Modulation of the host small GTPase Rab1 function by Legionella pneumophila effectors

Yunhao Tan, Purdue University

Abstract

The vacuolar pathogen Legionella pneumophila utilizes the Dot/Icm apparatus to deliver numerous effectors into the host cytosol where they function to establish a niche permissive for bacterial replication by subverting the host membrane trafficking. Since membrane trafficking events are highly conserved between mammalian cells and the budding yeast Saccharomyces cerevisiae, we performed a yeast-based screen to identify L. pneumophila effectors that modulate the host secretory pathways. This led to the identification of AnkX, which is toxic to yeast and disrupts the host membrane trafficking. Taking advantage of its yeast lethal phenotype, we have identified the N-terminal Fic domain is essential to the function of AnkX. Exemplified by Ypt1, the yeast homologue of the mammalian small GTPase Rab1, several yeast genes involved in vesicle transport could suppress the AnkX mediated yeast toxicity. Mass spectrometry analysis of Rab1 co-expressed with AnkX in yeast revealed that this small GTPase is covalently modified with the phosphorylcholine (PC) moiety at Ser76. In vitro biochemical assay verified that AnkX is a PC transferase that uses CDP-choline as a substrate to target Rab1 in a Fic dependent manner. Phosphorylcholination (PCylation) of Rab1 renders it less accessible to both guanine nucleotide exchange factor (GEF) and GTPase activation protein (GAP). While another bacterial effector Lem3 antagonizes the activity of AnkX by catalyzing the dephosphorylcholination (dePCylation) reaction to remove the PC moiety from Rab1 thereby restoring the normal function of this small GTPase. Together, our results imply that reversible PCylation is a novel virulence strategy used by L. pneumophila to modulate Rab1function. One hallmark during the biogenesis of the Legionella containing vacuole (LCV) is the recruitment of the host small GTPase Rab1 by the coordinated action of several effector proteins. Multifunctional effector SidM activates Rab1 and locks it into an active state with its guanine nucleotide exchange factor (GEF) and AMPylation activities. LepB is a bacterial GTPase activation protein (GAP) which participates in the Rab1 removal process by antagonizing the GEF activity of SidM. However, the inaccessibility of AMPylated Rab1 to LepB indicates the existence of an unknown factor functioning upstream of LepB to deAMPylate Rab1. Here we reported that SidD could suppress the yeast toxicity mediated by SidM. In vitro biochemical analysis and infection data revealed that SidD is a deAMPylase that removes the AMP moiety from Rab1 to facilitate its release from the LCV. Our results suggest that AMPylation is a reversible process controlling by dedicated enzymes. Overall, by studying L. pneumophila effectors targeting the host small GTPase Rab1, my thesis research led to the discovery of three novel post-translational modifications employed by the bacterium to modulate the function of this small GTPase, namely, PCylation, dePCylation and deAMPylation. These findings not only increase our understanding of L. pneumophila pathogenesis but also provide important insight into general host cell biology in that PCylation and AMPylation have been observed in eukaryotic cells under physiological condition. I therefore expect future studies on effector functions in the field of host and pathogen interaction would lead to more and more exciting discoveries with regards to the virulence strategies used by pathogens and the regulatory mechanisms in the eukaryotic cell biology..

Degree

Ph.D.

Advisors

Luo, Purdue University.

Subject Area

Biology|Microbiology

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