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Non-classical 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 B. 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 1- and 2-D NMR analysis. The results showed that the B. cereus ATCC 10987 HF-PS has a repeating oligosaccharide consisting of a 6)--GalNAc-(14)--ManNAc-(14)--GlcNAc-(1 trisaccharide that is substituted with -Gal at O3 of the -GalNAc residue, and non-stoichiometrically acetylated at O3 of the 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 HexNAc3 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

structure, repeating unit, Bacillus, cell wall polysaccharide, Bacillus cereus

NCBI PubMed ID: 18757856
Publication DOI: 10.1074/jbc.M803234200
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: rcarlson@ccrc.uga.edu
Institutions: Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30605
Methods: 13C NMR, 1H NMR, methylation, NMR-2D, HF solvolysis, 31P NMR, MALDI-TOF MS, composition analysis, NMR-1D, mild hydrazinolysis

Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc(O3-α-Gal)-(1→6)-α-GlcNAc(O3-α-Gal, O4-β-Gal)-(1→]6-12 The genes whose products promote the galactosylation of B. anthracis SCWP are not yet known. We show here that the expression of galE1, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for B. anthracis SCWP galactosylation. The galE1 mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-type B. anthracis, but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of galE1 diminishes the capsulation of B. anthracis with poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of the B. anthracis cell wall envelope and for the pathogenesis of anthrax disease. IMPORTANCE Unlike virulent Bacillus anthracis isolates, B. anthracis strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth in vitro in broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants of B. anthracis strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes galE1 and galE2 We identified galE1 as necessary for SCWP galactosylation. Deletion of galE1 decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of B. anthracis and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.

cell wall, capsule, S layer, Bacillus anthracis, GalE1, poly-d-γ-glutamic acid, secondary cell wall polysaccharide, UDP-glucose 4-epimerase

NCBI PubMed ID: 29229702
Publication DOI: 10.1128/JB.00562-17
Journal NLM ID: 2985120R
Publisher: American Society for Microbiology
Correspondence: oschnee@bsd.uchicago.edu
Institutions: Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USA, Department of Microbiology, University of Chicago, Chicago, Illinois, USA, Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA, Headquarters, United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Methods: PCR, MALDI-TOF MS, genetic methods, RP-HPLC, immunofluorescence labeling

Endolysins are bacteriophage enzymes that lyse their bacterial host for phage progeny release. They commonly contain an N-terminal catalytic domain that hydrolyzes bacterial peptidoglycan (PG) and a C-terminal cell wall-binding domain (CBD) that confers enzyme localization to the PG substrate. Two endolysins, phage lysin L (PlyL) and phage lysin G (PlyG), are specific for Bacillus anthracis. To date, the cell wall ligands for their C-terminal CBD have not been identified. We recently described structures for a number of secondary cell wall polysaccharides (SCWPs) from B. anthracis and B. cereus strains. They are covalently bound to the PG and are comprised of a -ManNAc-GlcNAc-HexNAc- backbone with various galactosyl or glucosyl substitutions. Surface plasmon resonance (SPR) showed that the endolysins PlyL and PlyG bind to the SCWP from B. anthracis (SCWPBa) with high affinity (i.e. in the muM range with dissociation constants ranging from 0.81 x 10-6 to 7.51 x 10-6 M). In addition, the PlyL and PlyG SCWPBa binding sites reside with their C-terminal domains. The dissociation constants for the interactions of these endolysins and their derived C-terminal domains with the SCWPBa were in the range reported for other protein-carbohydrate interactions. Our findings show that the SCWPBa is the ligand that confers PlyL and PlyG lysin binding and localization to the PG. PlyL and PlyG also bound the SCWP from B. cereus G9241 with comparable affinities to SCWPBa. No detectable binding was found to the SCWPs from B. cereus ATCC (American Type Culture Collection) 10987 and ATCC 14579, thus demonstrating specificity of lysin binding to SCWPs.

polysaccharide, bacteriophage, Bacillus anthracis, secondary cell wall polymer, endolysin

NCBI PubMed ID: 23493680
Publication DOI: 10.1093/glycob/cwt019
Journal NLM ID: 9104124
Publisher: IRL Press at Oxford University Press
Correspondence: elmark@ccrc.uga.edu
Institutions: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
Methods: GC-MS, HF solvolysis, SDS-PAGE, composition analysis, genetic methods, binding assays, SPR

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying "anthrax-like" symptoms in Cameroon (Bc CA) and Côte d'Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10-20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains.

NMR spectroscopy, Bacillus anthracis, Bacillus cereus, cell wall polysaccharides (SCWPs)

NCBI PubMed ID: 28832613
Publication DOI: 10.1371/journal.pone.0183115
Journal NLM ID: 101285081
Publisher: San Francisco, CA: Public Library of Science
Correspondence: sforsb@ccrc.uga.edu
Institutions: Centers for Disease Control and Prevention, Atlanta, GA, United States of America, Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States of America, Indian Institute of Engineering Science and Technology, Shibpur, West Bengal, India, Robert Koch-Institute, Center for Biological Threats and Special Pathogens, Berlin, Germany
Methods: 13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, sugar analysis, biological assays, immunochemical methods, HF treatment, reduction with NaBD4