Characterization of Legionella virulence mechanisms

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 was 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. Our latest studies revealed that the protein SidD from L. pneumophila is a novel modulator of the host protein Rab1. Rab1 is a molecular switch that can increase or reduce the amount of vesicles transported between membrane-bound compartments within eukaryotic cells. Rab1 is critical to cellular function and viability, and its misregulation can cause 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 Legionella proteins on the activity of Rab1. We discovered that the effector protein SidD has a novel enzymatic activity that allows Legionella to precisely control the timing of Rab1 exploitation during infection. The results from our studies not only increased our understanding of the complexity of bacterial infections but will also help to learn more about the mechanism how proteins may be regulated within our own cells, and how their misregulation causes disease.