Marcelo E. Guerin
Institute of Molecular Biology of Barcelona (IBMB) – Spanish National Research Council (CSIC), Spain
Molecular mechanisms of capsule priming and polymerization in pathogenic bacteria
Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules. In addition, using a multi-pronged approach, we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. We purified all requisite enzymes and demonstrated their potential for the fermentation-free production of Hib vaccine antigens. Moreover, we provide the X-ray crystal structure of the capsule polymerase Bcs3, the first multi enzyme machine for the synthesis of complex heteropolymers present in both Gram-negative and Gram-positive pathogens. Dimerization of Bcs3 allows two polymer binding domains to channel the nascent chains towards two separate catalytic centers, each formed by three enzymes: the ribofuranosyltransferase CriT, the phosphatase CrpP and the ribitol-phosphate transferase CroT. Supported by biochemical studies and comprehensive 2D NMR, our data explains how the basket-like shape of Bcs3 supports the concerted action of this unique multi-enzyme assembly.
Selected references
1. Litschko C, Di Domenico V, Schulze J, Li S, Ovchinnikova OG, Voskuilen T, Bethe A, Cifuente JO, Marina A, Budde I, Mast TA, Sulewska M, Berger M, Buettner FFR, Lowary TL, Whitfield C, Codée JDC, Schubert M, Guerin ME, Fiebig T. Transition transferases prime bacterial capsule polymerization. Nat. Chem. Biol. 2025 Jan;21(1):120-130.
2. Cifuente JO, Colleoni C, Kalscheuer R, Guerin ME. Architecture, Function, Regulation, and Evolution of α-Glucans Metabolic Enzymes in Prokaryotes. Chem. Rev. 2024 Apr 24;124(8):4863-4934.
3. Cifuente JO, Schulze J, Bethe A, Di Domenico V, Litschko C, Budde I, Eidenberger L, Thiesler H, Ramón Roth I, Berger M, Claus H, D'Angelo C, Marina A, Gerardy-Schahn R, Schubert M, Guerin ME, Fiebig T. A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule. Nat. Chem. Biol. 2023 Jul;19(7):865-877.
4. Trastoy B, Du JJ, Cifuente JO, Rudolph L, García-Alija M, Klontz EH, Deredge D, Sultana N, Huynh CG, Flowers MW, Li C, Sastre DE, Wang LX, Corzana F, Mallagaray A, Sundberg EJ, Guerin ME. Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases. Nat. Commun. 2023 Mar 27;14(1):1705.
5. Anso I, Naegeli A, Cifuente JO, Orrantia A, Andersson E, Zenarruzabeitia O, Moraleda-Montoya A, García-Alija M, Corzana F, Del Orbe RA, Borrego F, Trastoy B, Sjögren J, Guerin ME. Turning universal O into rare Bombay type blood. Nat. Commun. 2023 Mar 30;14(1):1765.
The organization of this seminar is supported by the SFR-B2S Federation.