Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Host organism: Sus scrofa
Associated disease: pleuropneumonia [ICD11:
CA40 
, ICD11:
CA40.Y ]
NCBI PubMed ID: 34076489Publication DOI: 10.1128/mBio.00897-21Journal NLM ID: 101519231Publisher: Washington, DC: American Society for Microbiology
Correspondence: Timm Fiebig <Fiebig.Timm
mh-hannover.de>
Institutions: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany, Fraunhofer International Consortium for Anti-Infective Research (iCAIR), Hannover, Germany, Department of Biosciences, University of Salzburg, Salzburg, Austria, Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, Germany
Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.
NMR, Neisseria meningitidis, polysaccharides, capsule, glycosyltransferase, teichoic acid, enzymatic synthesis, glycerol, glycoconjugate vaccine, antibiotic resistance, Recombinant Protein Expression, animal health, TagF-like polymerases, veterinary vaccine development
Structure type: polymer chemical repeating unit
Location inside paper: Fig. 8, App11
Compound class: CPS
Contained glycoepitopes: IEDB_136906,IEDB_137472,IEDB_141794,IEDB_142488,IEDB_146664,IEDB_151528,IEDB_190606,IEDB_983931,SB_192,SB_7
Methods: 13C NMR, 1H NMR, NMR-2D, SDS-PAGE, 31P NMR, Western blotting, cloning, enzymatic synthesis, HPLC-AEC, solid-phase synthesis, BLASTp
Synthetic data: enzymatic
Related record ID(s): 9683, 9684, 9685, 9686, 9688, 9689, 9690, 10897
NCBI Taxonomy refs (TaxIDs): 754348Reference(s) to other database(s): GTC:G15185GK
Show glycosyltransferases
There is only one chemically distinct structure:
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