Background: The 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. Results: Here, 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. Conclusions: Altogether, 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.
- Nucleic acid programmable protein array
ASJC Scopus subject areas
- Microbiology (medical)
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Additional file 1: Table S1. of Legionella effector AnkX interacts with host nuclear protein PLEKHN1
Machner, M. P. (Contributor), Allgood, S. C. (Contributor), Noll, R. R. (Contributor), Qiu, J. (Contributor), Romero Dueñas, B. P. (Contributor), Barker, K. (Contributor), Caplan, J. L. (Contributor), Neunuebel, M. R. (Contributor), Yu, X. (Contributor) & LaBaer, J. (Contributor), figshare Academic Research System, Jan 5 2018
Yu, X. (Contributor), Noll, R. R. (Creator), Romero Dueñas, B. P. (Contributor), Allgood, S. C. (Contributor), Barker, K. (Creator), Caplan, J. L. (Creator), Machner, M. P. (Creator), LaBaer, J. (Creator), Qiu, J. (Creator) & Neunuebel, M. R. (Contributor), figshare Academic Research System, 2018