Legionella effector AnkX interacts with host nuclear protein PLEKHN1

Xiaobo Yu,Matthias P Machner,M Ramona Neunuebel,Kristi Barker,Jeffrey L Caplan,Barbara P Romero Dueñas,Rebecca R Noll,Ji Qiu,Joshua Labaer,Samual C Allgood

doi:10.1186/s12866-017-1147-7

Abstract

BackgroundThe intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires’ disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX’s interaction with host proteins and the means through which it exerts its cellular function is limited.ResultsHere, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491–809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response.ConclusionsAltogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.

Highlights

The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires’ disease
nucleic acid programmable protein array (NAPPA)-based identification of host binding partners for L. pneumophila effector AnkX To identify potential interactions between AnkX and human proteins, we used a proteome array method we recently established to efficiently and reliably pinpoint human proteins bound by L. pneumophila effectors [18, 26]
As we have recently demonstrated, NAPPA efficiently identifies Ras in the brain (Rab) Guanosine triphosphatase (GTPase) bound by the L. pneumophila effector proteins SidM and LidA [18]

Summary

Introduction

The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires’ disease. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. Bacterial pathogens have the ability to thrive in cells of the innate immune system (macrophages, neutrophils, and dendritic cells), which are normally destined to ingest and digest microbes [1]. Survival and replication of L. pneumophila within the Legionella-containing vacuole (LCV) relies on the translocation of over 300 effector proteins into the host cytosol via a Type IVB secretion system (T4SS) encoded by the defect in organelle trafficking/ intracellular multiplication (dot/icm) genes [3, 6,7,8]. Such mutants reside in phagosomes that enter the canonical endosomal maturation pathway

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