Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

Xavier Charpentier,Jing W Zhu,Howard A Shuman,Joëlle E Gabay,Moraima Reyes,Arthur Weiss,Craig R Roy

doi:10.1371/journal.ppat.1000501

Abstract

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions.

Highlights

Legionella pneumophila is the causative agent of the acute pneumonia known as Legionnaires’ disease [1,2]
To identify the host cell function required for activity of the Icm/Dot system we used a small molecule-mediated perturbation strategy called chemical genetics
We identified host cell factors required for L. pneumophila phagocytosis

Summary

Introduction

Legionella pneumophila is the causative agent of the acute pneumonia known as Legionnaires’ disease [1,2]. The Icm/Dot Type IVB secretion system is required for avoiding phagosome-lysosome fusion and for intracellular multiplication [6,7]. The Icm/Dot system mediates translocation of multiple effector proteins that are responsible for transforming the nascent Legionella phagosome into a replicative compartment, called the Legionella-containing vacuole (LCV) [8]. Most effector proteins have uncharacterized functions, some have been studied in detail and target multiple host cell processes important for the intracellular survival of L. pneumophila [8,10]. The early requirement of a functional Icm/Dot system suggests that effectors must be rapidly translocated upon encounter of the host cell in order to alter trafficking of the newly-formed phagosome and prevent its fusion with the lysosome [21,22]

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Results

Discussion

Conclusion