The Structure of the Major Cell Wall Polysaccharide of Bacillus anthracis Is Species-specific (2006)

In this report we describe the structure of the polysaccharide released from Bacillus anthracis vegetative cell walls by aqueous hydrogen fluoride (HF). This HF-released polysaccharide (HF-PS) was isolated and structurally characterized from the Ames, Sterne, and Pasteur strains of B. anthracis. The HF-PSs were also isolated from the closely related Bacillus cereus ATCC 10987 strain, and from the B. cereus ATCC 14579 type strain and compared with those of B. anthracis. The structure of the B. anthracis HF-PS was determined by glycosyl composition and linkage analyses, matrix-assisted laser desorption-time of flight mass spectrometry, and one- and two-dimensional nuclear magnetic resonance spectroscopy. The HF-PSs from all of the B. anthracis isolates had an identical structure consisting of an amino sugar backbone of →6)-α-GlcNAc-(1→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→, in which the α-GlcNAc residue is substituted with α-Gal and β-Gal at O-3 and O-4, respectively, and the β-GlcNAc substituted with α-Gal at O-3. There is some variability in the presence of two of these three Gal substitutions. Comparison with the HF-PSs from B. cereus ATCC 10987 and B. cereus ATCC 14579 showed that the B. anthracis structure was clearly different from each of these HF-PSs and, furthermore, that the B. cereus ATCC 10987 HF-PS structure was different from that of B. cereus ATCC 14579. The presence of a B. anthracis-specific polysaccharide structure in its vegetative cell wall is discussed with regard to its relationship to those of other Bacillus species. In this report we describe the structure of the polysaccharide released from Bacillus anthracis vegetative cell walls by aqueous hydrogen fluoride (HF). This HF-released polysaccharide (HF-PS) was isolated and structurally characterized from the Ames, Sterne, and Pasteur strains of B. anthracis. The HF-PSs were also isolated from the closely related Bacillus cereus ATCC 10987 strain, and from the B. cereus ATCC 14579 type strain and compared with those of B. anthracis. The structure of the B. anthracis HF-PS was determined by glycosyl composition and linkage analyses, matrix-assisted laser desorption-time of flight mass spectrometry, and one- and two-dimensional nuclear magnetic resonance spectroscopy. The HF-PSs from all of the B. anthracis isolates had an identical structure consisting of an amino sugar backbone of →6)-α-GlcNAc-(1→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→, in which the α-GlcNAc residue is substituted with α-Gal and β-Gal at O-3 and O-4, respectively, and the β-GlcNAc substituted with α-Gal at O-3. There is some variability in the presence of two of these three Gal substitutions. Comparison with the HF-PSs from B. cereus ATCC 10987 and B. cereus ATCC 14579 showed that the B. anthracis structure was clearly different from each of these HF-PSs and, furthermore, that the B. cereus ATCC 10987 HF-PS structure was different from that of B. cereus ATCC 14579. The presence of a B. anthracis-specific polysaccharide structure in its vegetative cell wall is discussed with regard to its relationship to those of other Bacillus species. Bacillus anthracis is a Gram-positive, spore-forming bacterium that causes anthrax (1Mock M. Fouet A. Annu. Rev. Microbiol. 2001; 55: 647-671Crossref PubMed Scopus (866) Google Scholar). Cell wall carbohydrates such as capsular polysaccharides are well known virulence factors with regard to numerous bacterial pathogens, both Gram-negative and Gram-positive. However, relatively little is known about the carbohydrates in the vegetative cell walls of B. anthracis as well as other members of the B. cereus group of bacteria. Whereas there have been some glycosyl composition analyses, there have been no reported structures for carbohydrates from the vegetative cell wall of B. anthracis. Generally, the carbohydrate-containing components of the vegetative cell walls of Gram-positive bacteria consist of the extensive peptidoglycan layer, teichoic acids, lipoteichoic acids, capsular polysaccharides, and crystalline cell surface proteins known as S-layer proteins that are often glycosylated (2Messner P. Glycoconj. J. 1997; 14: 3-11Crossref PubMed Scopus (102) Google Scholar). However, the B. anthracis cell wall differs in several aspects from this generalized description. First, B. anthracis cells are surrounded by a poly-γ-d-glutamate capsule and not by a polysaccharide capsule. Second, their cell walls do not contain teichoic acid (3Molnar J. Pragai B. Acta Microbiol. Acad. Sci. Hung. 1971; 18: 105-108PubMed Google Scholar), and last, their S-layer proteins are not glycosylated (1Mock M. Fouet A. Annu. Rev. Microbiol. 2001; 55: 647-671Crossref PubMed Scopus (866) Google Scholar, 4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). However, glycosyl composition comparisons of the cell walls of B. anthracis, Bacillus cereus, and Bacillus thuringiensis show that they do contain glycosyl residues and that they differ from one another in their glycosyl compositions (5Fox A. Stewart C.G. Waller L.N. Fox K.F. Harley W.M. Price R.L. J. Microbiol. Methods. 2003; 54: 143-152Crossref PubMed Scopus (57) Google Scholar). To date, cell wall carbohydrates from the vegetative cells of members of the B. cereus group have been addressed only to a limited extent (6Ekwunife F.S. Singh J. Taylor K.G. Doyle R.J. FEMS Microbiol. Lett. 1991; 82: 257-262Google Scholar, 7Fox A. Steward G.C. Fox K. Rostovtseva S. J. Clin. Microbiol. 1993; 31: 887-894Crossref PubMed Google Scholar, 8Amano K. Hazama S. Araki Y. Ito E. Eur. J. Biochem. 1977; 75: 513-522Crossref PubMed Scopus (32) Google Scholar). All of these carbohydrates are rich in amino glycosyl residues but have variations in the type and amounts of these residues. The study of Ekwunife et al. (6Ekwunife F.S. Singh J. Taylor K.G. Doyle R.J. FEMS Microbiol. Lett. 1991; 82: 257-262Google Scholar) focused on the glycosyl composition of a carbohydrate polymer released from the cell wall through hydrogen fluoride (HF) treatment (HF treatment releases wall polysaccharides covalently bound via a phosphate bond to the peptidoglycan) of B. anthracis (Δ Sterne) and found that the HF-released polysaccharide (HF-PS) 3The abbreviations used are: PS, polysaccharide; TOCSY, total correlated spectra; NOESY, nuclear Overhauser effect correlation spectra; HSQC, quantum coherence experiment; NOE, nuclear Overhauser effect. contained Gal, GlcNAc, and ManNAc in an approximate ratio of 3:2:1. This HF-PS was also further investigated by Mesnage et al. (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). They reported the importance of a pyruvyl substituent with regard to the function of this polysaccharide in anchoring the S-layer proteins to the cell wall. Fox et al. (7Fox A. Steward G.C. Fox K. Rostovtseva S. J. Clin. Microbiol. 1993; 31: 887-894Crossref PubMed Google Scholar) investigated a number of B. anthracis and B. cereus strains for their total cell glycosyl compositions, which showed interesting differences between the different strains. For example, in contrast to the B. anthracis strains, all B. cereus strains investigated contained GalNAc, suggesting possible differences in cell wall architecture in the different Bacillus species cell walls and, possibly, the occurrence of strain- or species-specific carbohydrates. The possibility of species/strain-specific structures is of interest for at least two reasons: the taxonomy within the B. cereus group has recently a of M. J. PubMed Scopus Google Scholar, J. M. T. P. P. J. S. S. K. M. M. B. P. E. E. P. J. PubMed Scopus Google Scholar) and cell wall carbohydrates of B. cereus group members to their In the of cell wall carbohydrate structures in the of their importance in The function of one B. anthracis cell wall polysaccharide has been addressed in the This function is its for anchoring the S-layer proteins to the vegetative cell wall (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). The S-layer proteins contain a S-layer which is found also in other S-layer proteins from Gram-positive bacteria and in cell wall such as and from E. J. PubMed Google Scholar). is that S-layer to cell wall carbohydrates that are covalently to the cell wall peptidoglycan via phosphate (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar) and the S-layer proteins to the bacterial cell This function has been investigated in for B. anthracis (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). only a of about an isolated strain, B. cereus addressed the of a Bacillus species cell wall carbohydrate structure K. Hazama S. Araki Y. Ito E. Eur. J. Biochem. 1977; 75: 513-522Crossref PubMed Scopus (32) Google Scholar, Araki Y. Ito E. Eur. J. Biochem. PubMed Scopus Google Scholar, Araki Y. Ito E. Eur. J. Biochem. PubMed Scopus Google Scholar). The structures of and cell wall carbohydrates have been for that The carbohydrate was of GlcNAc, GalNAc, and in of Araki Y. Ito E. Eur. J. Biochem. PubMed Scopus Google Scholar), the carbohydrate was of GlcNAc, Gal, and in of Araki Y. Ito E. Eur. J. Biochem. PubMed Scopus Google Scholar). a in cell wall carbohydrate within members of the B. cereus we report the structures of the HF-PSs of a number of B. anthracis and B. cereus strains. were determined for these polysaccharides from B. anthracis Ames, B. anthracis and B. anthracis also compared these structures with those from a closely related B. cereus strain, ATCC and from the B. cereus type strain, ATCC 14579. The showed that all three B. anthracis strains contained the HF-PS structure that from that of B. cereus ATCC in from that of B. cereus ATCC 14579. and anthracis Ames, B. anthracis B. anthracis and B. cereus strains ATCC and ATCC 14579 were from the for The was by of were in of at In the of the were at for the were the in of and these cell used to of in was at or cereus and B. on a at was by the at were in by at for two in and by and surface on were by for at to further and carbohydrate of Cell bacterial cells were as and cell walls were by of a J. Scholar). the bacterial cells to were in on by a with a at a of The or of cells was cells were by The and were at Cell wall were by at at for The cell wall were by in by an as and and of the Cell Cell wall from the B. anthracis Ames, B. anthracis B. anthracis and the of B. anthracis were with at for The was by in an The was by was used as the and an was used to the from the The that a in the were and used for further composition was by the and of M. P. Scopus Google Scholar). The were and by to In the were at for to the by and in the presence of at for the by with the were with at for to The were in and on a acid was to the of T. S. Biochem. 1971; PubMed Scopus Google Scholar). This of and of the isolated polysaccharides were linkage was to a of the of and Scopus Google Scholar). the were in with by of a and for at was to the and for of was and for another The was on an and the polysaccharide was by between and The in the was and used for the of The polysaccharide was to at for by with acid at for The were to their by at The was acid and acid was as by and with and The were a at for and were by with and the were in and by a The linkage of each were from its mass and by its to that of polysaccharide were in of and this was to the and with The was in of and to a All one- and two-dimensional were at on a the by were a of and the were with to of to that of at correlated were a of in both a of of with total correlated and through nuclear Overhauser effect correlation were a of of and The used for and were and To the a quantum coherence was with and of and respectively, and a of with were used in the All the were and for matrix-assisted laser of flight mass was used to the mass for each polysaccharide was in of and at with acid as the of this was on each on a matrix-assisted laser and The were in and a laser with of and composition as E. P. P. and E. in showed that the HF-PSs from B. anthracis strains Ames, B. anthracis B. anthracis and B. anthracis all had the and in an approximate The composition of the HF-PS from B. cereus ATCC 10987 of Gal, GlcNAc, and in a and that of B. cereus ATCC 14579 of GlcNAc, and in a of the B. anthracis HF-PSs contained of the of acid as determined by the of T. S. Biochem. 1971; PubMed Scopus Google Scholar). of the B. anthracis HF-PSs showed that all of these polysaccharides contained the glycosyl Gal, GlcNAc, GlcNAc, GlcNAc, GlcNAc, GlcNAc, and The in the in these polysaccharides that there is in the of the residues. a matrix-assisted laser of flight mass that the B. anthracis HF-PSs were in the number of this residues. The mass of the B. anthracis polysaccharide is in and the compositions for the are in The mass a of The mass in each differ from those in the by a mass that is with a of three and three three that differ from one another by a in this For is with a composition of one Gal and two Gal residues. This in Gal with the in of the that the in these polysaccharides is to in of one or of the residues by Gal residues. The that each in only one or two Gal residues that these in only one of the for each the and are to and of polysaccharide from B. anthracis in a of the B. anthracis residue compositions of the HF-PSs from B. anthracis are different from those for B. cereus ATCC and the B. cereus type strain The HF-PS from B. anthracis with the two B. cereus strains is in clearly differs from the other in the of for their glycosyl and as is by the composition that the structure of the HF-PS from the B. anthracis is different from those of B. cereus ATCC 10987 and ATCC 14579 and, furthermore, that the B. cereus ATCC 10987 structure differs from that of strain ATCC 14579. The the HF-PSs of B. anthracis Ames, B. anthracis Sterne, and B. anthracis Pasteur are in are identical to one another and the that these polysaccharides all have the TOCSY, NOESY, and also identical for the HF-PSs from the B. anthracis strains. of the identical of these of the B. anthracis the are for the HF-PS isolated from B. anthracis 3The for the HF-PSs from the B. anthracis strains. The are for the HF-PSs from B. anthracis B. anthracis and B. anthracis Pasteur The for B. anthracis was identical to those in this The in for the B. anthracis polysaccharide at and the that this polysaccharide of a a that is with the composition and mass the and of these that three of these glycosyl residues are and at least two the of the glycosyl residue the glycosyl residue with at from the to its which that this residue is in the The presence of a of glycosyl residues is also by the which clearly also a resonance at that is with the of a pyruvyl substituent as reported by Mesnage et al. (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). However, the relatively of this resonance that the pyruvyl is in and is an and is that the of this was the aqueous treatment of the cell The of the and for the polysaccharides were determined by a of two-dimensional and The for these in is in the of the HF-PS from B. anthracis The structure and the and are The of the HF-PSs from B. anthracis Sterne, and Pasteur are identical to this and for the B. anthracis cell wall in a an at The through are from the and is from the that the resonance at has a to the and the that has a and, is an α-Gal To and was to the the of the to This showed that is to at and another at which was as The resonance in to in the which are the The that at are to a at which the presence of at this The for this and for the were also from the has an at with residue the through are from the and and the and of show that residue has a and is a α-Gal of the show that is to and a at which was to This was to with in the with residue to in this was not possible to the of the However, the in this are to that at about that these are The of residue has a of The through were from the and of the showed that was to and to at which were as the The showed that at were to a at which is with a glycosyl residue that is substituted at The also showed that was to a at with this an and, the that this is a composition the presence of only and ManNAc the from through the that this residue has glycosyl that are with a and, residue is as a α-GlcNAc The of residue has a of The and show that is to at The that only via that residue has a and, has a The that is to a at the that residue is a of the to from the of and The also showed that is to and to with at which were as were to a at with a The of a residue is to both and have However, the discussed further between and the that these all have and, that this residue has a is a The of residue has a of and, as both this and the of show that has a The and show that is to at also at at and at of the showed that is to at and which were as these are to a with a of The also that is to a at show that residue is the a β-GlcNAc has an with a and a of that has a The and from to and The also that has a total of that residue has a The also show that is to and as and also to a with a of which was to This in to at to which were as the The showed that these are to a at is a The and as with the and mass also in the There are of residue α-GlcNAc as by an glycosyl to an at and another glycosyl at there is an of residue as by another through an at and an of residue via a through at and glycosyl and as well as the α-GlcNAc residues and the and mass that show in the and in the of in this to of the α-GlcNAc residue by the Gal residues and The of the glycosyl residues was determined by has a from at to of residue a There is also a to of the β-GlcNAc and and to at and at the has a from at to of residue a to of residue was also and to and at and respectively, were also has a from to the of residue which a This with for residue that the has a There is also a from the of residue to its at has to at and The at and are to with and respectively, as for a ManNAc The to the at is an to of residue However, is that the ManNAc residue is to this of β-GlcNAc as is by a residue However, there is also a to a is that this is to a of an to and an to of residue The of at this on the β-GlcNAc in a to the of the β-GlcNAc residue for the between these two these the presence of a and, with the for residues and that the polysaccharide a structure The presence of a residue is also with the The residue also has an from to a at this residue is a is that this is the as that not in to However, is possible that one of the of the α-Gal residue the to to of the β-GlcNAc residue is in to the ManNAc to for this the β-GlcNAc has to at and The to and are to and respectively, which for a The at is to an to of residue the α-GlcNAc residue the β-GlcNAc is to of residue the α-GlcNAc the a for this with the of residue was also that the of residues and are in further The has a to of residue as well as to and at and The at also with an to at that residue is to α-GlcNAc residue at for residue a between the of residues and were also that the of these two residues are in these with the mass glycosyl and linkage show that the polysaccharide an report in this study the structure of the HF-PS from B. anthracis and that this structure is the for B. anthracis Ames, B. anthracis Sterne, B. anthracis and B. anthracis In a as well as composition that this structure is different from the HF-PS from a strain of B. cereus ATCC 10987 that is closely related to B. anthracis J. E. J. PubMed Scopus Google Scholar), and from the HF-PS of B. cereus ATCC type (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar, F.S. Singh J. Taylor K.G. Doyle R.J. FEMS Microbiol. Lett. 1991; 82: 257-262Google Scholar) reported the composition of the cell wall polysaccharide from B. anthracis and that of the polysaccharide released from the cell wall by treatment with aqueous This treatment the phosphate of the polysaccharide to the cell wall The composition of the HF-PS was reported to consist of Gal, and in a ratio (6Ekwunife F.S. Singh J. Taylor K.G. Doyle R.J. FEMS Microbiol. Lett. 1991; 82: 257-262Google Scholar), or in a ratio (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). as in this is with the report of Ekwunife et al. (6Ekwunife F.S. Singh J. Taylor K.G. Doyle R.J. FEMS Microbiol. Lett. 1991; 82: 257-262Google Scholar) that the B. anthracis HF-PS of these glycosyl residues in a was also reported that the HF-PS was (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar), and that the pyruvyl substituent is for the function of the as the for the S-layer of S-layer proteins (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). the presence of a pyruvyl substituent is further is that a number of reported polysaccharide structures from related strains have the of an amino sugar backbone substituted by glycosyl residues and The polysaccharide from B. cell walls of a backbone in which the residue is substituted at O-3 with a residue and the ManNAc residue is substituted at O-3 with a β-GlcNAc residue Araki Y. S. Ito E. Eur. J. Biochem. PubMed Scopus Google Scholar). The polysaccharide for the cell walls of B. of backbone in which the ManNAc residue is substituted with a pyruvyl residue at the S. Araki Y. Ito E. Eur. J. Biochem. PubMed Scopus Google Scholar). B. cereus cell wall polysaccharide from strain is reported to have a backbone in which the residue is substituted with an at the ManNAc with a β-GlcNAc at and the residue with a at K. Hazama S. Araki Y. Ito E. Eur. J. Biochem. 1977; 75: 513-522Crossref PubMed Scopus (32) Google Scholar). The structure we report for the HF-PS from the B. anthracis, as in also of an amino sugar backbone of in which the α-GlcNAc residue is substituted with and at O-3 and O-4, respectively, and the β-GlcNAc substituted with at O-3. The also that there is variability in the presence of the two Gal on the α-GlcNAc in the backbone structure of all of these polysaccharides, the B. anthracis to the presence of a The of the amino sugar backbone in all of these cell wall polysaccharides that this has an function for the of Bacillus species. the HF-PS from B. anthracis had is in a report by Mesnage et al. (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar). Whereas Mesnage et al. (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar) not the structure of this the they show of the HF-PS for B. anthracis is identical to the we show for the HF-PSs isolated from B. anthracis Ames, B. anthracis Sterne, and B. anthracis Mesnage et al. (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar) that is a of the showed a resonance with a pyruvyl group resonance at as reported by Mesnage et al. (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar), to in of the B. anthracis The for this is at this However, are to is possible that the of these were by treatment and, in in the the Mesnage et al. (4Mesnage S. Fontaine T. Mignot T. Delepierre M. Mock M. Fouet A. EMBO J. 2000; 19: 4473-4484Crossref PubMed Scopus (269) Google Scholar) reported that a was in the of the pyruvyl substituent to the polysaccharide in that the resonance at The was to in the S-layer and to its The also showed an in in of on and, on a in cell the to all of these were by the to the HF-PS with that the to the polysaccharide is for the of B. anthracis and, its is a for In to the function of the HF-PS in B. anthracis and cell a report by et al. A. M. P. Y. A. Scopus Google Scholar) that a for a cell wall polysaccharide in the of the They showed that the from the that vegetative B. anthracis cells were The are that are of the and are in the of a both Gram-negative and Gram-positive are and function by to carbohydrate components on the surface of the T. J. PubMed Scopus Google Scholar), possible that the of to B. anthracis cells on the of to a carbohydrate of the vegetative cell wall. is to that the the The of this composition and have that the polysaccharide from Bacillus species cell walls on the species have that this is in that glycosyl compositions B. cereus and also within a composition differences in the B. cereus group as by the different of the HF-PSs from B. anthracis, B. cereus ATCC and B. cereus ATCC 14579. The B. cereus ATCC 10987 strain is closely related to B. anthracis strains as reported by et al. J. E. J. PubMed Scopus Google Scholar) in that a that is to but the that for the In the of B. cereus ATCC 10987 is to B. anthracis, is to B. cereus ATCC 14579 J. E. J. PubMed Scopus Google Scholar). these the that the B. anthracis structure we report is to B. anthracis and different from that of closely related B. cereus strains. to this several isolates of B. cereus, which are to in J. M. J. T. Acad. Sci. PubMed Scopus Google Scholar). in that in these B. cereus strains, the cell wall polysaccharide compositions are to those of the B. anthracis that the cell wall polysaccharide have a function in the virulence of B. anthracis as well as of these B. cereus strains. in is focused on comparisons of the HF-PSs from the B. cereus strains that are closely related to B. anthracis. In we have the structure of the cell wall carbohydrate of B. anthracis. that this cell wall carbohydrate is for and for of B. anthracis, and, a for of for B. anthracis