Ehrlichia chaffeensis TRP120-mediated ubiquitination and proteasomal degradation of tumor suppressor FBW7 increases oncoprotein stability and promotes infection.

Ehrlichia chaffeensis (E. chaffeensis) exploits evolutionarily conserved Notch and Wnt host cell signaling pathways to downregulate innate immune host defenses and promote infection. The multifunctional E. chaffeensis TRP120 effector which has HECT E3 ubiquitin ligase activity, interacts with the host nuclear tumor suppressor F-BOX and WD domain repeating-containing 7 (FBW7). FBW7 is the substrate recognition subunit of the Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex (SCF) known to negatively regulate a network of oncoproteins (Notch, cyclin E, c-Jun, MCL1 and cMYC). In this study, we demonstrate that TRP120 and FBW7 colocalize strongly in the nucleus by confocal immunofluorescent microscopy and interactions between TRP120 and FBW7 FBOX and WD40 domains were demonstrated by ectopic expression and co-immunoprecipitation. Although FBW7 gene expression increased during E. chaffeensis infection, FBW7 levels significantly decreased (>70%) by 72 h post infection. Moreover, an iRNA knockdown of FBW7 coincided with increased E. chaffeensis infection and levels of Notch intracellular domain (NICD), phosphorylated c-Jun, MCL-1 and cMYC, which are negatively regulated by FBW7. An increase in FBW7 K48 ubiquitination was detected during infection by co-IP, and FBW7 degradation was inhibited in infected cells treated with the proteasomal inhibitor bortezomib. Direct TRP120 ubiquitination of native and recombinant FBW7 was demonstrated in vitro and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. This study identifies the tumor suppressor, FBW7, as a TRP120 HECT E3 Ub ligase substrate, and demonstrates that TRP120 ligase activity promotes ehrlichial infection by degrading FBW7 to maintain stability of Notch and other oncoproteins involved in cell survival and apoptosis.

Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response.

Ehrlichia chaffeensis is an obligately intracellular bacterium that establishes infection in mononuclear phagocytes through largely undefined reprogramming strategies including modulation of host gene transcription. In this study, we demonstrate that the E. chaffeensis effector TRP47 enters the host cell nucleus and binds regulatory regions of host genes relevant to infection. TRP47 was observed in the nucleus of E. chaffeensis-infected host cells, and nuclear localization was dependent on a variant MYND-binding domain. An electrophoretic mobility shift assay (EMSA) demonstrated that TRP47 directly binds host DNA via its tandem repeat domain. Utilizing chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) with E. chaffeensis-infected cells, TRP47 was found to bind at multiple sites in the human genome (n = 2,051 at p < 10-30). Ontology analysis identified genes involved in functions such as immune response, cytoskeletal organization, and signal transduction. TRP47-bound genes included RNA-coding genes, many of these linked to cell proliferation or apoptosis. Comparison of TRP47 binding sites with those of previously-identified E. chaffeensis nucleomodulins identified multiple genes and gene functional categories in common including intracellular transport, cell signaling, and transcriptional regulation. Further, motif analysis followed by EMSA with synthetic oligonucleotides containing discovered motifs revealed a conserved TRP47 DNA-binding motif. This study reveals that TRP47 is a nucleomodulin that enters the nucleus via a MYND-binding domain and appears to play a role in host cell reprogramming by regulation of transcription.

Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation.

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 auto-activates 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 tri-tyrosine 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 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=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, 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.