A mass spectrometry approach to unveil cytoplasmic bacteria interactomes

BBSRC：Gema Kate Banister：BB/T007222/1免疫系统是为识别感染而构建的。因此，在不同的环境中有不同的系统来识别病原体。一种被称为非典型炎症体的途径负责识别设法进入人体细胞的细菌。它识别一些细菌表面的一种关键成分，称为脂多糖。这导致了爆炸性的免疫信号，在错误的情况下可能是有益的，但也可能是有害的。非典型炎症体途径参与了脓毒症和慢性疾病的进展。败血症不仅占全球死亡人数的近20%，而且在英国和加拿大也是一个巨大的经济负担。因此，了解可能导致脓毒症和其他疾病(如慢性疾病)的生化途径是非常重要的。在这个项目中，我将在细菌进入细胞内后识别细菌外部的人类蛋白质。这将告诉我们在感染期间人类细胞和细菌之间的相互作用。这对于了解我们的免疫系统如何保护我们免受细菌侵袭非常重要。我还将重点介绍非典型炎症体途径，因为该途径与一系列自身免疫性疾病和脓毒症有关。我正在开发一种新技术，在细菌感染后将它们从人类细胞中分离出来。这项技术将使我能够在不破坏细菌表面发生的任何信号的情况下抓住细菌。然后，我将使用一种名为质谱学的技术来鉴定细菌表面的蛋白质。质谱仪将蛋白质分成几小块，并告诉你每一块的重量，这样你就可以识别和量化你所拥有的蛋白质。这将使我们能够确定参与人类细胞-细菌相互作用的未知因素。我将使用缺乏关键的非典型炎症体途径成分的上皮细胞系来了解这些蛋白质如何影响宿主和细菌表面之间的信号传递。我假设这些蛋白质将影响细菌表面被识别的方式。这将为我们提供有关这些蛋白质如何对细菌的免疫反应做出贡献的关键细节。更深入地了解人体细胞内细菌的免疫反应可能为药物靶向提供潜在的途径，这将对加拿大和英国有利。它将指导进一步研究当免疫反应出错并导致败血症或自身免疫性疾病时会发生什么。该项目将与加拿大实验室建立联系，并为未来的合作打开大门。我目前的实验室在使用生化方法研究免疫反应方面拥有专业知识，主办实验室在质谱学和宿主-病原体相互作用方面拥有专业知识，这使得这一协同合作将很好地为这个项目工作，并为未来的合作提供途径。

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.