Legionella effector AnkX interacts with host nuclear protein PLEKHN1

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.Electronic supplementary materialThe online version of this article (10.1186/s12866-017-1147-7) contains supplementary material, which is available to authorized users.

Bacterial Factors Targeting the Nucleus: The Growing Family of Nucleomodulins

“…L. pneumophila also secretes AnkX, which may have other functions related to the manipulation of nuclear factors. Under ectopically expressed conditions, mCherry-AnkX exhibits a punctuated distribution in the nucleus and co-localizes with PLEKHN1, a poorly characterized protein that localizes in nuclear speckles and cytosolic puncta [104] (Figure 2A).…”

Bacterial nucleomodulins and cancer: An unresolved enigma

“… 29 AnkH [84] L. pneumophila Interferes with host cell transcription (T4SS). 30 AnkX [91] L. pneumophila Interacts with host nuclear protein PLEKHN1, manipulating inflammation (T4SS). 31 SnpL [74] L. pneumophila Targets host RNA polymerase II and regulates host gene expression (T4SS) 32 LntA [ 50 ] Listeria monocytogenes Targets host cell chromatin repressor BAHD1 and stimulates interferon genes.…”

Bacterial nucleomodulins: A coevolutionary adaptation to the eukaryotic command center

“…Recently, AnkX was discovered to colocalize with host cell PLEKHN1 in the nucleus. PLEKHN1 is an endogenous cell protein found to speckle the inside of HEK293T cell nuclei [ 3 , 146 ]. During ectopic expression, the central region of AnkX was necessary for the targeting and binding of PLEKHN1 [ 146 ].…”

“…PLEKHN1 is an endogenous cell protein found to speckle the inside of HEK293T cell nuclei [ 3 , 146 ]. During ectopic expression, the central region of AnkX was necessary for the targeting and binding of PLEKHN1 [ 146 ]. While little is known regarding the consequences of AnkX-PLEKHN1 interactions, PLEKHN1’s association with various proteins involved in the inflammatory response has led to the hypothesis that AnkX potentially functions as a novel nucleomodulin capable of simultaneously preventing LCV-lysosomal fusion in the cytosol and manipulating host inflammatory response from within the nucleus [ 146 ].…”