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.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2011
Total Cost
$704,580
Indirect Cost
City
State
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Zip Code
Tang, Yanyang; Qiu, Ji; Machner, Matthias et al. (2017) Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays. Curr Protoc Cell Biol 74:15.21.1-15.21.14
Lin, Yi-Han; Machner, Matthias P (2017) Exploitation of the host cell ubiquitin machinery by microbial effector proteins. J Cell Sci 130:1985-1996
Romano-Moreno, Miguel; Rojas, Adriana L; Williamson, Chad D et al. (2017) Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL. Proc Natl Acad Sci U S A 114:E11151-E11160
Machner, Matthias P; Storz, Gisela (2016) Infection biology: Small RNA with a large impact. Nature 529:472-3
Lin, Yi-Han; Doms, Alexandra G; Cheng, Eric et al. (2015) Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX. J Biol Chem 290:25766-81
Morrissette, Naomi S; Machner, Matthias P (2015) Ingenious strategies of microbial pathogens. Mol Biol Cell 26:1007
Yu, Xiaobo; Decker, Kimberly B; Barker, Kristi et al. (2015) Host-pathogen interaction profiling using self-assembling human protein arrays. J Proteome Res 14:1920-36
Lucas, MarĂ­a; Gaspar, Andrew H; Pallara, Chiara et al. (2014) Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5. Proc Natl Acad Sci U S A 111:E3514-23
Gaspar, Andrew H; Machner, Matthias P (2014) VipD is a Rab5-activated phospholipase A1 that protects Legionella pneumophila from endosomal fusion. Proc Natl Acad Sci U S A 111:4560-5
Hammond, Gerald R V; Machner, Matthias P; Balla, Tamas (2014) A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 205:113-26

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