Stereoisomer-specific reprogramming of a bacterial flagellin sialyltransferase

Summary of the publication:

Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages, for example bacterial flagella. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament in bacteria. The group of GCIR member Professor Patrick Viollier showed that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. They established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in another bacterium. Finally, by rewiring FlmG glycosylation using chimeras, they defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.

The work has been published in The EMBO Journal and the funding support was from the Swiss National Science Foundation, the University of Geneva, UNITEC and Swisslife Foundation.

Link to full article: https://doi.org/10.15252/embj.2022112880

What does it mean?

Sialic acids are carbohydrate-based sugar molecules involved in a wide range of functions across all branches of life. Bacteria are able to biosynthesize them and incorporate them into cell-surface structures. They have specific roles in motility, biofilm formation, and host immune avoidance, depending on which surface structure they are added to. Bacteria are careful to select the correct component and add the correct sialic acid to it. How is this the correct surface carrier and the correct sugar chosen? Here the authors unlock this mystery, showing that a specific component of bacterial surface structure (the flagellum) is modified with a specific-type of sialic acid, like a chimney sweeper that first select the right brush for the matching chimney. The key in this work was to find the "chimney sweeper" of bacterial cells and then to re-engineer it so it could sweep same chimney with another brush. Now that the researchers understand how the chimney sweeper works, they will try to engineer versions that will sweep other chimneys with other brushes. Ultimately this property can be exploited in biotechnology, for example designing new types of bacterial vaccines. It turns out that the flagellum component that is modified with the sialic acid has properties that make it suitable as vaccine carrier. This potential application will be developed by a grant jointly funded by SNF and Innosuisse through the Bridge-Discovery program, showcasing how fundamental research can unexpectedly open the door (or chimneys)  for biotechnological applications.

BRIDGE Discovery Grant:

GCIR Members Professors Patrick Viollier and Arnaud Didierlaurent have been awarded with the Bridge-Discovery grant for their project: "FlagSyl, Polyvalent mucosal adjuvant and vaccine carriers based on flagellin glycosylation systems"

Protein glycosylation is the most abundant form of protein modification in eukaryotes, modulating protein-protein interactions, activity and/or stability. Specific enzymes (glycosyltransferases, GTs), ubiquitous in all domains of life, execute an enzymatic reaction, transferring either simple monosaccharides or polysaccharides en bloc via covalent linkage to OH groups (O-glycosylation on serine or threonine residues) or to NH2-groups (N-glycosylation on asparagine) by O-GTs or N-GTs, respectively. As almost a third of all human peptide hormones are O-glycosylated, O-GTs are attractive biotechnological tools for therapeutic proteins that must be produced in soluble form. Therefore, ideally O-GT-based reactions should be also be soluble, yet most donor sugars and enzymatic reactions are executed by (naturally) membrane-linked O-GTs.

Bacterial O-GTs are attractive biotechnological tools because of their apparent simplicity in design, small molecular mass and amenability for bacterial (E. coli-based) protein overexpression systems. While many bacterial O-GTs and donor sugars are also membrane-anchored, one class of simple and soluble O-GTs has recently been studied: the FlmG O-GT that O-glycosylates flagellin (the subunit of the flagellar filament), with (soluble) sialic acids in the cytoplasm. This reaction can be reconstituted in vivo in heterologous hosts, requiring FlmG, flagellin and the sugar biosynthesis operon. Flagellin is an immune adjuvant that binds and engages the TLR5 innate immune receptor on mucosal surfaces, triggering the release of pro-inflammatory cytokines and chemokines. Because of its excellent solubility and adjuvating properties, flagellin may act as booster to combat microbial infections, including pneumoniae caused by (antibiotic resistant) bacteria that are not eliminated by conventional chemotherapy.

The goal of this project is to leverage the adjuvant properties of bacterial flagellin and its ability to be specifically glycosylated with sialic acids to develop it into a dual-function and highly soluble (all-in-one) bio-glyco-conjugate carrier. Chemical conjugation, although successfully used for vaccine development against bacterial pathogens is still challenging for bacterial antigens such as LPS and capsules due to structural modifications and solubility issues that can be bypassed by bio-glyco-conjugation. Along with input industrial partners having expertise in vaccine technologies, vaccine formulations and glyco-engineering, Prof. Patrick Viollier and Prof. Arnaud Didierlaurent, will benefit from a partnership with Prof. Jean-Claude. Sirard (University of Lille, France) via the FAIR project to test nebulization of glycosylated flagellins.

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