Deciphering the molecular mechanisms and physiological consequences of macrophage polarisation during Salmonella infection

Lead Research Organisation: Imperial College London
Department Name: Infectious Disease

Abstract

Macrophages are a key type of immune cell that fights disease-causing microorganisms, such as bacteria. They act by 1) directly fighting the infection by forming a hostile environment to kill the pathogen and 2) producing molecules to alert other immune cells to the danger and ultimately create an inflammatory response. As well as adopting this "killing" state, macrophages can also adopt a "repairing" state, in which they initiate events to reduce inflammation, resolve infection and repair tissues damaged during the inflammatory response. The process by which macrophages adopt either state, known as macrophage polarisation, requires communication within a cell, which is referred to as cell signalling.

Proteins are large molecules that carry out critical functions in our cells, from chemical reactions (enzymes) to the regulation of transcription. Transcription makes RNA from the hereditary material (DNA) contained within the cell and can be controlled by proteins called transcription factors. RNA is then the code to make new proteins. Every protein is made up by a unique string of smaller building blocks called amino acids. The sequence of amino acids determines the 3-dimensional structure and function of a protein. During cell signalling, protein modification by small chemical groups can increase, decrease or change their function. During phosphorylation, something called a phosphoryl group is added to specific amino acids of the protein. This reaction is carried out by enzymes called kinases. Some kinases only modify specific types of amino acids called serine and threonine amino acids whereas others can also modify the amino acid tyrosine.

Disease-causing bacteria, like Salmonella, use their own proteins to interfere with host cell signalling and thereby host immunity. We have recently found that a protein called SteE, delivered from Salmonella into macrophages, binds a host kinase that normally only modifies serine and threonine amino acids. When together with SteE the kinase now modifies a tyrosine amino acid on a new target, which is a transcription factor. Ultimately, this prompts macrophages to inappropriately adopt the "repair" state rather than the "killing" state. This promotes Salmonella survival and long-term persistence inside the host.

This project will 1) define the changes in macrophage DNA transcription mediated by SteE during Salmonella-infection and test whether additional host proteins are required to instruct the "Salmonella-friendly" state of macrophages. 2) Investigate host changes in small molecules (metabolites) during Salmonella infection. 3) Study how the 3D arrangement of SteE and the host kinase are altered in order to allow novel substrates to be modified. Collectively, these findings will reveal the mechanism of how the Salmonella protein SteE promotes disease and provide valuable insight into host immune processes.

Salmonella is a major human health challenge; causing a wide range of diseases in humans, from self-limiting diarrhoeal disease, to typhoid fever, a life-threatening systemic disease. Our findings will enable us to gain profound understanding on the pathogenesis of a global, disease-causing bacterium. Ultimately, this may promote the development of novel ways to combat bacterial infections, something which is of vast importance with the rise of antibiotic-resistant bacterial strains.

Technical Summary

As a key cell of the immune system, macrophages are equipped for pathogen destruction, yet they serve as a niche for the human disease-causing pathogen Salmonella enterica. By delivering virulence factors (effectors) across host cell membranes, Salmonella interferes with immune signalling, promoting its replication, persistence and systemic dissemination within the host. We recently showed that Salmonella effector SteE alters the amino acid and substrate specificity of the host serine/threonine kinase GSK3, enabling it to tyrosine-phosphorylate the anti-inflammatory and M2-polarising transcription factor, STAT3.

We will now use transcriptomics in genetically modified macrophages to define SteE-regulated genes that are STAT3 dependent and independent. Then, ChIP-seq will be used to distinguish SteE-induced genes directly regulated by STAT3. Finally, after identifying novel targets for the kinase activity of the SteE/GSK3 complex we will investigate the role of these in mediating M2-like polarisation and Salmonella persistence. M2-polarised macrophages exhibit a distinct metabolic state compared to inflammatory M1 cells and Salmonella shifts the host metabolome; however, whether this represents an effector-mediated virulence mechanism is unknown. We will therefore study key metabolites in primary macrophages upon infection and determine whether they are regulated by SteE. Finally, we will use structural biology and biochemistry to understand the profound conformational changes occurring in SteE and GSK3 that enable the switch in GSK3 kinase specificity and elucidate the atomic mechanism of STAT3 phosphorylation by GSK3.

This will provide valuable insight into key immune signalling processes and immunometabolism as well as pathogenesis mechanisms for this global pathogen. This may promote the development of novel anti-bacterial or immunoregulatory therapeutics, which are urgently needed with the rise of antibiotic-resistant bacterial strains.
 
Description Meeting with Science Minister to discuss early career researchers
Geographic Reach National 
Policy Influence Type Participation in a guidance/advisory committee
 
Description Collaboration with Dennis Ko 
Organisation Duke University Medical Centre
Department Department of Molecular Genetics
Country United States 
Sector Academic/University 
PI Contribution We have purified proteins and conducted a kinase assays as part of this collaboration which has been used in a recently preprinted publication on the Salmonella effector SteE.
Collaborator Contribution Our collaborator has a shared interest in the Salmonella effector SteE, they have led a story on how this mimic host cytokine signaling.
Impact The work has resulted in a preprint, published 15th Feb 2024. https://www.biorxiv.org/content/10.1101/2024.02.14.580367v1
Start Year 2023
 
Description Dr Peter Hill - SteE collaboration 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution My laboratory is leading the project and providing PDRA - IDDE and students to conduct the experiments
Collaborator Contribution Partner - Dr Hill is providing intellectual input and data he has generated that is of overlapping interest.
Impact Dr Hill is providing expertise in RNAseq, combining this with our expertise in cell and molecular biology the collaboration is multi-disciplinary.
Start Year 2022
 
Description Rittinger 
Organisation Francis Crick Institute
Department Mill Hill Laboratory
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution I provided expertise in the area of host pathogen interaction, innate immune signalling and infection biology as well as the leading the research direction of the project and provided the lead training to the PhD student who worked on this collaboration.
Collaborator Contribution Our collaborators provided critical advice, support, reagents and access to equipment in the area of structural biology and x-ray crystallography. The collaborating group helped train my PhD student in the use of software and provided essential experimental guidance as well as data that was used in publication.
Impact Under this award, the collaboration resulted in one publication to date DOI 10.1074/jbc.RA118.004255 The collaboration was multi-disciplinary, bringing my expertise in host cell signaling, innate immunity and infection biology together with that of structural and biochemical experts from the Francis Crick Institute.
Start Year 2017
 
Description British Council funded mentoring women in STEM in Brazil 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact A PDRA on the grant participated in a British Council-funded programme between King's College London & 2 Brazilian unis led by Prof Andrea Streit (KCL) to support & mentor women in STEM in Brazil. The programme involves film evenings followed by discussions for female STEM undergraduate students in Brazil. The PDRA prepared a video accompanying a film about Marie Curie, where she talked about the challenges she faced as a women in STEM and the situation today, encouraging young women to pursue a career in STEM.
Year(s) Of Engagement Activity 2022
 
Description Career panel member 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Activity involved either short presentation about career progress followed by questions (2021; University of Birmingham) or panel career event with Q&A (2022; Imperial College London).
Year(s) Of Engagement Activity 2021,2022
 
Description Cracking Crick Science Spectacular 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Here the event showcases science in a show hosted by the Crick Public Engagement team - named PDRA on the grant attended and helped on the day. Children in years 5-7 had the opportunity to see the Crick and learn about science and research.
Year(s) Of Engagement Activity 2022
 
Description Crick Discovery Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This is a discovery day showcasing the science at the Crick and targeted families and young children. There are different activities and discussion about research. Named PDRA on the grant attended and answered questions about the Crick and their research from the general public.
Year(s) Of Engagement Activity 2022
 
Description Host school student in the laboratory 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A school pupil attended the laboratory to conduct experiments over a period of 3 days. The student discussed this visit with her sixth form class afterwards, explaining about basic research and the need for this to understand disease.
Year(s) Of Engagement Activity 2023
 
Description Participation in half term outreach activity 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Families attended workshop like sessions with activities for children centered on learning about bacteria, how they cause disease and spread. Questions about research and bacteria were discussed. Children were engaged.
PDRA- IDDE - represented the laboratory at this event.
Year(s) Of Engagement Activity 2023
 
Description Women in science discussion groups 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Several activities were planned with the intended purpose of supporting women in science and generating discussion about how to retain women in science. Lots of discussion was sparked and women are keen to support each other.
Year(s) Of Engagement Activity 2023