Galactosylation of the Secondary Cell Wall Polysaccharide of Bacillus anthracis and Its Contribution to Anthrax Pathogenesis

Abstract

, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. SCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc(3-α-Gal)-(1→6)-α-GlcNAc(3-α-Gal, 4-β-Gal)-(1→] The genes whose products promote the galactosylation of SCWP are not yet known. We show here that the expression of, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for SCWP galactosylation. The 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 , but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of diminishes the capsulation of 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 cell wall envelope and for the pathogenesis of anthrax disease. Unlike virulent isolates, strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth 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 strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes and We identified as necessary for SCWP galactosylation. Deletion of decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.

Functional Characterization of Enzymatic Steps Involved in Pyruvylation of Bacterial Secondary Cell Wall Polymer Fragments

“…In P. alvei , the SCWP consists of pyruvylated [→3)-β- D -Man p NAc-(1→4)-β- D -Glc p NAc-(1→] disaccharide repeats ( Schäffer and Messner, 2005 ; Janesch et al, 2013a ). The SCWP repeats of B. anthracis ( Mesnage et al, 2000 ; Forsberg et al, 2012 ) and B. cereus strains ( Choudhury et al, 2006 ; Forsberg et al, 2011 ) as analyzed from hydrofluoric acid-treated material are extensions of the disaccharide motif by one α- D -GlcNAc residue yielding [→4)-β- D -Man p NAc-(1→4)-β- D -Glc p NAc-(1→6)-α- D -Glc p NAc-(1→] and include additional non-stoichiometric galactosyl ( Chateau et al, 2018 ) and acetyl modifications; in these SCWPs, exclusively the non-reducing-end β- D -ManNAc residue carries a 4,6-linked pyruvate ketal modification.…”

The Bacillus cereus Group: Bacillus Species with Pathogenic Potential

“…Depletion of the glycosyltransferases results in irregularly shaped non-viable cells (132). In addition, a mutant lacking a UDP-glucose 4-epimerase required for SCWP galactosylation exhibits reduced encapsulation and decreased virulence in a murine model for anthrax (133). Recent investigations of B. cereus isolates causing anthrax-like disease indicate SCWP with structure similar to that of B. anthracis , but with an additional α-Gal substitution at O3 of ManNAc (134).…”

Pyruvate Substitutions on Glycoconjugates

“…The S-layer gene cluster [99,112,113,114] encodes, among others, components for pyruvylation ( B. anthracis CsaB) and O -acetylation of the terminal SCWP trisaccharide, and a Wzy-like protein. The other gene clusters play predicted roles in the formation of lipid-linked precursors of the murein linkage unit and the trisaccharide repeat [112,114,115,116]. The SCWP biosynthesis model proposes the separate assembly of different undp-PP-linked building blocks in the cytoplasm—the murein linkage unit, trisaccharide repeat, and terminal modified trisaccharide [113]—followed by the individual translocation across the cytoplasmic membrane via Wzx, followed by SCWP polymerization at the outer face of the membrane involving Wzy [113].…”