Secondary cell wall polysaccharides from Bacillus cereus strains G9241, 03BB87 and 03BB102 causing fatal pneumonia share similar glycosyl structures with the polysaccharides from Bacillus anthracis.

Secondary cell wall polysaccharides (SCWPs) are important structural components of the Bacillus cell wall and contribute to the array of antigens presented by these organisms in both spore and vegetative forms. We previously found that antisera raised to Bacillus anthracis spore preparations cross-reacted with SCWPs isolated from several strains of pathogenic B. cereus, but did not react with other phylogenetically related but nonpathogenic Bacilli, suggesting that the SCWP from B. anthracis and pathogenic B. cereus strains share specific structural features. In this study, SCWPs from three strains of B. cereus causing severe or fatal pneumonia (G9241, 03BB87 and 03BB102) were isolated and subjected to structural analysis and their structures were compared to SCWPs from B. anthracis. Complete structural analysis was performed for the B. cereus G9241 SCWP using NMR spectroscopy, mass spectrometry and derivatization methods. The analyses show that SCWPs from B. cereus G9241 has a glycosyl backbone identical to that of B. anthracis SCWP, consisting of multiple trisaccharide repeats of: →6)-α-d-GlcpNAc-(1 â†’ 4)-ß-d-ManpNAc-(1 â†’ 4)-ß-d-GlcpNAc-(1→. Both the B. anthracis and pathogenic B. cereus SCWPs are highly substituted at all GlcNAc residues with α- and ß-Gal residues, however, only the SCWPs from B. cereus G9241 and 03BB87 carry an additional α-Gal substitution at O-3 of ManNAc residues, a feature lacking in the B. anthracis SCWPs. Both the B. anthracis and B. cereus SCWPs are pyruvylated, with an approximate molecular mass of ≈12,000 Da. The implications of these findings regarding pathogenicity and cell wall structure are discussed.

A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies.

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested.

The immunoreactivities of hydrogen fluoride (HF)-released cell wall polysaccharides (HF-PSs) from selected Bacillus anthracis and Bacillus cereus strains were compared using antisera against live and killed B. anthracis spores. These antisera bound to the HF-PSs from B. anthracis and from three clinical B. cereus isolates (G9241, 03BB87, and 03BB102) obtained from cases of severe or fatal human pneumonia but did not bind to the HF-PSs from the closely related B. cereus ATCC 10987 or from B. cereus type strain ATCC 14579. Antiserum against a keyhole limpet hemocyanin conjugate of the B. anthracis HF-PS (HF-PS-KLH) also bound to HF-PSs and cell walls from B. anthracis and the three clinical B. cereus isolates, and B. anthracis spores. These results indicate that the B. anthracis HF-PS is an antigen in both B. anthracis cell walls and spores, and that it shares cross-reactive, and possibly pathogenicity-related, epitopes with three clinical B. cereus isolates that caused severe disease. The anti-HF-PS-KLH antiserum cross-reacted with the bovine serum albumin (BSA)-conjugates of all B. anthracis and all B. cereus HF-PSs tested, including those from nonclinical B. cereus ATCC 10987 and ATCC 14579 strains. Finally, the serum of vaccinated (anthrax vaccine adsorbed (AVA)) Rhesus macaques that survived inhalation anthrax contained IgG antibodies that bound the B. anthracis HF-PS-KLH conjugate. These data indicate that HF-PSs from the cell walls of the bacilli tested here are (i) antigens that contain (ii) a potentially virulence-associated carbohydrate antigen motif, and (iii) another antigenic determinant that is common to B. cereus strains.

Structural elucidation of the nonclassical secondary cell wall polysaccharide from Bacillus cereus ATCC 10987. Comparison with the polysaccharides from Bacillus anthracis and B. cereus type strain ATCC 14579 reveals both unique and common structural features.

Nonclassical secondary cell wall polysaccharides constitute a major cell wall structure in the Bacillus cereus group of bacteria. The structure of the secondary cell wall polysaccharide from Bacillus cereus ATCC 10987, a strain that is closely related to Bacillus anthracis, was determined. This polysaccharide was released from the cell wall with aqueous hydrogen fluoride (HF) and purified by gel filtration chromatography. The purified polysaccharide, HF-PS, was characterized by glycosyl composition and linkage analyses, mass spectrometry, and one- and two-dimensional NMR analysis. The results showed that the B. cereus ATCC 10987 HF-PS has a repeating oligosaccharide consisting of a -->6)-alpha-GalNAc-(1-->4)-beta-ManNAc-(1-->4)-beta-GlcNAc-(1--> trisaccharide that is substituted with beta-Gal at O3 of the alpha-GalNAc residue and nonstoichiometrically acetylated at O3 of the N-acetylmannosamine (ManNAc) residue. Comparison of this structure with that of the B. anthracis HF-PS and with structural data obtained for the HF-PS from B. cereus type strain ATCC 14579 revealed that each HF-PS had the same general structural theme consisting of three HexNAc and one Hex residues. A common structural feature in the HF-PSs from B. cereus ATCC 10987 and B. anthracis was the presence of a repeating unit consisting of a HexNAc(3) trisaccharide backbone in which two of the three HexNAc residues are GlcNAc and ManNAc and the third can be either GlcNAc or GalNAc. The implications of these results with regard to the possible functions of the HF-PSs are discussed.

Cell wall carbohydrate compositions of strains from the Bacillus cereus group of species correlate with phylogenetic relatedness.

Members of the Bacillus cereus group contain cell wall carbohydrates that vary in their glycosyl compositions. Recent multilocus sequence typing (MLST) refined the relatedness of B. cereus group members by separating them into clades and lineages. Based on MLST, we selected several B. anthracis, B. cereus, and B. thuringiensis strains and compared their cell wall carbohydrates. The cell walls of different B. anthracis strains (clade 1/Anthracis) were composed of glucose (Glc), galactose (Gal), N-acetyl mannosamine (ManNAc), and N-acetylglucosamine (GlcNAc). In contrast, the cell walls from clade 2 strains (B. cereus type strain ATCC 14579 and B. thuringiensis strains) lacked Gal and contained N-acetylgalactosamine (GalNAc). The B. cereus clade 1 strains had cell walls that were similar in composition to B. anthracis in that they all contained Gal. However, the cell walls from some clade 1 strains also contained GalNAc, which was not present in B. anthracis cell walls. Three recently identified clade 1 strains of B. cereus that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241, had cell wall compositions that closely resembled those of the B. anthracis strains. It was also observed that B. anthracis strains cell wall glycosyl compositions differed from one another in a plasmid-dependent manner. When plasmid pXO2 was absent, the ManNAc/Gal ratio decreased, while the Glc/Gal ratio increased. Also, deletion of atxA, a global regulatory gene, from a pXO2- strain resulted in cell walls with an even greater level of Glc.

Rhizobium etli CE3 bacteroid lipopolysaccharides are structurally similar but not identical to those produced by cultured CE3 bacteria.

Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin.

The structure of the major cell wall polysaccharide of Bacillus anthracis is species-specific.

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)-alpha-GlcNAc-(1-->4)-beta-ManNAc-(1-->4)-beta-GlcNAc-(1-->, in which the alpha-GlcNAc residue is substituted with alpha-Gal and beta-Gal at O-3 and O-4, respectively, and the beta-GlcNAc substituted with alpha-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.

A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis.

l-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 30 degrees C. The apparent K(m) for NADPH is 90 microm and 16.9 microm for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-beta-l-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.