Structural basis of bacterial polysaccharide biosynthesis and priming in pathogenic bacteria
[eng] This doctoral thesis is focused on the understanding, from a structural and biochemical view point, of the enzymatic mechanisms involved in the biosynthesis of the capsule of pathogenic bacteria. We elucidate the mechanisms governing the priming and polymerization of bacterial capsule polysacc...
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| Format: | doctoral thesis |
| Status: | Published version |
| Publication Date: | 2025 |
| Country: | España |
| Institution: | Universidad de Barcelona |
| Repository: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/228367 |
| Online Access: | https://hdl.handle.net/2445/228367 http://hdl.handle.net/10803/697044 |
| Access Level: | Embargoed access |
| Keyword: | Bacteris patògens Radiocristal·lografia Pathogenic bacteria X-ray crystallography |
| Summary: | [eng] This doctoral thesis is focused on the understanding, from a structural and biochemical view point, of the enzymatic mechanisms involved in the biosynthesis of the capsule of pathogenic bacteria. We elucidate the mechanisms governing the priming and polymerization of bacterial capsule polysaccharides (CPS) by using a combination of X-Ray crystallography, single particle cryo electron microscopy (cryoEM), NMR and biochemical assays. We discovered two enzymes, named transition transferases, involved in the biosynthesis of the linker between the conserved glycolipid anchor Kdo and the serotype specific CPS. In Actinobacillus pleuropneumoniae serotypes 3 and 7, the two transition transferases enzymes are CpsA and CpsC. Specifically, CpsA is a glycerol-3-phosphate transferase and CpsC is a glycerol-3-phosphate polymerase. Furthermore, we solved the crystal structure of Cps3D, the multienzyme capsule polymerase of A. pleuropneumoniae serotype 3, demonstrating how its activity is boosted in the presence of Cps3A. We identified the presence of such enzymatic machinery in both group 2 Gram-negative and WTA I Gram-positive bacteria. We also solved crystal structures of the O-acetyl transferase Cps11E of A. pleuropneumoniae serotype 11 system, an enzyme involved in the decoration of the CPS core. Finally, we provided the crystal and the cryoEM structures of Bcs3, the polymerase involved in the biosynthesis of the Haemophilus influenzae type b (Hib) CPS. Bcs3 exhibits a unique dimeric architecture in which each subunits contains three different enzymes. Its concave shape offers an optimal environment for the synthesis of the CPS. Very importantly, we can produce length controlled capsule oligomers of Hib through chemoenzymatic reaction for the use in glycoconjugates vaccine formulations. This thesis greatly contributes to the understanding of the molecular mechanism regulating the biosynthesis of bacterial capsules. |
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