Exploring and exploiting group 2 capsule biosynthesis enzymes

• Applicant: Dr. Timm Fiebig
• Subject Area: Biochemistry
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 412824531

The expression of a capsule is a successful strategy of pathogenic bacteria to evade the host immune response. However, the polysaccharides that form the capsule are also the first structures encountered by the host immune system and can thus be utilized as antigens in highly effective glycoconjugate vaccines. For vaccine manufacturing, capsule polymers are typically harvested from pathogen cultures. This laborious, biohazardous and cost-intensive step prevents the global distribution of glycoconjugate vaccines and hinders their use in the animal health sector, where they could help reduce the overuse of antibiotics and the associated rise of antibiotic resistance. Acknowledging the importance of pathogen-free provision of capsule polymer, we develop protocols for the chemo-enzymatic synthesis of glycoconjugate vaccines that are based on the in vitro use of recombinant enzymes from group 2 capsule biosynthesis complexes. Centerpieces of these protocols are the so-called capsule polymerases (CPs), which build-up the polymer from nucleotide activated precursors. We recently discovered the novel TagF-like CP family, whose members combine glycosyltransferase and hexose-/polyol-phosphate transferase activity to assemble highly complex polymers containing saccharides, phosphates and polyols. An even higher level of structural diversity is added to the polymer backbone by polymer-modifying glycosyl- (GTs) and/or O-acetyl-transferases (OATs). TagF-like CPs are expressed, among others, by the pig-specific pathogen Actinobacillus pleuropneumoniae (App), which causes major economic losses worldwide, and the human pathogen Haemophilus influenzae (Hi), an important cause of respiratory disease, especially in infants. During the first funding period we characterized and engineered the elongation mechanism of TagF-like CPs to enable the production of milligram amounts of tailor-made polymers. We further developed synthesis cascades to generate commercially unavailable nucleotide activated polyol precursors required as substrates for TagF-like CPs. To drive the biotechnological application of TagF-like CPs and associated enzymes forward, we identified (i) seven novel capsule polymer modifying enzymes (two OATs and five GTs) from App and (ii) the previously unknown TagF-like CP from Hi serotype b (Hib). The latter enzyme comprises a ribosyltransferase domain with an as yet undescribed fold. In the frame of this grant application, we aim to exploit the aforementioned enzymes to develop a solid-phase synthesis pipeline for a penta-valent anti-App vaccine and an enzyme cascade for the up-scaled synthesis of the Hib polymer starting from ribose. Based on promising preliminary crystallization results, we propose the first biochemical and structural characterization of one polymer-modifying GT, one OAT, and the novel ribosyltransferase domain from Hib.

• DFG Programme: Research Grants