens are linked to the core region remained unanswered. Here we present data that describe the complete structures of the B. bronchiseptica and B. parapertussis LPS carbohydrates. NMR Spectroscopy—NMR spectra were recorded at 30 °C in D2Oon Varian UNITY INOVA 500, 600, or 800 instruments, using acetone as a reference for proton (2.225 ppm) and carbon (31.5 ppm) spectra. Varian standard programs COSY, NOESY (mixing time of 200 ms), TOCSY (spinlock time of 120 ms), HSQC, and gHMBC (long-range transfer delay of 100 ms) were used with digital resolution in the F2 dimension of <2 Hz/pt. Spectra were assigned using the Pronto program (10Kjaer M. Andersen K.V. Poulsen F.M. Methods Enzymol. 1994; 239: 288-308Crossref PubMed Scopus (164) Google Scholar). Monosaccharide Analysis—Hydrolysis was performed with 4 m trifluoroacetic acid (110 °C, 3 h). Monosaccharides were conventionally converted into alditol acetates and analyzed by gas chromatography on an Agilent 6850 chromatograph equipped with a DB-17 (30 m × 0.25 mm) fused silica column using a temperature gradient of 180–240 °C at 2 °C/min. Mass Spectrometry—Electrospray ionization-MS spectra were obtained using a Micromass Quattro spectrometer in 50% MeCN with 0.2% HCOOH at a flow rate of 15 μl/min with direct injection. For capillary electrophoresis-MS analysis, a Prince CE system (Prince Technologies, The Netherlands) was coupled to a 4000 QTRAP mass spectrometer (Applied Biosystems/MDS Sciex, Canada). A sheath solution (isopropanol-methanol, 2:1) was delivered at a flow rate of 1.0 μl/min. Separations were obtained on an ∼90-cm bare, fused silica capillary using 15 mm ammonium acetate, pH 9.0. An electrospray ionization voltage of 5 kV was used. Tandem mass spectra were obtained using an in-source fragmentation strategy with a decluster voltage of 300 V (11Li J. Wang Z. Altman E. Rapid Commun. Mass Spectrom. 2005; 19: 1305-1314Crossref PubMed Scopus (44) Google Scholar). Chromatography—Anion exchange chromatography was performed on HiTrap Q column (5 ml; Pharmacia) in water (A) and 1 m NaCl (B) gradient from 0% B for 20 min and then linear gradient to 100% B with UV detection at 220 nm. Reverse phase chromatography was performed using C18 column (Aqua, 250 × 9 mm, Phenomenex) using a 0.1% trifluoroacetic acid-90% MeCN gradient (0% MeCN for the first 20 min, followed by a linear gradient to 100% MeCN over 1 h) with UV detection at 220 nm. Size exclusion chromatography was performed on a Sephadex a G-15 column (1.6 × 80 cm) or Sephadex a G-50 column (2.5 × 80 cm) in pyridine-acetic acid buffer (4 ml of pyridine and 10 ml of acetic acid in 1 liter of water) using a refractive index detector (Waters). Bacterial Strains and Cultures—B. bronchiseptica BAA-588 (RB50), 10580, and B. parapertussis 15311, 15989, and BAA-587 (12822) were obtained from ATCC. B. bronchiseptica 512 and 110H have been described previously (7Di Fabio J.L. Caroff M. Karibian D. Richards J.C. Perry M.B. FEMS Microbiol. Lett. 1992; 76: 275-281Crossref PubMed Scopus (75) Google Scholar). Bacteria were grown on Bordet-Gengou agar plates and then grown in Stainer-Scholte broth (12Stainer D.W. Scholte M.J. J. Gen. Microbiol. PubMed Scopus Google Scholar). Bacteria were and for LPS LPS was performed using the as described (7Di Fabio J.L. Caroff M. Karibian D. Richards J.C. Perry M.B. FEMS Microbiol. Lett. 1992; 76: 275-281Crossref PubMed Scopus (75) Google Scholar). of the LPS were with acetic acid at 100 °C for 3 The was by and were by chromatography on a Sephadex G-50 In most a of was from the column the of LPS was and core were and by and core two on with the and were in core and in the acid and it in of of the was in liquid hydrogen and at temperature for 1 or was by on The was in was by and the on Sephadex G-50 to a and several in the of the The were by phase 1 and 2 were in the region of the were analyzed by and similar were 3 in and several other oligosaccharides that were not analyzed in the of the from 512 was in water and were the solution was at temperature for were by and the solution was on a Sephadex G-50 were by C18 as described 4 as in and several other strains of B. bronchiseptica BAA-588 (RB50), 10580, and and three strains of B. parapertussis 15311, 15989, and BAA-587 were used in the present The of B. bronchiseptica and B. parapertussis have been and analyzed (5Parkhill J. Sebaihia M. Preston A. Murphy L.D. Thomson N. Harris D.E. Holden M.T. Churcher C.M. Bentley S.D. Mungall K.L. Cerdeno-Tarraga A.M. Temple L. James K. Harris B. Quail M.A. Achtman M. Atkin R. Baker S. Basham D. Bason N. Cherevach I. Chillingworth T. Collins M. Cronin A. Davis P. Doggett J. Feltwell T. Goble A. Hamlin N. Hauser H. Holroyd S. Jagels K. Leather S. Moule S. Norberczak H. O'Neil S. Ormond D. Price C. Rabbinowitsch E. Rutter S. Sanders M. Saunders D. Seeger K. Sharp S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Unwin L. Whitehead S. Barrell B.G. Maskell D.J. Nat. Genet. 2003; 35: 32-40Crossref PubMed Scopus (758) Google Scholar), for the and to be of the LPS were and are in The NMR spectra of obtained of the LPS were complex to be This is a of the of several in at the of of FucNAc4N, and of The O chain of Bordetella LPS is a polymer of 2,3-diacetamido-2,3-dideoxy-α-galacturonic acid. of are to with was used to the with the first at the of all the LPS with to the of between all between which are present at the of the O (8Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google Scholar), and of the that the of the O the of the were on Sephadex G-50 and the oligosaccharides were by C18 phase This large oligosaccharides that between strains in the the from strains and 512 structure 1 in or the from strains 10580, 15311, 15989, and structure 2 in These oligosaccharides the complete O of the LPS, the monosaccharides at the and several monosaccharides to the end previously not as of the O The oligosaccharides from different strains in the of of uronic acids, as be A of NMR spectra HSQC, of the oligosaccharides obtained from each was and all proton and carbon were assigned This the of acid with previously described (8Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google end groups However, an three of acid, acid, and at the end were oligosaccharides 1 and 2 were from the LPS by the of the of with The of the was by gas of alditol other monosaccharides not be by chemical and were identified only by NMR NMR data acids, in a new The of the monosaccharides were identified on the of proton and NMR chemical which were in with the standard for each were from the and chemical of and as as of for and between the monosaccharides were on the of the and heteronuclear multiple bond are not but they are all as for the All for structures 1 and 2 were in to the previously structures of the and end the the trisaccharide at the This was present in the oligosaccharides obtained from all strains. of the of of electrospray mass spectra of 1 and 2 were with the of of acid with the structures in The mass that with were the most for each O For in the of B. bronchiseptica in 4 the at to with the and end monosaccharides mass However, the for these structures all uronic the acid at the which was present in the were for each in the mass spectra. This to the of the number of uronic acid of the in the mass spectra that they are and of which is the of an between the and from the species with different of each O antigen comprises a of the with different of amidated and it was only to determine the number of amidated of different from the mass of the as in with for polymers of in all strains. This in of the most polymer a of the biosynthesis of these the of amidated and to determine the the pH of NMR chemical of the oligosaccharides 1 and 2 was analyzed using spectra. The spectra were recorded at two different first and then of 5 of 20 in to the of and of uronic This that the from and is the first of the O not and these three were The and from the of the into two that and to free and the other that not of was not to how of the NMR and data complete of the and and the of several amidated in the O were used to the of free and amidated uronic to of in free acid or and were as as of and The of the the of of and of acids, whereas were This the of the three in a and the of free in the of the A and were present in the spectra of oligosaccharides 1 and as as of of A and B The from the of the different strains in mass on B was present as an acid or an B was in in all B. parapertussis strains but in acid in all B. bronchiseptica strains in with the of by in B. parapertussis strains 15311, and The of B was in NMR the of A was to the in by or to high to not of the end of the in a new of the between 1 and 2 and the of B. bronchiseptica LPS with for a time to the of a large number of which were by phase of the structures of these to the of 3 was analyzed by NMR and of all NMR and to the structure in obtained from structure was the of two of can have a free or amino at The was by the of the of and that 3 was by the O chain 1 and at of in the LPS. B. bronchiseptica LPS was to in an to the fragment at from the This several to of with free amino groups at of or of All of with a free amino at were by but with amino groups were converted into at as previously Perry M.B. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar). The were by phase HPLC, and the structure was structure was analyzed using NMR and In the NMR spectra the from all of the of 1 were The the end in oligosaccharides 1 and had the was linked to of the acid, The fragment was the as for that of both and was by with This a of the of by not the end of 4 was a of all monosaccharides had the and with the structure in The most obtained from the of 4 was that it the band A trisaccharide by a similar trisaccharide of the and but with an of The NMR from of and from were in the spectra of between to and to which were in the spectra of the and heteronuclear multiple bond between of and of and between of and of were the structure in 4 was a of oligosaccharides 1 and both the O chain and a fragment of the these data the structure of the of B. bronchiseptica LPS be This in the of spectra from all other strains. of the data from of 4 it to the structure of the of B. bronchiseptica of the in these complex spectra were assigned in and with the structure from the described were The capillary electrophoresis-MS of groups of by to The most at to the with and in the and was with the in each were to and of spectra of the from the other strains of B. bronchiseptica the of structure with the or different and However, as was from previously data on the structure of B. parapertussis LPS core (9Caroff M. Aussel L. Zarrouk H. Martin A. Richards J.C. Therisod H. Perry M.B. Karibian D. J. Endotoxin. Res. 2001; 7: 63-68Crossref PubMed Scopus (52) Google Scholar), B. parapertussis had different the fragment was was linked to the of The and were present in and were only in core with the O were in B. parapertussis O These were the only differences between the B. parapertussis and B. bronchiseptica Mass spectra of B. parapertussis were of to with the of the and K. B. pertussis, B. parapertussis, and B. bronchiseptica share of and yet have host and cause different in (2Mattoo S. Cherry J.D. Clin. Microbiol. Rev. 2005; 18: 326-382Crossref PubMed Scopus (863) Google Scholar). This is related to the differences in the these bacteria with which in is likely to be by the The LPS of these bacteria Preston A. Maskell D.J. Infect. 2000; PubMed Scopus Google but a number of as For B. bronchiseptica and B. parapertussis, B. pertussis not an O antigen to of the that contains the O antigen biosynthesis A. J. R. Stevens K. Churcher C.M. K.L. J. Barrell B. Maskell D.J. Infect. PubMed Google Scholar). B. parapertussis and B. bronchiseptica both similar O antigens but different Preston A. Maskell D.J. Infect. 2003; PubMed Scopus (52) Google Scholar). we have a of the carbohydrate backbone of B. bronchiseptica and B. parapertussis LPS with the complete LPS structures of these bacteria. We have that the of these LPS are in to and The B. bronchiseptica core is identical to that described for a of B. pertussis M. J. Martin A. Karibian D. Lett. 2000; PubMed Scopus Google Scholar). The B. parapertussis LPS core in two We have that it is in the distal trisaccharide that to the band A of LPS. This trisaccharide is by the A. Maskell D. Microbiol. 19: PubMed Scopus Google Scholar). B. parapertussis contains a and the of the trisaccharide is not to the of the the of band A LPS from B. parapertussis was to be to a in the to the that to band A LPS Maskell D.J. Microbiol. PubMed Scopus Google Scholar, A. R. Maskell D.J. 2002; 33: PubMed Scopus Google Scholar). it was that the O antigen was to the core by a to the of the trisaccharide and that of or O antigen to the biosynthesis of band A LPS or O antigen LPS. The was to or that all of the LPS core was by the O in the of band A LPS that is with B. data that band A trisaccharide is from B. The is at present of in B. pertussis in the of the first two of the trisaccharide to the core A. R. Maskell D.J. 2002; 33: PubMed Scopus Google Scholar), and it is not for the trisaccharide to be present for transfer from to the The B. parapertussis core from of B. bronchiseptica and B. pertussis in that the and the are not present on all molecules. the for is The the that to the core of the (5Parkhill J. Sebaihia M. Preston A. Murphy L.D. Thomson N. Harris D.E. Holden M.T. Churcher C.M. Bentley S.D. Mungall K.L. Cerdeno-Tarraga A.M. Temple L. James K. Harris B. Quail M.A. Achtman M. Atkin R. Baker S. Basham D. Bason N. Cherevach I. Chillingworth T. Collins M. Cronin A. Davis P. Doggett J. Feltwell T. Goble A. Hamlin N. Hauser H. Holroyd S. Jagels K. Leather S. Moule S. Norberczak H. O'Neil S. Ormond D. Price C. Rabbinowitsch E. Rutter S. Sanders M. Saunders D. Seeger K. Sharp S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Unwin L. Whitehead S. Barrell B.G. Maskell D.J. Nat. Genet. 2003; 35: 32-40Crossref PubMed Scopus (758) Google are present and in B. the B. parapertussis core A. Maskell D. J. Endotoxin. Res. 2001; 7: Google that contains the the not differences from the B. bronchiseptica and B. pertussis to explain the of these in B. that core structure are currently An question is the B. parapertussis core from of the other This be to host B. parapertussis from a B. bronchiseptica very and at a time B. pertussis was present in the host B. parapertussis in the of pertussis and have for B. parapertussis that not the antigens as B. pertussis in Microbiol. 2005; PubMed Scopus Google Scholar). band A LPS is J. Infect. 1994; PubMed Google Scholar), and that the and are of host pertussis O have the of the O antigen to the which had remained until In B. bronchiseptica the O antigen is to of the band A In B. parapertussis, in the of trisaccharide it is to of the core The O by in these bacteria are identical at the amino acid the amino or of these are not of the of the or it a that it to transfer the O antigen to or to This band A trisaccharide to the core all three The is linked to the core in the of both band A and O antigen In both the is antigen in B. or A trisaccharide in B. bronchiseptica or O antigen in B. and the in each of these are very identified that Bordetella O antigen is linked to the core a novel that was not identified in The is the in B. bronchiseptica and B. contains a structure similar to the band A trisaccharide, but in which and a and a The that is only in O LPS and not in B. pertussis that not O antigen that it is a of the O antigen and not an of the we that the O antigen are to on an and that is to the The of with the of the genetics of O antigen biosynthesis in The of B. bronchiseptica and B. parapertussis of which are almost identical between the with the that O antigens the region and the A. J. R. Stevens K. Churcher C.M. K.L. J. Barrell B. Maskell D.J. Infect. PubMed Google Scholar). The regions of the and was to the that synthesize the of the strains and different The of most of the are but the for the of the We that some to synthesize the to be by the A. Maskell D. J. Endotoxin. Res. 2001; 7: Google Scholar), and these to the The of is at of were identified in the B. bronchiseptica whereas all B. parapertussis strains studied the This is with B. bronchiseptica genetically strains whereas B. parapertussis comprises strains with the of species C.A. S. J. PubMed Scopus Google Scholar, C.A. 2005; PubMed Scopus Google Scholar). The of these to is of O that some of the uronic in the O chain are only some to be for the and of the and and the first of the The of the amidated the O chain of is that of the to be species as it is amidated in all the B. parapertussis strains studied but is not amidated in all the B. bronchiseptica strains. The the of the O antigen and can with the of the and the of the O antigen by the host This differences between LPS of and a for of and The of the that the O antigen to the core is an for the of LPS biosynthesis in these A identified that some B. bronchiseptica strains synthesize different O antigens C.A. 2005; PubMed Scopus Google Scholar), and the a for of the LPS of these strains. We M. B. Perry for LPS and of and J. B. and R. for the The NMR spectra at 800 were obtained at the Varian spectrometer of the for NMR of

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