Ehrlichia chaffeensis TRP32 Nucleomodulin Function and Localization Is Regulated by NEDD4L-Mediated Ubiquitination.

Tierra R Farris,Jere W Mcbride,Jennifer Y Wang,Bing Zhu

doi:10.3389/fcimb.2017.00534

Tierra R Farris, Jere W Mcbride + Show 2 more

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https://doi.org/10.3389/fcimb.2017.00534

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Abstract

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate various host cell processes. In a previous study, we reported that the E. chaffeensis nucleomodulin TRP32 regulates transcription of host genes in several biologically relevant categories, including cell differentiation and proliferation. In this study, we investigate the effect of ubiquitination on TRP32 function and localization within the host cell. TRP32 is both mono- and polyubiquitinated on multiple lysine residues during infection and when ectopically expressed. Despite lacking a canonical PPxY motif, TRP32 interacted with, and was modified by the human HECT E3 ubiquitin (Ub) ligase NEDD4L. TRP32 ubiquitination was not by K48-linked polyUb chains, nor was it degraded by the proteasome; however, TRP32 was modified by K63-linked polyUb chains detected both in the cytosol and nucleus. HECT ligase inhibitor, heclin, altered the subnuclear localization of ectopically expressed TRP32 from a diffuse nuclear pattern to a lacy, punctate pattern with TRP32 distributed around the periphery of the nucleus and nucleoli. When a TRP32 lysine null (K-null) mutant was ectopically expressed, it exhibited a similar phenotype as single lysine mutants (K63R, K93R, and K123R). However, the K-null mutant showed increased amounts of cytoplasmic TRP32 compared to single lysine mutants or heclin-treated cells ectopically expressing TRP32. These alterations in localization corresponded to changes in TRP32 transcriptional repressor function with heclin-treated and single lysine mutants unable to repress transcription of a TRP32 target genes in a luciferase assay.

Highlights

Ehrlichia chaffeensis is a gram-negative, obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis
Ub-positive bands that coincided with detection of TRP32 (Figure 2B, panel 1) were detected at ∼37, 40, ∼60, and ∼100 kDa by the FK2 antibody (Figure 2B, panel 2); polyubiquitinated TRP32 was detected at ∼60 and 100 kDa by the FK1 antibody
We identified TRP32 as a nucleomodulin that regulated transcription of host genes related to cellular differentiation and proliferation (Farris et al, 2016)

Summary

Introduction

Ehrlichia chaffeensis is a gram-negative, obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. E. chaffeensis TRP32 Regulated by Ubiquitination reprograms various host cell processes is not fully understood; a group of type 1 secreted, tandem repeat protein (TRP) effectors similar to the repeats-in-toxin family of exoproteins are involved. The most well-characterized TRP effectors, TRP120 and TRP32, interact with many host cell targets, directly activate cell signaling pathways, and activate/repress host cell transcription. TRP120 interactions with ADAM17 on the host cell surface activate the Notch pathway, resulting in the downregulation of innate immune toll-like receptors (Lina et al, 2016a). TRP120 binds a GC-rich motif, leading to upregulation of specific host genes involved in transcriptional regulation, signal transduction, and apoptosis (Zhu et al, 2011). TRP32 binds a G-rich motif consisting of imperfect GGTGGC-like sequence repeats, but preferentially targets genes regulating cell proliferation and differentiation. TRP32 was shown to activate and repress expression of targets in a gene-specific manner during infection and in a luciferase reporter assay (Luo and McBride, 2012; Farris et al, 2016)

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