Publishable Summary
Summary description of project context and objectives
We targeted 2 areas of intracellular pathogen biology; initial uptake into phagocytic cells, which may impact on subsequent trafficking, and phagosome-lysosome fusion events, which are manipulated by Mycobacterium tuberculosis and Salmonella to promote survival. We used state-of-the-art cell-based screening with siRNA libraries and pharmaceutically active compounds to identify potential macrophage targets, and systems biology to integrate and analyse the data. We identified genetic targets manipulated by siRNA, and enzymes modulated with small molecules, and improved systems biology techniques to allow the interrogation of complex data sets.
Initial phagocytosis models, based on existing knowledge and data collected within the project or already published, used novel automated modelling methods to combine data and expert knowledge. Personnel issues meant the scope of the models was more limited than planned, but we used automated methods to model endosome maturation and LDL trafficking, with time-course data on protein concentrations. Structured output prediction on high-throughput datasets was done by machine learning and predictive clustering. We used mathematical modelling to investigate an interaction between apparently antagonistic enzymes that regulate phagosome maturation. Complex formation produced novel forms of switch-like and bell-shaped responses that could have a functional role in the temporal regulation of phosphoinositides during phagosome maturation.
We experimentally characterised the bacteria-containing compartments, and the kinetics of intracellular phagosomal and endosomal trafficking, using a combination of high throughput fluorescence microscopy and novel quantitative multiparametric analysis. We developed high-throughput, high-content screening assays using both murine and primary human macrophages. The mycobacterial infection assay was optimized, combining recombinant BCG expressing GFP, with lysosome staining, providing a direct readout of the release of the phagosomal maturation block and degradative delivery of bacteria to lysosomes when the system was perturbed with either siRNA or small molecules.
Optimised assays and protocols were used to identify key regulators of the host cellular machinery, e.g. kinases, small GTP-binding proteins and their effectors, which are required to control infection of host cells, intracellular trafficking and growth or clearance of non-pathogenic and pathogenic Salmonella and mycobacteria. Using targeted chemical and genetic approaches we identified a number of hits that act on the host-cell and not the bacterium; these are now being taken forward in studies beyond this project.
We attempted to identify bacterial components that mediate interactions with macrophages, preventing phagosome-lysosome fusion, and therefore promoting bacterial survival. We used mycobacterial mutants selected for their inability to block phagosome-lysosome fusion during macrophage infection in vitro. A panel of mutants was tested in human type 1 and type 2 macrophages, but only 4 mutants showed the original phenotype, probably due to differences in macrophages used; murine or transformed cell lines appear to differ fundamentally in the way they traffic intracellular mycobacteria. The next step will be to compare the transcriptional response of the macrophage to infection with wild type and mutant bacteria.
Significant progress was made towards the main objectives, using high-throughput genetic and chemical screens to identify host-cell targets that can be manipulated to overcome the phagosome-lysosome fusion block put in place by intracellular pathogens. We optimised screening protocols, data analysis methods and systems biology techniques with which to integrate and interrogate the data generated. Target and pathway identification is underway and will continue beyond the life of this project.
Description of work performed and main results
This project targeted two areas of intracellular pathogen biology: the initial uptake into phagocytic cells, which may impact on the subsequent trafficking, and the later phagosome-lysosome fusion events, which are manipulated by M. tuberculosis and Salmonella to promote their survival. We have used state-of-the-art cell-based screening techniques with libraries of small-inhibitory RNA (siRNA) molecules and pharmaceutically active compounds to identify potential macrophage targets, and combined this with the power of systems biology to analyse and integrate the different data sets. We have identifying genetic targets that can be manipulated by siRNA and enzymes that can be modulated with small molecules, which will be taken forward in studies to investigate their suitability as targets and drugs for antimicrobial therapy. A spin-off has been the identification of compounds that interfere with eukaryotic cell proliferation, and may have a role in cancer therapy. We identified bacterial mutants with altered trafficking in human macrophages, and these will form the basis of future work to examine the host macrophage transcriptional response to mutant and wild type bacteria.
We further developed methods for the automated modelling of dynamic systems (in the form of ordinary differential equation) using experimental data and expert knowledge, a typical task in systems biology modelling. We used these automated methods to model endosome maturation and LDL trafficking, using time-course data on protein concentrations. We also applied machine learning methods for structured output prediction and predictive clustering to a large number of datasets generated from high-throughput screens carried out during the project.
Molecules including small GTPases and phosphoinositides regulate phagosome maturation. We identified a surprising interaction between apparently antagonistic enzymes, and used mathematical modelling to investigate this. We found that complex formation can produce novel forms of switch-like and bell-shaped responses, and postulate this could have a functional role in the temporal regulation of phosphoinositides during phagosome maturation.
Expected final results and potential impacts
As envisaged in the original project aims we have identified a number of hits that may have impact, subject to further development, as therapeutic agents. These may have impact in the treatment of a number of diseases, including cancer, autoimmune disease, and bacterial infection, and the results will be exploited by the respective partner institutions.
We identified inhibitors of MHC class II expression, specifically one that down-regulated gamma-interferon mediated increase in MHC class II expression. The exact molecular mechanism is as yet unknown, this approach may be of interest for controlling autoimmune responses that are mediated and maintained by MHC class II expression, such as multiple sclerosis and colitis. The current hits have the advantage of not affecting normal MHC class II expression, making side effects less likely.
Partners previously identified the host kinase Akt1 as key in determining the fate of intracellular bacteria. In this project inhibitors of Akt1 with increased potency and specificity were developed. These Akt1 inhibitors could have application in the control of bacterial infections but are also important in oncology where Akt1 is often activated by other factors. A spin-off of this was the identification of another kinase not only essential in the normal activation and proliferation of myeloid cells but also often upregulated in some leukemias. Compounds were identified that interfere with kinase activity and cell proliferation. Some of these activities will be take forward in Spin-out companies.
Significant advances were also made in data analysis tools that have application in other areas of systems biology, where disparate types of date need to be integrated and interrogated together to leverage the maximum output.
The project has resulted in 14 publications so far, with several more in preparation. These cover all aspects of the project, and it is anticipated that some will have significant impact in their respective fields. The systems biology methods are applicable to a broad range of biomedical fields. We have used open access publishing where possible to maximize the availability of our findings to interested researchers.
We identified 31 dissemination activities involving over 2,000 people at which various aspects of the work were presented to national and international audiences of scientists and the general public.