Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Regulates Host Gene Expression and Post-Translational Modifications Determine its Localization and Function

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. Previously, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also auto-activates reporter gene expression in yeast. In these studies, I demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription and which is regulated by various post-translational modifications including phosphorylation and ubiquitination. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal tri-tyrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. I also show that TRP32 is mono and poly-

ubiquitinated on multiple lysine residues during infection by the host E3 Ub ligase NEDD4L. When TRP32 poly-ubiquitin chains were examined by immunoblotting K63-linked chains were detected but not K48 or K11-linked chains and TRP32 was not responsive to treatment with the proteasome inhibitor carfilzomib. An electromobility shift assay (EMSA) demonstrated TRP32 binding to host DNA 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 ChIP-Seq, I 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=1223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and non-coding RNA genes, miRNA 21 and miRNA 142. TRP32 target genes were differentially regulated during infection, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay. Additionally, I show that treatment with the HECT-ligase inhibitor, heclin, alters TRP32 subnuclear localization and impairs TRP32’s ability to repress transcription of target genes in a luciferase assay and that this can be phenocopied using K63R and K123R mutants of TRP32.

Description
Keywords
Ehrlichia chaffeensis, effectors, post-translational modification, nucleomodulin, ubiquitin, cellular microbiology, microbiolgy,
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