Ehrlichia chaffeensis TRP32 Is a Nucleomodulin That Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation

Abstract

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also autoactivates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal trityrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA-binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we determined that TRP32 binds a G-rich motif primarily within ؎500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing as primary TRP32 targets. TRP32-bound genes (n ‫؍‬ 1,223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as those encoding FOS, JUN, AKT3, and NRAS and noncoding RNA genes microRNA 21 (miRNA 21) and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

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

“…Neither of these phenotypes were due to accumulation of undegraded cellular protein as treatment with the proteasome inhibitor bortezomib did not alter cellular localization of TRP32 (Figure 4B ). In order to test this possibility, we performed a cellular assay using a firefly luciferase reporter expressed under control of the TRP32 target promoters in the presence or absence of these inhibitors (Farris et al, 2016 ). We found that Pyr41 caused drastic decreases in expression of both the constitutively transcribed Renilla luciferase and the firefly luciferase suggesting that this inhibitor broadly dysregulated transcription (data not shown).…”

“…TRP32 also binds a G-rich motif consisting of imperfect GGTGGC-like sequence repeats, but preferentially targets genes regulating cell proliferation and differentiation. TRP32 was also 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 ).…”

“…A common theme among bacterial pathogens is the hijacking of host post-translational machinery to modify effectors (Ribet and Cossart, 2010 ; Ravikumar et al, 2015 ; Popa et al, 2016 ). E. chaffeensis effectors are phosphorylated, ubiquitinated, and SUMOylated by host enzymes (McBride et al, 2011 ; Wakeel et al, 2011 ; Dunphy et al, 2014 ; Farris et al, 2016 ; Zhu et al, 2017 ), and these PTMS are important for effector function. TRP120 ubiquitination and SUMOylation is required for interactions with host proteins, and TRP32 tyrosine phosphorylation plays a role in its nuclear localization.…”

Bacterial Factors Targeting the Nucleus: The Growing Family of Nucleomodulins

“…These observations support the hypothesis of large-scale transcriptional alteration induced by Ank200 via mechanisms associated with the regulation of Alu-Sx elements. TRP32 and TRP120 bind to host-cell DNA at G-rich and G + C-rich motifs, respectively, as well as chromatin-associated proteins, such as histone methylases and demethylases, polycomb-group (PcG) proteins and other components of chromatin-remodeling complexes [43][44][45][46][47][48]. TRP120 acts as a transcriptional activator for host genes [44], while TRP32 activates or represses expression of its target genes [46,47].…”

“…Ehrlichia TRP32 is regulated by different modifications. Tyrosine phosphorylation controls its nuclear localization [47], while ubiquitination on lysine 63, catalyzed by the host cell protein NEDD4L, is required for TRP32 transcription factor function and subnuclear localization [111]. For TRP120, ubiquitination and sumoylation are critical in establishing its eukaryotic target landscape and thus, function [51,52].…”