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Machner Lab: Unit on Microbial Pathogenesis

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The goal of our research is to obtain a detailed understanding of the molecular mechanisms used by the pathogenic bacterium Legionella pneumophila to manipulate host cell processes during infection.

L. pneumophila is a Gram-negative bacterium that is commonly found within freshwater reservoirs as a natural parasite of amoeba. When inhaled by humans L. pneumophila can cause a severe pneumonia known as Legionnaires' disease. The pathogen establishes a replication vacuole within infected cells that avoids lysosomal fusion. The organism hijacks cargo vesicles of the infected host cell and transforms its phagosome into specialized membrane compartment that resembles host cell rough endoplasmic reticulum (ER). Within this protective niche L. pneumophila can replicate to high numbers, eventually lyse the spent host cell and infect neighboring cells.

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Figure 1. Infection Cycle of L. pneumophila

L. pneumophila delivers a large number of effector proteins into the host cell via a type IV secretion system (T4SS) named Dot/Icm. Little is known about the molecular function of these translocated substrates. Bacterial mutants with a defective Dot/Icm system are avirulent, underscoring the importance of the effector proteins for L. pneumophila pathogenesis. The main goal of our research is to characterize the function of L. pneumophila effector proteins and their effect on host cell processes.

Upon uptake by the host cell, L. pneumophila rapidly recruits cargo vesicles to the surface of its Legionella-containing vacuole (LCV) to create a camouflaged compartment that supports bacterial replication. One of the vesicle routes intercepted by L. pneumophila is the early secretory pathway between the endoplasmic reticulum (ER) and the Golgi compartment. Rab1, a key regulator of this pathway, transiently localizes to the LCV, suggesting that L. pneumophila utilize Rab1 in order to redirect secretory vesicle traffic to its LCV.

We recently discovered that the effector protein SidM is critical for Rab1 exploitation. SidM combines the activity of a GDI displacement factor (GDF) and a guanine nucleotide exchange factor (GEF). SidM mediates dissociation of Rab1 from its chaperone GDP dissociation inhibitor (GDI) and Rab1 activation by catalyzing exchange of GDP against GTP in Rab1. Bacterial mutants lacking SidM are defective for Rab1 recruitment, indicating that SidM is essential to exploit the pool of GDI-bound Rab1 during infection. SidM is the first example of a protein shown to combine GDI displacement and nucleotide exchange activity. It remains to be seen if other prokaryotic and eukaryotic proteins exist that share this remarkable ability with SidM.

In addition to its GEF/GDF activity, SidM also catalyzes the covalent attachment of adenosine monophosphate (AMP) to tyrosine-77 of Rab1, a process known as AMPylation. AMPylated Rab1 is locked in the active conformation because it cannot be inactivated. This process may allow L. pneumophila to efficiently exploit cellular cargo vesicle transport without interference by host proteins that would otherwise deactivate Rab1.

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Figure 2. Model for Rab1 Exploitation by Legionella Effector Proteins

Two to three hours after bacterial uptake the host factor Rab1 eventually disappears from the LCV. The molecular details underlying this removal were unclear. The Legionella effector LepB was believed to inactivate Rab1 by triggering GTP hydrolysis, followed by extraction of Rab1 from the LCV membrane by GDI (which binds only inactive Rab1). LepB, however, is unable to inactivate Rab1 in its AMPylated form, leaving open the question of how L. pneumophila can remove AMPylated Rab1 from its LCV.

We now discovered that L. pneumophila, in addition to encoding the AMPylase SidM, also produces the effector protein SidD which catalyzes AMP removal from Rab1, a process known as de-AMPylation (or de-adenylylation). Once AMP has been removed by SidD, de-AMPylated Rab1 is accessible for inactivation by LepB. L. pneumophila mutants lacking SidD showed a prolonged colocalization with host cell Rab1, demonstrating that SidD is required for the timely removal of Rab1 from LCVs.

The identification of SidD as Rab1 de-AMPylase not only provides the missing link in a complex chain of Rab1 modulation events during L. pneumophila infection, but also preresents an intriguing example for how prokaryotic and eukaryotic cells could regulate protein function through reversible AMPylation.

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Last Updated Date: 11/30/2012
Last Reviewed Date: 11/30/2012

Contact Information

Name: Dr Matthias Machner
Tenure Track Investigator
Unit on Microbial Pathogenesis
Phone: 301-435-8417
Fax: 301-402-0078
Email: machnerm@mail.nih.gov

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