Identification, characterization and exploitation of the enzyme repertoire that connects structurally variable capsule polymers to their conserved glycolipid anchor

• Applicant: Dr. Timm Fiebig
• Subject Area: Biochemistry ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 553100037

Capsules are important virulence factors for bacterial pathogens. They protect the bacterial cell from the host immune system, from desiccation and physical stresses. They consist of so called capsular polysaccharides or capsule polymers, which are structurally highly divers long chain-carbohydrates that can additionally comprise polyols and phosphates, and give rise to antigenic epitopes which are the foundation of capsule serotypes within a species. Capsular polysaccharides have been very successfully used as antigens in glycoconjugate vaccines, in which they are coupled to a carrier protein to induce a T-cell dependent immune response. The enzymes that generate capsules are promising tools for the development of pathogen-free vaccine synthesis protocols and interesting drug targets. Gram-negative capsules are often assembled using an enzyme complex known as ABC-transporter-dependent assembly systems, which is expressed by important human and animal pathogens like extraintestinal pathogenic Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, Campylobacter jejuni, Pasteurella multocida and Actinobacillus pleuropneumoniae. In all of these pathogens, the capsular polysaccharide is anchored to the cell surface by a conserved glycolipid consisting of phosphatidylglycerol and a polymer of β-linked 3-deoxy-D-manno-oct-2-ulosonic acid (poly(Kdo)). While the enzymes assembling the glycolipid as well as many capsule polymerases are well understood, the enzyme repertoire (transition transferases) and thus the linker connecting both components remain elusive, leaving the question how the dense capsule layer is attached to the cell surface unanswered. As of now, the identification and study of transition transferases has been hindered by an absence of suitable, synthetic poly(Kdo) acceptor substrates. We developed an enzyme-based synthesis protocol for fluorescently labeled poly(Kdo) and identified a set of transition transferases. Herein, the poly(Kdo) synthesis will be up-scaled to allow a comprehensive structural and biochemical characterization of theses enzymes. As a novel concept in capsule biosynthesis, we observed that the transition transferases stimulate the capsule polymerases to produce more and longer capsule polymers. This activating effect will be analyzed in molecular detail, as it implies that more enzymes than the polymerase might be needed to successfully study and understand a capsule biosynthesis system and / or effectively produce capsule polymer enzymatically for biotechnological purposes such as vaccine development. Finally, we aim to translate our findings into novel approaches for enzyme-based vaccine synthesis protocols.

• DFG Programme: Research Grants