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

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.

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.