Characterization of Legionella effector proteins

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

In order to determine the function of uncharacterized L. pneumophila effector proteins a protocol for their recombinant production and purification had to be established. For protein production, the open reading frames of selected L. pneumophila effector proteins were introduced into the bacterial strain Escherichia coli, an expression host commonly used in laboratories for the production of recombinant proteins. The L. pneumophila effector proteins were subsequently isolated from E. coli lysate through binding of their tag to an affinity resin. Yield and stability of the harvested effector proteins was determined by gel matrix separation (SDS PAGE) and the proteins were stored in a frozen state for further analyses. The L. pneumophila effector LidA has previously been shown to interact with various host cell proteins of the Rab family of GTPases. These Rab proteins are molecular switches that can increase or reduce the amount of vesicles transported between membrane-bound compartments within eukaryotic cells. Many of these pathways are critical to cellular function and viability, and their misregulation has been identified as cause for various genetic disorders in humans. L. pneumophila has been found to benefit from host cell vesicle transport processes. The pathogen hijacks transport vesicles from selected trafficking routes in order to transform its surrounding vacuole into a compartment that mimics host cell organelles. To understand the molecular details of vesicle hijacking we studied the effect of LidA on Rab GTPases in vitro and determined the importance of Rab function for L. pneumophila virulence using tissue culture infection studies combined with fluorescence microscopy. The results from our ongoing studies will increase our understanding of the complexity of bacterial infections and will help to learn more about the role of certain host-pathogen interaction for L. pneumophila virulence.