Dataset Information

Structure-Immunogenicity Relationship of ?- and ?-Tetrasaccharide Glycoforms from Bacillus anthracis Exosporium and Fragments Thereof.

ABSTRACT: The tetrasaccharide (2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-?-d-glucopyranosyl-(1?3)-?-l-rhamnopyranosyl-(1?3)-?-l-rhamnopyranosyl-(1?2)-l-rhamnopyranose) from the major exosporium protein (BclA) of Bacillus anthracis has been proposed as a target for development of diagnostics and immune therapy or prophylaxis. While the immunodominant character of the anthrose residue has been previously elucidated, the role of the stereochemical configuration of the downstream rhamnose is unknown. Because the linkage of this residue to the GlcNAc bridging the glycan and the protein is lost during isolation of the tetrasaccharide, its ?- and ?-glycoforms have been synthesized. Herein, we prepared neoglycoconjugates from a series of fragments of the tetrasaccharide, including the complete ?- and ?-tetrasaccharide glycoforms, a 2-demethoxylated version of the ?-tetrasaccharide, and the ?- and ?-trirhamnosides and CRM197. By immunization of mice, we showed that the anti ?- and ?-tetrasaccharide serum equally recognized both glycoforms. In contrast the sera produced following immunization with the ?- and ?-trirhamnoside fragments exhibited higher recognition for their own antigens than for their anomeric counterparts. The anti ?- and ?-tetrasaccharide sera recognized Sterne spores in a comparable fashion. ?BclA spores not expressing the major exosporium protein were also recognized by the same sera, while mutants that produced the carbohydrate antigen with deletion of either rhamnose or anthrose were not. The tetrasaccharide could, therefore, be expressed in proteins other than BlcA. This work proves that ?- and ?-tetrasaccharide are equally potent immunogens.

SUBMITTER: De Ricco R

PROVIDER: S-EPMC6222408 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Structure-Immunogenicity Relationship of α- and β-Tetrasaccharide Glycoforms from Bacillus anthracis Exosporium and Fragments Thereof.

De Ricco Riccardo R Ventura Christy L CL Carboni Filippo F Saksena Rina R Kováč Pavol P Adamo Roberto R

Molecules (Basel, Switzerland) 20180820 8

The tetrasaccharide (2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-α-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose) from the major exosporium protein (BclA) of Bacillus anthracis has been proposed as a target for development of diagnostics and immune therapy or prophylaxis. While the immunodominant character of the anthrose residue has been previously elucidated, the role of the stereochemical configuration of the downstream rha ...[more]

PMID: 30127242

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Project description:In preliminary experiments, the absence of anthrose in our genetic Sterne knockout was found to decrease pagA expression in BHI broth containing glucose (Fig 5A and B). This contrasted with the large spike in pagA expression when the Sterne antC mutant is grown in protein rich HIB media which does not contain sugars. When exogenous anthrose was added during growth in BHI broth, expression from PpagA increased in both WT (purple line in Fig 5A) and antC Sterne (purple line in Fig 5B). Many bacteria respond to levels of metabolic intermediates and modulate gene expression accordingly. We hypothesized intracellular or extracellular anthrose levels could impact the vegetative cells through modification of gene expression during vegetative growth. Wildtype B. anthracis Sterne was grown in the presence and absence of exogenously added pure anthrose to avoid any possibility of confounding data in a deletion mutant. Bacteria were harvested at two timepoints post-addition of anthrose, 30 min and 2 h, and compared to diluent only treated cultures (Fig 5C). After 30 min treatment, there were 6 genes significantly upregulated more than two-fold while 17 genes were downregulated (Fig 5D). These genes were mainly involved in generating metabolic intermediates such as pyruvate and trehalose. The most upregulated gene was a putative membrane protein (log2FC=10.98) followed by a gene encoding a GerPF homologue at a log2FC of 10.98. Mutation of GerPF family proteins have been linked to a super-dormant spore phenotype[10]. The gene experiencing the highest downregulation was a putative lipoprotein (log2FC = -20.02). No genes located on pXO1 were detected at 30 min. A subset of differentially transcribed genes from the 30 min timepoint are listed in Fig 5F. After 2 h of anthrose treatment, 52 genes experienced significant up regulation, 18 of which were hypothetical proteins (Fig 5E). Forty-five genes experienced significant down regulation across replicates, 13 of which were hypothetical proteins.

Project description:The outermost exosporium layer of Bacillus anthracis spores, the causative agents of anthrax, is comprised of a basal layer and an external hair-like nap. The nap includes filaments composed of trimers of the collagen-like glycoprotein BclA. Essentially all BclA trimers are attached to the spore in a process in which part of the 38-residue amino-terminal domain (NTD) of BclA forms an extremely stable interaction with the basal layer protein BxpB. Evidence indicates that the BclA-BxpB interaction is direct and requires trimeric BxpB. To further investigate the nature of the BclA-BxpB interaction, we determined the crystal structure of BxpB. The structure was trimeric with each monomer consisting of 11 β strands with connecting loops. The structure did not include apparently disordered amino acids 1-19, which contain the only two cysteine residues of the 167-residue BxpB. The orientation of the structure reveals regions of BxpB that could be involved in interacting with the BclA NTD and with adjacent cysteine-rich proteins in the basal layer. Furthermore, the BxpB structure closely resembles that of the 134-residue carboxyl-terminal domain of BclA, which forms trimers that are highly resistant to heat and detergent. We demonstrated that BxpB trimers do not share this resistance. However, when BxpB trimers are mixed with a peptide containing residues 20-38 of BclA, they form a complex that is as stable as BclA-BxpB complexes extracted from spores. Together, our results provide new insights into the mechanism of BclA-BxpB attachment and incorporation into the exosporium. IMPORTANCE The B. anthracis exosporium plays major roles in spore survival and infectivity, but the complex mechanism of its assembly is poorly understood. Key steps in this process are the stable attachment of collagen-like BclA filaments to the major basal layer structural protein BxpB and the insertion of BxpB into an underlying basal layer scaffold. The goal of this study is to further elucidate these interactions thereby advancing our understanding of exosporium assembly, a process shared by many spore-forming bacteria including important human pathogens.

Dataset Information

Structure-Immunogenicity Relationship of ?- and ?-Tetrasaccharide Glycoforms from Bacillus anthracis Exosporium and Fragments Thereof.

Publications

Structure-Immunogenicity Relationship of α- and β-Tetrasaccharide Glycoforms from <i>Bacillus anthracis</i> Exosporium and Fragments Thereof.

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