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PHAGOSYS Final Report 2012

Executive Summary

One of the first lines of defence against bacterial pathogens that enter our bodies are phagocytes such as macrophages and monocytes, that literally eat the bacteria and destroy them. However some pathogenic bacteria, including Salmonella and Mycobacterium tuberculosis, have evolved mechanisms whereby they can survive inside these very cells; one way in which they do this is to inhibit the endosomal fusion processes inside the macrophage that expose the bacteria to lethal factors. The aim of this project was to identify the mechanisms bacteria use to block killing by macrophages, and by doing so find ways to overcome these blocks and promote intracellular killing. Helping the macrophage to do its job in this way could be the basis for therapeutic treatments that would be added to conventional anti-microbial drug treatment. This could help shorten treatment times for diseases like tuberculosis, which currently requires 6-9 months of therapy. Since this type of therapy would enhance macrophage function as opposed to killing bacteria, it would be unlikely to lead to the emergence of further microbial drug resistance.

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 Mycobacterium 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.

Summary description of project context and objectives

Project context: Most bacteria that enter a host organism, by ingestion or inhalation, are engulfed and killed by phagocytic cells such as macrophages. However some pathogenic bacteria, including Salmonella and Mycobacterium tuberculosis, have evolved mechanisms whereby they can survive inside the very cells that our bodies have developed to kill the pathogens. The overall aim of this project was to try and identify how these bacteria prevent killing by macrophages, and by so doing find ways to overcome this block and promote intracellular killing. Helping the macrophage to do its job in this way could be the basis for therapeutic treatments that would be added to conventional anti-microbialdrug treatment. This could help shorten treatment times for diseases like tuberculosis, which currently requires 6-9 months of therapy, and since this would enhance macrophage function as opposed to killing bacteria, would be unlikely to lead to the emergence of further drug resistance.

This project was designed to target 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 used the power of systems biology to analyse and integrate the different data sets. We report here on our success in identifying genetic targets that can be manipulated by siRNA and enzymes that can be modulated with small molecules, coupled with improved systems biology techniques that allow the interrogation of complex data sets.

Project objectives: The first objective of the project was to develop models of phagocytosis, based on existing expert knowledge and on data collected within the project or already available in the literature. This required the development of methods for automated modelling able to make use of both data and expert knowledge. Major progress was made towards achieving these goals, but some changes were required due to unforeseeable personnel issues, which meant that the scope of the models constructed was more limited than originally planned. 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 by the project partners. 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.

The experimental objectives of the project ran in parallel to the modelling and we set out to characterise the bacteria-containing compartments, and determine the kinetics of intracellular phagosomal and endosomal trafficking. This was done using a combination of high throughput fluorescence microscopy cell-based assays and a novel quantitative multiparametric analysis developed at MPI-CBG. We successfully developed the required high-throughput, high-content screening assays using both murine and the more physiologically relevant primary human macrophages. The mycobacterial infection assay was optimized, combining recombinant Mycobacterium bovis bacillus Calmette-Guerin (BCG) expressing Green Fluorescent Protein, with LysoTracker® Red DND-99 staining of host-cell lysosomes. This provided a direct readout of the release of the phagosomal maturation block and degradative delivery of the bacteria to lysosomes when the system was perturbed with either siRNAs or small molecules.

The optimised assays and protocols were then used to identify, using RNAi or small molecules, the 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. It proved impracticable to cover the entire druggable genome of ~7000 targets, but using more targeted chemical and geneti c approaches we have identified a number of promising hits that act on the host-cell and not the bacterium, and which are now being taken forward in studies that will continue beyond this project.

We also set out to identify the bacterial components that mediate the interactions with the macrophage, preventing phagosome-lysosome fusion, and therefore promoting bacterial survival. The approach was to make use of the mycobacterial mutants that have been selected on the basis of their inability to block phagosome-lysosome fusion during macrophage infection in vitro, and reported in the literature. A panel of mutants was collected and tested in human type 1 and type 2 macrophages, but only four mutants showed the same phenotype as originally reported. This is probably due to differences in the type of macrophage used in the original screens; murine or transformed cell lines are commonly used, but 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.

Overall we made significant progress towards achieving the main objective of the program, which was to use 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. To do this we optimised screening protocols, data analysis methods and systems biology with which to integrate and interrogate the large volumes of data generated. Target and pathway identification is underway and will continue beyond the life of the current project.

Description of main S & T results/foregrounds

Potential impact and main dissemination activities and exploitation results

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 have 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. 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.

Address of project public website and relevant contact details

PHAGOSYS Periodic Report 2012

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