Rheinische Friedrich-Wilhelms-Universität Bonn

 AG  Haas

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The Projects - an Overview

This lab is studying phagosome biogenesis in health and disease. As for disease, our ‘favourite pathogens’ are currently Rhodococcus equi (causes severe bronchial problems in foals and can be life-threatening to immunosuppressed humans), Salmonella typhimurium and Mycobacterium bovis BCG (the former tuberculosis vaccination stran, a model bacterium for M. tuberculosis). These bacteria interfere with the development of a phagolysosome from their surrounding phagosome. The following questions are of particular interest to us:

(1) How does phagolysosome formation normally occur, i.e., when the ingested microorganism is not interfering? Even here, many basic questions are still unsolved! For example, which roles do the pathogen’s surface molecules have in signalling from the outside of the macrophage? How is the phagocytic cup closed, how are these organelles transported, what are the regulatory proteins, how precisely is the phagosome acidified?

(2) Which macrophage compartment does each of the selected intracellular bacteria inhabit? What is its composition and how can the pathogens cause abnormal phagosome development? What do these pathogens produce, so that their phagosomes mature abnormally? How do such bacterial factors interact with the host cell?

(3) What happens to a phagosome once the host cell is activated by factors that stimulate immune responses (such as interferon-gamma)? In some cases, it has been reported that "inhibited" phagosomes are then turned into phagolysosomes. This is possibly the reason why the immune system activation can, eventually, cure most infectious diseases. Yet, enhanced phagolysosome formation as the killing mechanism no. 1 in activated macrophages does not eradicate all pathogens!

(4) How can we dissect phagosome biogenesis in a minimalist system? We have reconstituted cell-free phagosome-lysosome and phagosome-endosome fusion in a test tube and this allows us to ask questions like: Which proteins and lipids are involved in this particular fusion step? Which microbial molecules can interfere with this fusion? In which order do these factors function in phagolysosome formation?

Our Experimental Approaches

We are using a truly multidisciplinary, problem-oriented approach to solve the questions above. We are combining the tools of cell biology (analysis of the intracellular localization of bacteria by immunofluorescence, electron microscopy and immuno-EM) as well as of biochemistry (isolation of phagosomes and analysis of their composition; fusion of phagosomes with endocytic organelles in the test tube, determination of the presence of certain subcellular markers on phagosomes, cytosol fractionation), as well as of microbiology (biochemical and genetic manipulation of the bacteria and study of how that affects interaction with host cells) with tools of immunology (does "activation" of macrophages by immunomodulators change the rate of phagolysosome formation? How are microbial antigens presented? How are intracellular bacteria killed?) and we include biophysical tools in cooperations (interaction of bacterial proteins with target membranes; purification and mass spectrometry of bacterial surface lipids which divert regular phagosome trafficking).

Financial support for our research

The current financial support of our studies by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereiche 645 and 670) and the Bonner Forum Biomedizin is gratefully acknowledged.