Structural Elucidation of the Nonclassical Secondary Cell Wall Polysaccharide from Bacillus cereus ATCC 10987

Abstract

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 twodimensional NMR analysis. The results showed that the B. cereus ATCC 10987 HF-PS has a repeating oligosaccharide consisting of a 36)-␣-GalNAc- (134) The Bacillus cereus is a group of Gram-positive bacteria that includes Bacillus anthracis, B. cereus, and Bacillus thuringiensis strains. Members of this group are very closely related. In fact, on the basis of detailed phylogenetic analysis, it has been suggested that they all may belong to a single species (1). Despite the very close relatedness of B. cereus group members, there is considerable pathogenic variability. Some members of this group are not pathogenic, whereas others are opportunistic pathogens causing a range of conditions on a variety of hosts. Bacillus thuringiensis is an insect pathogen, and B. cereus is normally a soil-dwelling bacterium, which in rare cases, causes, in humans, usually nonfatal cases of food poisoning, sepsis, endophthalmitis, and occasionally severe or fatal pneumonia. One member, B. anthracis, causes anthrax in animals and humans and is considered a high threat bioterrorism agent.This variability in pathogenicity and host range is largely attributed to the plasmid content of the B. cereus group members, which can vary in size and number (2). For example, the crystal toxin genes of B. thuringiensis are carried on a plasmid (3), and plasmids pXO1 and pXO2 contain the genes required for the production of the B. anthracis toxin proteins and ␥-polyglutamate capsule, respectively (4, 5). Further, recent B. cereus isolates from cases of severe and fatal pneumonia were found to have the pXO1 plasmid (6, 7), and another report showed that B. cereus or B. thuringiensis isolates from cases of "anthrax-like" disease in gorillas contain both pXO1 and pXO2 (8, 9).For numerous bacterial pathogens, both Gram-positive and Gram-negative, cell wall polysaccharides are known virulence factors. However, little work has been done on the cell wall polysaccharides from members of the B. cereus group. We recently showed that the polysaccharides released from the cell walls from members of the B. cereus group using aqueous hydrogen fluoride (HF) 3 have carbohydrate compositions that vary qualitatively in a manner that is correlated, at least in part, to phylogenetic relatedness as determined by multilocus

Environmental and Biofilm-dependent Changes in a Bacillus cereus Secondary Cell Wall Polysaccharide

“…8). In their previous work, Leoff et al (23,24) did report the presence of Ne HF-PS polysaccharide in the B. cereus ATCC14579 without providing its full structure but conclusively established the fine structures of its equivalent in a wide panel of B. cereus strains (41). In contrast, these authors never mentioned the presence of an equivalent of Ch HF-PS in any B. cereus strain, including Bc 14579, or in B. anthracis (23-25, 27, 41).…”

“…We first established that the polysaccharide fraction released by HF from B. cereus strain ATCC 14579 grown in planktonic conditions was composed of two distinct polysaccharides presenting very different structural features. Whereas one presented strong similarities with HF-released polysaccharide (HF-PS) isolated from B. cereus 10987 (24), the other one exhibited totally original structural features. Then, we demonstrated that these two polysaccharides were differentially regulated along the growth of bacteria in the biofilm phase but not in the planktonic phase.…”

“…In particular, the backbone of the secondary cell wall polysaccharides isolated from B. anthracis Ames and B. cereus 10987 were shown to be composed of structurally related trisaccharide repeating units differentially substituted by a strain-specific pattern of ␣-and ␤-D-galactosidase residues. In comparison, B. cereus 14579 strain appears to synthesize similar but more complex polysaccharides containing the consensus trisaccharide sequence, the structure of which is, however, still elusive (24).…”

“…The exact structures of the secondary cell wall polysaccharides from two B. cereus strains were recently compared with those of B. anthracis Ames after their release from the cell wall using aqueous hydrogen fluoride (HF). The detailed analysis of the peptidoglycan HF-labile fractions established the occurrence of the consensus sequence (␣-HexNAc-␤-ManNAc-␤-GlcNAc) in all three strains (24,27). In particular, the backbone of the secondary cell wall polysaccharides isolated from B. anthracis Ames and B. cereus 10987 were shown to be composed of structurally related trisaccharide repeating units differentially substituted by a strain-specific pattern of ␣-and ␤-D-galactosidase residues.…”

“…cereus cell surface displays teichoic acids and secondary cell wall polysaccharides (23)(24)(25)(26). These secondary cell wall polysaccharides were suggested to represent in this bacterium and its close relatives a virulence-associated carbohydrate anti-gen motif (23).…”