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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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.”
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