How does the immune system respond to bacterial infections? The surface of virtually all bacteria are coated with sugar polymers, called glycans. We spoke to Professor Emma Slack, Dr Milad Radiom, Dr Yagmur George Turgay and Suwannee Ganguillet about the SNUGly project’s work in investigating how the immune system recognises these glycans, which could inform the development of vaccines against certain pathogens. The salmonella enterica pathogen is a common cause of food poisoning, usually after being transferred from farm animals or rodents via contaminated food. This particular pathogen is widely used in laboratories as an animal infection model, as researchers have a lot of very good handles on how to work with it. “With this bacterium, we understand what quite a lot of its genome does, we understand the structures on its surface, and we understand how it invades,” explains Professor Emma Slack. As Group Leader in the Laboratory for Food Immunology at ETH Zurich, Professor Slack is the Principal Investigator of the SNUGly project, in which researchers are looking at how the immune system responds to bacterial infections like salmonella enterica. “We’re looking at how the glycans that coat the surface of basically all bacteria are recognised by the immune system,” she says. “We’re investigating the constraints on how you produce an effective immune response that targets these sugars, and the effect of that on the bacteria.”
Immune response This research is focused primarily on salmonella enterica and e.coli at this stage, with Professor Slack and her colleagues mainly using mice in their work, but it could hold wider relevance to other pathogens and hosts. Most of what is currently understood about the immune response against pathogens, particularly protective antibody responses, comes from viruses. “A lot of work has been done on the affinity of antibodies binding to proteins or small molecules,” outlines Professor Slack. Previous work using immunological tools called haptens has shown how to select for higher affinity B-cells – which play an important role in the immune system – while further important insights have also been gained. “It’s been shown how high-affinity antibodies against nitrophenol can be made. This hapten has also been used to show how B-cells get selected to get into immune responses in the first place, while very interesting work has been done looking at how B-cells bind to antigens, and how B-cells selectively take up antigen from different types of membranes among cells,” continues Professor Slack.
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These proteins are taken up in the B-cell, then digested and presented to a T-cell, a process which is not fully understood. The nature of the pull on the glycan, and the proteins that are then taken along, depends on many physico-chemical parameters. “It depends on the stiffness of the presenting membrane for example. It makes a difference whether the thing that the glycan is attached to is stiff or soft,” points out Dr Turgay. The fact that the mice have a B-cell receptor specific for a glycan also enables researchers to track how B-cells behave in a whole animal when exposed to antigens that bind with different affinities, a topic PhD student Suwannee Ganguillet is investigating. “We’re using lipid bilayers, in which we integrate lipidlinked O-glycans. We’re trying to simplify what happens in vivo into an in vitro model, that we can analyse under a microscope,” she explains. “This is dynamic, live microscopy. We can look at B-cells specific to that glycan, and see how the cell responds. We can see how much antigen is taken up, how that antigen is processed and can look at the T-cells and measure the activation strength.”
Vaccine effectiveness
Figure. 1. Atomic force microscopy (AFM) is used to probe antibody-bacterial glycan interactions. (A) An AFM image of S. Tm SB300. The surface is fully exposed with O-antigen molecules. (b) Schematic of AFM molecular level measurement of interaction forces between antibody and O-antigen. (c) A series of interaction forces between STA121 Fab and O5:O12 O-antigen giving an unbinding force 70–110 pN. The unbinding force is measured on retraction cycle (red).
These earlier findings are based on having a very small, well-behaved, rigid antigen however, which is not the case with the glycans associated with salmonella, called O-antigens. This particular antigen has a highly flexible structure that is radically different to nitrophenol. “You can think of nitrophenol as being a bit like a door handle on a solid door which you can grab onto, whereas the O-antigen is like a big bucket of slime,” says Professor Slack. Researchers in the project are investigating how these O-antigens bind to antibodies; Dr Milad Radiom, a Senior Assistant at the Laboratory, is using atomic force microscopy (AFM) to look at antibody-bacterial glycan interactions on the single molecule level. “The surface of the bacteria is effectively a carpet of these glycans. With AFM we can look at the carpet, and at individual molecules within the carpet,” he explains. “We can look at different effects, like O-antigen-antibody binding, opening O-antigen coil and pulling it out of the membrane, with great precision, examining forces in the range of tens to hundreds of piconewtons (10 -12 of a newton).”
This enables researchers to probe and understand the underpinnings of binding affinity, which may then affect the immunogenicity of an antigen, essentially its ability to stimulate an immune response. As Senior Scientist at the Laboratory, Dr Yagmur George Turgay is investigating the relationship between the strength of binding and immunogenicity. “We’ve gathered the tools that we need to perform certain experiments, including the glycans, the pathogen and the antibodies. We have antigens which are completely different in terms of binding affinity,” he outlines. In vitro antigen sampling assays will then be conducted using knock-in mice which express these different antibodies as B-cell receptors, from which Dr Turgay hopes to gain deeper insights. “We will check whether the stronger-binding molecule would sample more antigen, compared to the weaker binding antibody or B-cell receptor. We’ll also look at whether this would then lead to a stronger immune response,” he continues. “In order to get a good immune response you not only need to pull at the glycan, but also to drag along certain proteins.”
EU Research
The wider aim here is to understand the fundamental biology that affects the effectiveness of a vaccine. All of the reagents and genetically modified animals are in place, and a lot of in vitro work has been done looking at the biophysics of antibody-glycan interactions. “We’ve been
using AFM, as well as other techniques like surface plasmon resonance, so we know the overall affinities of the antibodies we’re looking at,” says Professor Slack. The next major step will be to get in vivo animal experiments up and running, and to look at transgenic B-cell responses to different types of vaccine. “We can do almost infinite permutations of these experiments, so we’re going to have to be quite selective,” continues Professor Slack. “We’re also interested in extending this and starting the process of generating reagents for other bacterial glycans. So we want to extend the scope of this research beyond salmonella and have a tool-set that we can compare for different glycans and different pathogens.” One possibility is extending this research into capsular polysaccharides, sugar polymers which have a very different structure to glycans. These polysaccharides are often charged, whereas O-antigens are typically neutral. “The capsules we’re interested in are mainly anionic, containing things like sialic acids,” says Professor Slack. This is a technically challenging area, and so Professor Slack and her colleagues are very busy as they seek to build a fuller picture. “The problem with many of the capsular polysaccharides is getting an antibody response in order to make monoclonal antibodies and generate reagents in the first place, because these are often very non-immunogenic structures. This requires quite a lot of synthetic biology or carbohydrate chemistry, which is also very challenging,” she outlines.
SNUGly Systems-level novel understanding of anti-glycan immunity Project Objectives
Protective immunity against bacterial infections relies heavily on antibody responses that target bacterial surface glycans. However, the molecular constraints on induction of high-affinity glycan-targeting antibody responses remain poorly understood. We aim to better understand the influence of glycan structure on each stage of B cell activation, with a particular focus on intestinal immunity.
Project Funding
This project has received funding from the European Union’s Horizon 2020 research and innovation programme, Starting Grant.
Project Partners
• Professor Raffaele Mezzenga, D-HEST, ETH Zürich, Switzerland (Expertise on soft materials and atomic force microscopy) • Professor Beth Stadtmueller, University of Illinois, USA (Expertise in recombinant antibody production and structural analysis)
Contact Details
Milad Radiom Senior Assistant Institute for Food, Nutrition and Health Vladimir-Prelog-Weg 1-5, 8093 Zürich E: milad.radiom@hest.ethz.ch W: https://slacklab.ethz.ch/ Yagmur Turgay E: yagmur.turgay@hest.ethz.ch Emma Slack E: emma.slack@hest.ethz.ch Suwannee Ganguillet E: suwannee.ganguillet@hest.ethz.ch The SNUGly project team
Emma Slack is a British/Swiss scientist who trained in Cambridge, London, Hamilton (Ontario) and Bern before joining the ETH Zürich as an Ambizione fellow. Milad Radiom studied mechanical and chemical engineering in AmirKabir University of Technology, Nanyang Technological University, and Virginia Tech. He is fascinated by mechanobiology across the length scales. Yagmur Turgay studied biochemistry in Frankfurt, Germany, before completing his PhD at ETH Zürich. He is interested in modalities of antibody-glycan interactions for the development of oral vaccines. Suwannee Ganguillet completed her Bachelors Degree in Biology at the University of Lausanne before moving to Zürich for her Master’s in Microbiology and Immunology at ETHZ, where she carried out her thesis in Professor Slack’s lab.
Figure. 2. STA121 and STA5 IgG binding kinetics for S.Tm O-antigens. Binding of the indicated S. Tm O-antigen derivatives to immobilised STA121 and STA5 recombinant IgG was analysed by Biacore T200 SPR measurements. Sensograms from real time single-cycle kinetic measurements (black curves) were fitted using heterogeneous ligand kinetic model (red curve). KD1 and KD2 are shown on top of each sensogram.
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