A mass spectrometry approach to unveil cytoplasmic bacteria interactomes

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BBSRC : Gemma Kate Banister : BB/T007222/1The immune system is built to recognise infections. As such there are different systems to recognise pathogens in different environments. One pathway, called the non-canonical inflammasome, is responsible for recognising bacteria that have managed to get inside human cells. It recognises a key component of the surface of some bacteria called lipopolysaccharide. This leads to explosive immune signalling that can be both beneficial but harmful in the wrong context. The non-canonical inflammasome pathway is involved in the progression of sepsis and Chron's disease. Sepsis not only accounts for nearly 20% of deaths worldwide but is also a massive financial burden in the UK and Canada. Therefore, understanding the biochemical pathway which may contribute sepsis and to other diseases such as Chron's disease is very important. In this project I will identify human proteins on the outside of the bacteria after they have made their way inside cells. This will tell us about the interaction between the human cell and the bacteria during infection. This is important for understanding how our immune system protects us from the bacteria. I will also focus on the non-canonical inflammasome pathway as this pathway is involved in a range of autoimmune diseases and sepsis. I am developing a new technique to separate the bacteria from the human cells after they have infected them. This technique will allow me to grab the bacteria without disrupting any signalling occurring on the surface of the bacteria. I will then use a technique called mass spectrometry to identify the proteins on the surface of the bacteria. Mass spectrometry splits the proteins into small pieces and tells you how much each piece weighs, allowing you to identify and quantify which proteins you have. This will allow us to identify unknown involved in the human cell- bacteria interaction. I will use epithelial cell lines lacking key non-canonical inflammasome pathway components to see how these proteins affect the signalling between the host and the bacterial surface. I hypothesise that these proteins will affect how bacterial surface are recognised. This will give us critical details about how these proteins contribute to the immune response to bacteria. A deeper understanding of the immune response to bacteria inside human cells could provide potential routes for drug targeting which will be beneficial for Canada and UK. It will direct further research into what happens when the immune response goes wrong and causes sepsis or autoimmune diseases.This project will form connections with the Canadian lab and open doors for future collaborations. My current lab has expertise in using a biochemical approach to study the immune response and the host lab has expertise with mass spectrometry and host-pathogen interactions making this a synergistic collaboration that will work well for this project and provide avenues for future collaborations.