The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins.

Andree Hubber,Camille Hardiman,Kohei Arasaki,Craig R Roy,Pietro De Camilli,Hiroki Nagai,Fubito Nakatsu,David Lambright,Zhao-Qing Luo

doi:10.1371/journal.ppat.1004222

Abstract

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.

Highlights

Intracellular pathogens co-opt host processes to facilitate pathogen survival and replication within the eukaryotic host
We show that one mechanism used to control the localization of early effectors containing PI4Pbinding domains is to utilize the host machinery at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites that is involved in regulating PI4P-levels on the PM
Our data indicate that the host enzyme PI4KIIIa is involved in generating a pool of PI4P that mediates the localization of multiple effectors with a conserved PI4P-binding motif, and that the ER-localized

Summary

Introduction

Intracellular pathogens co-opt host processes to facilitate pathogen survival and replication within the eukaryotic host. Many bacterial pathogens possess secretion systems that transport bacterial proteins into host cells to orchestrate their intracellular survival. After uptake by phagocytic host cells, the gram-negative bacterium Legionella pneumophila prevents transport of the vacuole in which it resides through the canonical endocytic pathway, and actively transforms this vacuole into an organelle that supports bacterial replication. Endoplasmic reticulum (ER)- derived vesicles are rapidly recruited to the plasma membrane (PM) -derived vacuole in which L. pneumophila resides after uptake by host cells [1]. These early secretory vesicles originating from the ER appear to fuse with the L. pneumophila vacuole [2,3,4]. Functional interactions between PM-localized tSNAREs present on the L. pneumophila-containing vacuole (LCV) and ER-localized SNARE Sec22b likely contribute towards this remodeling process [5,6]

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Results

Conclusion