Rheinische Friedrich-Wilhelms-Universität Bonn

 AG  Haas

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Our Current Studies

1. Maturation of Phagosomes Containing Rhodococcus equi

One major interest of this lab is the study of macrophages that have been infected with the Gram-positive bacterium Rhodococcus equi. R. equi is closely related to mycobacteria which cause such important diseases as leprosy or tuberculosis. In fact, R. equi can cause a tuberculosis-like disease in AIDS patients with a high mortality. While R. equi is a very appealing model for tuberculosis research, it is also a very interesting pathogen in its own right. In horse breeding, R. equi-caused bronchopneumonia of very young foals is a severe problem and a few thousand inhaled bacteria suffice to cause, often deadly, disease.

Importantly, R. equi also multiplies in an unusual macrophage compartment . As this laboratory has shown, this subcellular compartment is a phagosome that has been arrested in its maturation between an early endocytic and a late endocytic stage. R. equi-containing phagosomes do not acidify and do not fuse with lysosomes and both these features likely contribute to bacterial multiplication in the macrophages of affected individuals. Interestingly, some of the bacterial genes that are required for this pathogenic behaviour, are located on a virulence plasmid. Current work in the lab aims at taking apart the relevant genes on this plasmid and testing for their relevance in the infection process. Similarly, we are analysing mutated rhodocococci for loss of their virulence. We have already produced and identified several relevant mutants which are, e.g., mutated in production of unusual cell wall compounds and which, different from the normal pathogen, are frequently delivered to a phagolysosome. This more genetic and cellular study is complemented by biochemical analysis of purified rhodococcal surface lipids for their effects on phagosome biogenesis when presented on harmless particles to a macrophage.

In addition, we have shown that R. equi kills murine macrophages during infection by a process called ‘necrosis’ rather than by apoptosis (‘programmed cell death’). We are currently investigating the pathway by which the microbes kill mammalian cells and, again, have a look at the plasmid genes that promote host cell killing.

Transmission electron microscope photography showing a macrophage phagosome with two R. equi bacteria. Note abundant vesicular and membraneous material in the phagosome space (many of which can likely serve as food for the bacteria). Furthermore, host cell mitochondria can be seen on the right side next to the phagosome membrane. Picture taken from our publication Fernandez-Mora et al. (2005) Traffic 6: 635.

2. Cell-free Reconstitution of Membrane Fusion Events During Phagosome Biogenesis

We have recently reconstituted the biochemical fusion of isolated phagosomes with isolated endosomes and lysosomes in a test tube. The idea of such a system is to describe the processes during phagosome maturation in molecular terms and to analyze the factors which contribute to the inhibition of phagosome maturation by the specialized bacteria. We have recently shown that this cell-free fusion faithfully reproduced many of the features of this fusion seen in complete cells which demonstrates that we can really re-construct this membrane fusion reaction in vitro. We are now performing biochemical purification steps for host factors which help usually non-fusogenic phagosomes to become able to fuse in the cell free system. This will be most interesting not only from a basic research point-of-view, but might also lead to new strategies to fight intracellular bacteria by forcing maturation of their phagosomes to phagolysosomes using pharmacological means. Furthermore, we are very interested to analyse the contribution of special, rare lipids, the phosphoinositides, in the various fusion steps.

Taken from: U. Becken et al. (2010) PNAS USA 107: 20726