Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

ABSTRACT Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

IMPORTANCE E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.
nents of the Notch and Wnt signaling pathways (9,11). Recently, we reported that E. chaffeensis activates canonical and noncanonical Wnt signaling to facilitate host cell entry and exploits Wnt signaling to promote intracellular survival (10).
The Notch signaling pathway is evolutionarily conserved in eukaryotes and plays important roles in cell proliferation, differentiation, and apoptosis, thereby influencing cell fate (12)(13)(14)(15). Three proteolytic cleavage steps are essential for the production of fully functional Notch receptor signaling. The first occurs at site 1 (S1) by furin in the trans-Golgi (16,17), resulting in translocation of the heterodimer to the cell surface. The canonical Notch pathway is activated when the extracellular domain of Notch receptor (NECD) binds to the ligand (DLL1, -3, and -4 and Jagged 1 and -2) expressed on the membrane of neighboring cells. This receptorligand interaction results in the exposure of site 2 (S2) in Notch for cleavage by ADAM metalloproteases (18), resulting in NECD shedding and subsequent cleavage of the intracellular domain (NICD) by ␥-secretase enzyme (S3 cleavage) (19). NICD translocates to the nucleus, where it forms a triprotein complex with DNA-binding transcription factor RBPj (CSL) and transcriptional coactivator Mastermind (MAM), activating Notch target gene transcription (20,21). We previously demonstrated that TRP120 interacts with the ADAM17 metalloprotease (11) and also acts as a nucleomodulin, binding target genes associated with the Notch signaling pathway, including notch1 (9).
The Notch pathway is most often functionally associated with cell development and cancer but was recently recognized as an important regulator of innate and adaptive immune responses. The role of Notch signaling in inflammation, autophagy (22), apoptosis (23), Toll-like receptor (TLR) expression (24), T and B cell development (14), and major histocompatibility complex (MHC) class II expression (25) in different cells, including macrophages, has been reported. A role for Notch signaling during bacterial infection has been reported for Salmonella enterica serovar Typhimurium, Mycobacterium bovis, Bacillus anthracis, and Clostridium difficile, in which either the bacteria or the secreted toxins altered the Notch pathway to regulate inflammation in the host cell (26,27).
TLRs are type I transmembrane proteins, which play critical roles in the innate immune response by sensing a diverse set of microbial ligands known as pathogen-associated microbial patterns (PAMPs). TLR2 and TLR4 detect peptidoglycan (PGN) and lipopolysaccharide (LPS) and are the most well-characterized pattern recognition receptors (PRRs) (28). Interaction of PAMPS with the TLRs causes activation of multiple signaling pathways, including NF-B, extracellular signal-regulated kinase 1/2 (ERK1/ 2), Jun N-terminal protein kinase (JNK), and p38 mitogenactivated protein kinase (MAPK), which leads to induction of proinflammatory cytokine production and monocyte maturation (29,30). TLR2 and TLR4 play protective roles against. E. chaffeensis infection (25,31), and E. chaffeensis causes decreased expression of TLR2/4 by inhibiting the ERK1/2 and p38 MAPK pathways followed by downregulation of activity of PU.1, a transcription factor required for the expression of TLR2/4 (32)(33)(34). However, a mechanistic understanding of E. chaffeensis inhibition of ERK1/2 and p38 MAPK pathways and PU.1 is unknown. The TLR, ERK1/2, and p38 MAPK pathways are tightly regulated by multiple signaling pathways, such as integrin CD11b and immunoreceptor tyrosine-based activation-associated receptors (35,36). Recently, association of Notch signaling in modulation of ERK1/2 and regulation of TLR4-triggered cytokine production was reported (24).
The present study reveals a novel host-pathogen interaction whereby E. chaffeensis exploits the Notch signaling pathway to downregulate innate PRRs. We determined that the Notch signaling is activated by E. chaffeensis, and Notch activation is directly induced by the T1S effector TRP120. We further analyzed the underlying survival mechanism and showed that E. chaffeensis and TRP120-mediated activation of the Notch pathway causes inhibition of the ERK1/2 and p38 MAPK signaling pathways and expression of transcription factor PU.1, which represses TLR2/4 expression. This investigation is the first to demonstrate pathogen exploitation of Notch signaling to modulate PRR expression and to promote intracellular survival.

RESULTS
E. chaffeensis activates the canonical Notch pathway during infection. Using Y2H and chromatin immunoprecipitation sequencing (ChIP-seq), we previously reported that E. chaffeensis TRP120 interacts with ADAM17 and binds to the promoter region of notch1 (9,11). Since E. chaffeensis interacts with a component of the Notch signaling receptor complex, we sought to investigate whether E. chaffeensis exploits this pathway. Activation of Notch receptor following interaction with its ligand and proteolytic cleavage by the ADAM17 and ␥-secretase enzyme involves nuclear translocation of NICD (18,19). Immunofluorescence microscopy was used to measure NICD expression and localization in uninfected and E. chaffeensis-infected cells. NICD translocation to the nucleus was observed within 2 h of E. chaffeensis infection (Fig. 1A). Since nuclear translocation of NICD results in activation of specific Notch target genes (20,21), next the expression of different Notch signaling components and target genes were examined in E. chaffeensis-infected cells. The most important and well-characterized Notch target genes are the families of basic helix-loop-helix proteins, hairy and enhancer of split (Hes) and hairy and enhancer of split with YRPW motif (Hey) (37). These DNA binding proteins function as transcriptional repressors and are the primary effectors of Notch signaling. Reverse transcription-PCR (RT-PCR) data showed notch1, hes1, hes5, and hey2 mRNA expression was significantly increased as early as 2 h postinfection (p.i.), reaching a maximum at 72 h p.i. (Fig. 1B). Consistent with RT-PCR data, increased expression of Hes1 and ADAM17 protein was also observed by Western immunoblotting after 2 days p.i. The housekeeping protein ␣-tubulin was unchanged (Fig. 1C). Collectively, these results demonstrate that E. chaffeensis activates the canonical Notch signaling pathway during infection.
Analysis of Notch signaling pathway gene expression during E. chaffeensis infection. To understand the global effect of E. chaffeensis on the Notch signaling pathway and to examine ehrlichial activation of this pathway, a transcriptional analysis was performed to examine Notch-regulated gene expression. A human Notch signaling PCR array consisting of 84 genes, including Notch binding and receptor processing genes, the putative Notch target genes, genes from Sonic Hedgehog and Wnt receptor signaling pathways that cross talk with the Notch signaling pathway (see Fig. S1 in the supplemental material for gene table) was used. Heat maps were constructed depicting the differential expression of the Notch signaling pathway genes in the E. chaffeensis-infected and uninfected cells ( Fig. 2A). The intensities of the red and green in the heat map represent the levels of induction and repression, respectively. PCR array data identified activation of canonical Notch signaling pathway by E. chaffeensis at 12, 24, 48, and 72 h p.i. The expression patterns of genes that were consistently upregulated throughout all different time points included the Notch target genes hes1, hey2, NFB1, NFB2, nr4a2, pax5, fosl1, chuk, and ccne1 and Notch pathway component genes-e.g., notch1 (receptor), dll4 (ligand), and maml2 (transcription complex protein). The transcription factor gene rbpj and E3 ubiquitin ligase gene dtx1, which play important roles in Notch pathway activation and regulation, were upregulated at 48 and 72 h p.i. Only a small percentage of genes were downregulated during the infection, including genes for the Notch pathway components dll1 and mmp7 ( Fig. 2A). In Fig. 2B, the scatter plot shows the comparison of the normalized expression of all genes in the Notch PCR array between infected and uninfected cells. The central line indicates unchanged gene expression (2-fold regulation cutoff), the red dots represent the genes that were upregulated, the green dots represent genes that were downregulated, and the black dots represent genes with no significant difference in expression level. Although the gene expression patterns were similar throughout the different time points, maximum changes in Notch gene expression occurred at 24 h p.i. When the differential expression of these individual genes was analyzed, 38 genes showed significant differential expression (P Ͻ 0.05), including 28 (33.33%) that were upregulated and 10 (11.90%) that were downregulated (Fig. 2C). These results support canonical Notch signaling pathway activation during early and late phase of E. chaffeensis infection.
The Notch signaling pathway is required for E. chaffeensis survival. Notch signaling not only is required for cell growth and proliferation, but also plays important role in determining the fate of mature immune cells (14,15). Since the Notch pathway regulates both innate and adaptive immune responses, and this path-way is activated during E. chaffeensis infection, the role of this pathway in ehrlichial survival was examined. To that end, cells were treated with Notch signaling transcription complex inhibitor SAHM1 and ␥-secretase inhibitor DAPT {N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester}. SAHM1 is a cell-permeable small peptide that targets critical protein-protein interaction in the Notch transcription complex and prevents their assembly (38). The ␥-secretase inhibitor DAPT is a dipeptide and targets the C-terminal fragment of presenilin that is a component of ␥-secretase protein (39). THP-1 cells were treated with different concentrations of SAHM1 (1, 5, and 10 M) and DAPT (0.5, 1, and 5 g/ml), and cells were infected with cell-free E. chaffeensis (at a multiplicity of infection [MOI] of 50). A dose-dependent effect of both of inhibitors on bacterial load was observed as the percentage of ehrlichia-infected cells was determined using Diff-Quik staining (see Fig. S2A to S2D in the supplemental material). A significant decrease in bacterial load in cells treated with 10 M SAHM1 and 5 g/ml DAPT was observed; therefore, this concentration was used in additional experiments. There was a Ͼ50% decrease in percentage of infected cells after inhibition of the Notch signaling pathway at day 1 p.i. and Ͼ80% inhibition at day 2 p.i. (Fig. 3A), and real-time qPCR detected añ 90% decrease in bacterial load in the presence of Notch inhibitors (Fig. 3B). No apparent cell death or toxicity was observed with inhibitor treatment within the experimental window, as cell viability measured by trypan blue (data not shown). To obtain additional evidence supporting the role of Notch pathway in E. chaffeensis survival, small interfering RNAs (siRNAs) were used to knock down expression of the receptor Notch1, metalloprotease ADAM17, and transcription factor RBPj in THP-1 cells, and then the cells were infected with E. chaffeensis. These components were selected since they play critical role in canonical Notch signal transduction. Bacterial load was measured using real-time quantitative PCR (qPCR) by amplification of the integral ehrlichial gene dsb. Consistent with Notch pathway inhibitor experiments, significant reduction of bacterial load was also found in cells in which Notch component genes were knocked down (Fig. 3C). Protein expression of Notch1, ADAM17, and RBPj was reduced in siRNA-transfected cells compared with that in the control siRNA-transfected cells (Fig. 3D). Overall, the results obtained from pharmacological inhibitors and siRNA experiments support the importance of Notch signaling in E. chaffeensis survival.
E. chaffeensis TRP120 protein interacts with Notch receptor complex. E. chaffeensis activates canonical Notch signaling, which is requisite for survival. However, the mechanism of Ehrlichiainduced Notch activation remains undefined. E. chaffeensis TRP effectors are among the major immunoprotective proteins and contain species-specific epitopes (40). TRP120 functions as an adhesin, facilitating ehrlichial entry, and is a nucleomodulin (7). Moreover, our Y2H data showed TRP120 interacts with ADAM17 and binds to the promoter region of notch1 (9,11). We hypothesized that TRP120 interaction with the Notch receptor complex components is required for activation of the Notch pathway. In order to further examine the distribution and colocalization of ADAM17 with the TRP120-expressing ehrlichial inclusions, cells were stained with anti-ADAM17 and anti-TRP120 antibody. Immunofluorescence microscopy showed diffused cytoplasmic localization of ADAM17 in uninfected THP-1 cells; however, consistent with previous Y2H data, colocalization of ADAM17 with morulae expressing TRP120 was observed (Fig. 4A). Since Notch1 and ADAM17 are components of the receptor complex, colocalization of Notch1 with E. chaffeensis morulae was also examined. In contrast to the diffused cytoplasmic localization of Notch1 in uninfected cells, colocalization of Notch1 with TRP120-expressing morulae was observed (Fig. 4B). These data were further validated by transfecting HeLa cells with green fluorescent protein (GFP)-tagged TRP120 or GFP control plasmids. Colocalization of ADAM17 and TRP120 was observed (Fig. 4C), demonstrating that Notch receptor components are associated with ehrlichial vacuoles and confirms previous TRP120-ADAM17 interaction data.

TRP120 activates the canonical Notch signaling pathway.
To further examine the role of TRP120 in Notch pathway activation, TRP120-coated FluoroSpheres (sulfate microsphere beads) were used to stimulate the THP-1 cells. Notch activation involves proteolytic release of NICD from Notch by the furin, ADAM17, and ␥-secretase enzymes and translocation of NICD to the nucleus. Thus, the expression of NICD was used to monitor activation of Notch pathway. Since human monocytes constitutively express Notch receptors and ligands at basal levels, we confirmed NICD basal-level expression in the cytoplasm of untreated cells. However, within 5 min of stimulation, condensed expression of NICD was observed near the nucleus, which then translocated to nucleus within 15 min of stimulation (Fig. 5A). Since thioredoxin tag was used as the TRP120 fusion protein, cells were treated with thioredoxin as a control. Nuclear translocation of NICD was not observed in untreated cells or in cells treated with thioredoxin. To further delineate the role of TRP120 modulating Notch pathway gene expression levels, cells were stimulated with TRP120-coated beads for 2 h, and the gene expression levels of notch1 and the Notch pathway target genes hes1 and hes5 were examined. A significant increase in expression of all of the selected genes in TRP120-treated compared to thioredoxin-treated cells was detected (Fig. 5B). To further establish the role of TRP120 in induction of the Notch signaling pathway, we globally analyzed the expression pattern of genes involved in Notch pathway using the human Notch signaling PCR array. The heat map in Fig. 5C shows the graphical representation of gene expression patterns of all 84 genes involved in Notch signaling, in cells stimulated with TRP120 and normalized to control cells treated with thioredoxin. Examination of individual gene expression identified 33 (39.3%) genes were differentially expressed (P Ͻ 0.05). The scatter plot in Fig. 5D shows differential expression of Notch pathway genes during TRP120 stimulation compared to the thioredoxin control, where the up-and downregulation of genes are represented as red and green dots (2-fold cutoff), respectively. Figure 5E shows the list of genes and the fold change during the TRP120 stimulation. TRP120-stimulated cells showed increased expression of 16 Notch pathway components (19%) including receptors (notch1 and notch3), ligands (dll1, -3, and -4), transcription factor complex proteins (maml2 and rbpj), ␥-secretase protein (psen1), and the TRP120-interacting enzyme (adam17). Significant induction of 12 target genes (14%), including hes1, hes5, hey2, NFB 1, NFB 2, il2ra, and lor, and downregulation of 4 genes (5%) (lmo2, id1, fosl1, and lrp5) was also observed. Previously, we demonstrated that ehrlichial TRPs, including TRP120, are secreted during infection (3); therefore, we were also interested in exploring whether soluble TRP120 could potentially modulate Notch signaling in neighboring cells. Approximately 65% of the Notch signaling pathway component and target gene exhibited significant differential expression when stimulated with soluble TRP120 (see Fig. S3A and B in the supplemental material). Together these data demonstrate that TRP120 independently and efficiently activates the canonical Notch signaling pathway. Moreover, these findings also suggest that ehrlichial infection not only manipulates the host Notch signaling pathway through direct interactions between ehrlichiae and host cells but potentially involves uninfected neighboring cells through the release of soluble TRP120 during the exit phase.
E. chaffeensis represses the ERK1/2 and p38 MAPK pathways through Notch signaling. Previous studies reported downregulation of PU.1 and TLR2/4 expression during E. chaffeensis infection and demonstrated that host cells become progressively less responsive to LPS-mediated stimulation. Moreover, the underlying mechanism involved inhibition of the ERK1/2 and p38 MAPK pathways (34). Since recent studies linked the Notch signaling pathway with inhibition of TLR-triggered inflammation and inhibition of ERK1/2 (24), we sought to determine the role of E. chaffeensis-mediated activation of Notch signaling in inhibition of the ERK1/2 and p38 MAPK pathways. Therefore, the phosphorylated and total levels of ERK1/2 and p38 MAPK protein were examined in response to E. chaffeensis infection and LPS stimulation in the presence and absence of Notch transcription factor inhibitor SAHM1. Decreased levels of phosphorylated ERK1/2 in response to LPS were detected within 3 h of ehrlichial infection. In contrast, inhibition of ERK1/2 phosphorylation was blocked in the absence of Notch signaling (Fig. 6A). However, the total level of ERK1/2 remained unchanged (Fig. 6B), and the level of phosphorylated p38 MAPK decreased beginning at 3 h p.i. and was significantly downregulated at 1 dpi, resulting in decreased responsiveness of p38 MAPK to LPS. However, phosopho-p38 MAPK levels were not decreased when the cells were pretreated with SAHM1 (Fig. 6C), and the total level of p38 MAPK remained unchanged (Fig. 6D). These results suggest that activation of Notch signaling plays a key role in downregulation of the ERK1/2 and p38 MAPK pathways during E. chaffeensis infection.
To investigate the role of Notch signaling in regulation of PU.1 expression, RBPj (Notch transcription factor) expression was silenced in THP-1 cells with specific siRNA. Control siRNA and RBPj siRNA-transfected cells were infected with E. chaffeensis 1 day posttransfection and stimulated with LPS (100 ng/ml) for 1 h after 1 day p.i. Using immunofluorescent microscopy, high levels and predominant localization of PU.1 in the nucleus of uninfected and control siRNA-treated THP-1 cells were observed, but there was a reduction of expression in E. chaffeensis-infected cells. However, we observed reconstitution of the PU.1 expression level in the nucleus of THP-1 cells which were treated with RBPj siRNA to inhibit Notch signaling before infection (Fig. 7A). Protein expression of RBPj was reduced in specific siRNA-transfected cells compared to control siRNA-treated cells (Fig. 7B). To independently confirm the results seen in immunofluorescence microscopy, PU.1 protein levels during E. chaffeensis infection in whole-cell lysates in the presence or absence of the Notch transcription factor inhibitor SAHM1 were determined by Western blotting. As shown in Fig. 7C, the level of PU.1 was reduced in E. chaffeensisinfected cells compared to that in controls. However, no inhibition was seen in E. chaffeensis-infected cells, which were treated with Notch inhibitor SAHM1. Densitometry data generated by the software ImageJ and normalized with the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed significant differences in the level of PU.1 in E. chaffeensis infected cells compared to those in control and inhibitor-treated cells (Fig. 7D). Thus, the Western blot and immunofluorescence assay (IFA) data were consistent and indicated that the Notch signaling was responsible for E. chaffeensis mediated inhibition of PU.1.
To demonstrate that the Notch-induced inhibition of PU.1 expression resulted in downregulation of TLR2/4 expression,  THP-1 cells were infected with E. chaffeensis in the presence or absence of Notch inhibitors (e.g., DAPT and SAHM1). After 24 h p.i., cells were treated with LPS (100 ng/ml) for 1 h. As shown in Fig. 8A and B, E. chaffeensis infection caused significant decrease in TLR2 and -4 expression compared to the level in uninfected cells even after LPS stimulation. However, E. chaffeensis was unable to downregulate TLR2/4 expression when Notch signaling was blocked. Western blot analysis of THP-1 cells treated under  the same conditions provided an independent approach to validate differential expression of these PRRs in the whole-cell lysate of E. chaffeensis-infected cells (2 days p.i.) in the presence or absence of Notch signaling. Immunoblot data correlated with gene expression results, showing reduced expression of TLR2/4 proteins in E. chaffeensis-infected cells compared to uninfected cells, whereas in the presence of Notch signaling inhibitor, differences in TLR expression compared to the uninfected control were not observed (Fig. 8C). In Fig. 8D, densitometry data show quantitative comparison of the Western blot where the levels of TLR2/4 were normalized with the housekeeping protein GAPDH. Collectively, these data support a key role of Notch signaling in TLR2/4 downregulation during E. chaffeensis infection.
TRP120 effector protein plays a crucial role in inhibition of TLR2/4 response. We determined that E. chaffeensis activates the canonical Notch signaling pathway, and this pathway suppresses TLR2/4 expression during infection. Since TRP120 plays a critical role in activation of Notch components and target gene expression, we sought to evaluate their effect on PU.1 inhibition and TLR2/4 expression. To test this, THP-1 cells were treated with 1 g/ml of either TRP120 or thioredoxin (control) in soluble form, or latex beads were coated with TRP120 and incubated at 37°C for 24 h. TRP120-treated and control cells were then stimulated with LPS (100 ng/ml) for 1 h, and expression of PU.1, TLR2, and TLR4 was determined using immunofluorescence microscopy. Figure 9A shows strong PU.1 expression in the nucleus of thioredoxin-treated cells in response to LPS stimulation. However, after LPS treatment, reduction in the expression level of PU.1 in response to TRP120 stimulation (both bead bound and in sus-pension) was observed. Since PU.1 transcription factor regulates TLR2/4 expression, we also determined the effect of TRP120 in TLR expression using the same method. Thioredoxin-treated cells showed strong TLR2 expression in both cytoplasm and nucleus of the cells in response to LPS; however, cells stimulated with TRP120 showed reduced expression of TLR2 (Fig. 9B). Similar results were found when TLR4 expression was measured in response to TRP120 stimulation (Fig. 9C). Western blotting was done to analyze the protein expression of PU.1, TLR2, and TLR4 in control and TRP120-stimulated (bead-bound and soluble) THP-1 cells in response to LPS. Decreased expression of all three proteins was observed, which further validated our IFA data (Fig. 9D). These studies demonstrate that TRP120 effector protein plays a direct role in modulating TLR expression during E. chaffeensis infection.

DISCUSSION
In recent years, the essential role of Notch signaling in determining the fate of T cells, B cells, dendritic cells (DCs), monocytes, and macrophages at different stages of life has been widely studied (24,(41)(42)(43)(44)(45). In this study, we demonstrated that E. chaffeensis and more specifically the TRP120 effector activates Notch signaling and exploits this evolutionarily conserved pathway for its survival. In addition, we have gained an understanding of the mechanisms behind E. chaffeensis-mediated activation of Notch signaling and how it affects PRR expression and ehrlichial survival in monocytes.
E. chaffeensis successfully evades innate immune recognition and natural phagocytic killing, but the mechanisms are unknown. However, the TRP effectors play important roles in immune subversion by chromatin manipulation or by directly interacting with host proteins to modulate cellular processes. More recently, we demonstrated that TRP effectors cause activation of the Wnt signaling pathway, which facilitates host cell entry by inducing phagocytosis and promotes intracellular survival (10). Previously, we reported that TRP120 interacts with ADAM17 metalloprotease and binds to the promoter region of notch1 (9,11). Since Notch is a critical signaling pathway in monocytes and macrophages (24,44,(46)(47)(48), we hypothesized that E. chaffeensis TRP120 directly activates the Notch signaling to promote survival. During E. chaffeensis infection, nuclear localization of transcriptionally active NICD and induction of a panel of Notch genes expressed in monocytes were observed. We demonstrated comprehensive molecular modulation of Notch signaling pathway genes and downstream target genes at different times after infection. This suggests that activation of the canonical Notch signaling pathway occurs at both early and late phases of ehrlichial infection. Induction of TRP120-interacting protein ADAM17 was also observed during ehrlichial infection. ADAM17, also known as tumor necrosis factor-converting enzyme, or TACE, is an important regulator of Notch signaling and plays a critical role in regulation of different cellular events, including proliferation and migration. Thus, ADAM17 is implicated in different human diseases, including cancer, and is a promising target for treatment (49). Several studies reported induction of ADAM17 and its anti-inflammatory effect during bacterial infection (50). Both Staphylococcus aureus and Pseudomonas aeruginosa induce ADAM17 in airway epithelial cells and regulate inflammation by neutralizing interleukin-6 (IL-6) (51) and tumor necrosis factor alpha (TNF-␣) (52). Based on our previous study showing direct TRP120-ADAM17 interaction and the colocalization studies herein, we propose that this interaction could activate ADAM17, resulting in Notch1 cleavage and pathway activation. It is not clear if TRP120 interacts directly with Notch1, but it is possible that such an interaction occurs. Moreover, TRP120-induced nuclear translocation of NICD and induction of genes associated with Notch signaling demonstrate that this effector protein plays important role in canonical Notch pathway activation. Compared to the bacterial infection, the TRP120, especially the soluble form, showed a stronger effect on modulation of Notch pathway gene expression. It might be because of the higher concentration of purified TRP120 used in the experiments and also because the soluble protein has more surface area for interaction with the receptor complex than the beadbound protein. This study revealed a novel mechanism whereby E. chaffeensis uses TRP120 to activate the Notch signaling pathway by interacting with and inducing Notch receptor complex components.
Notch plays critical role in regulating the maturation and function of different immune cells, including monocytes and macrophages. Therefore, Notch signaling-mediated changes in the properties of these cells may also affect bacterial growth. Pharmacological inhibitors against Notch transcription factor protein (SAHM1), ␥-secretase enzyme (DAPT), and siRNAs against Notch1, ADAM17, and RBPj confirmed that the canonical Notch signaling pathway is required for ehrlichial survival. Differences in ehrlichial loads observed between different siRNA treatments (at day 1 p.i.) might occur because the role of these targets varies at different stages of infection. Also, the siRNAs might vary in their efficacy, and it is possible that the ADAM17 and RBPj siRNAs required more time for efficient knockdown. Together, the gene expression studies correlated strongly with inhibitor data supporting the conclusion that canonical Notch signaling is required for ehrlichial survival and may be a novel target for developing therapeutics.
TLRs, the key PRR expressed on monocytes and macrophages, play critical roles in host defense against any invading pathogens, including E. chaffeensis (25,31,53). Interaction of TLR with PAMPs causes activation of a signaling transduction cascade and elicits a proinflammatory cytokine and chemokine response. This profound innate immune response helps in the elimination of invading organisms (29,54,55). E. chaffeensis lacks the genes required for biosynthesis of LPS and peptidoglycan, which presumably prevents detection by PRRs (56). However, this unusual cell wall structure does not protect them fully from recognition by immune cells. Using TLR4 Ϫ/Ϫ mice, it has been shown that inhibition of TLR4 causes decreased nitric oxide and IL-6 secretion by macrophages, which results in short-term persistence of ehrlichial infection (25). Recently, another study reported that absence of TLR2 impaired ehrlichial elimination and the TLR2-dependent immune response was protective against the infection (31). E. chaffeensis causes decreased expression of TLR2/4 in monocytes, which involves inhibition of ERK1/2, p38 MAPK, and PU.1 activity (2). However, the specific mechanism that E. chaffeensis utilizes to downregulate TLR expression and the upstream signaling molecule that inhibits the ERK1/2 and p38 MAPK pathways were unknown.
Multiple studies have reported cross talk between TLR and Notch signaling pathways (44,47). TLRs have been shown to activate Notch signaling through a JNK-dependent pathway to regulate inflammatory response (57). However, TLRs can also cause suppression of Notch signaling in macrophages (58). Combined effects of Notch and TLR signaling in the induction of the hes and hey genes have been shown to function as part of a feedback loop and attenuate the production of cytokines (e.g., IL-6 and IL-12). Association of Notch signaling in modulation of ERK1/2 and regulation of TLR4-triggered cytokine production has recently been reported (24). However, to our knowledge, no previous studies have reported Notch-mediated regulation of TLR response during bacterial infection. Herein, we demonstrated that E. chaffeensis causes inhibition of PU.1 and TLR2 and -4 expression by directly activating the Notch pathway. The underlying mechanism involves Notch-mediated inhibition of ERK1/2 and p38 MAPK activation. Based on our observation and previous findings, we also expected less NF-B (p65/50) activation by LPS stimulation in E. chaffeensis-infected cells (34). Although Notch PCR array data showed induction of NF-B1 (p105) and NF-B2 (p100) genes in THP-1 cells infected with E. chaffeensis or stimulated with TRP120, this finding does not contradict the previous findings, which reported inhibition of NF-B during infection. NF-B1 and NF-B2 proteins serve as both NF-B precursors and inhibitors. The classical inhibitors IB␣, -␤, -␦, and -sequester the transcription factor NF-B in the cytoplasm by masking their nuclear localization signal (NLS) (59). Unlike these inhibitors p105 and p100 proteins assemble into high-molecular-weight complexes and bind NF-B subunits (60). Previous studies reported M. bovis upregulation of Notch1 expression and activation of Notch signaling leading to activation of SOCS3 (suppression of cytokine signaling 3), which is a negative regulator of signaling of multiple cytokines and TLR (26,61). Herein, we provide data to support a different mechanism, whereby E. chaffeensis T1S effector protein TRP120 can manipulate Notch signaling to regulate immune recognition through inhibition of PRR expression to promote survival. Although we demonstrated that TLR2 and TLR4 expression is regulated during E. chaffeensis infection, it is possible that other TLRs can also be regulated by the Notch signaling during infection. TRP120 is found on the surface of the infectious dense-core form, but is also secreted into the host cell, where it interacts with a variety of host cell targets and DNA (9,11,62). TRPs can also be released from the E. chaffeensis-infected cell during infection (63). Data presented in this study demonstrated that TRP120 bound to a substrate or in soluble form can activate Notch signaling pathway and modulate PU.1 and TLR2/4 expression. These findings are significant as they suggest that the effects of E. chaffeensis TRP120 on Notch pathway activation not only occur through direct bacterium-host interactions but may have systemic effects through the release of soluble TRP120 that could interact with uninfected cells to downregulate innate immunity and promote infection.
In addition to TLR-mediated innate immune responses, another important mechanism involved in the elimination of intracellular bacteria is autophagy, which appears to be inhibited during ehrlichial infection (64). Studies indicate that Notch and Wnt signaling plays a crucial role in inhibition of autophagy through activation of the mTOR pathway and regulation of autophagy receptor p62 expression (65)(66)(67)(68), and preliminary data (T. T. Lina, and J. W. McBride, unpublished data) from our laboratory suggest Wnt and Notch signaling is involved in inhibition of autophagy during ehrlichial infection. Hence, Ehrlichia inhibition of TLR recognition is likely not the only innate immune mechanism that is affected by Notch signaling. Further studies are needed to fully understand the cross talk between the Wnt and Notch signaling pathways and how these pathways act synergistically to inhibit host innate immune responses such as autophagy to promote ehrlichial survival.
Our limited understanding of the molecular pathogen-host interactions and cellular pathways usurped by Ehrlichia as well as those of other obligate intracellular microbes is a major impediment to defining the mechanisms that enable ehrlichial intracellular survival and development of next-generation therapeutics aimed at mechanistically defined targets. This study reveals a novel effector-dependent mechanism, which involves interaction with the ADAM17 and Notch1 and activation of canonical Notch signaling pathway in monocytes, a primary target of E. chaffeensis, to modulate ERK1/2 and p38 MAPK pathways and regulate TLR2/4 expression (Fig. 10). Hence, this study demonstrates the importance of Notch pathway in ehrlichial survival and provides a new target for the development of a novel therapeutic approach against ehrlichial infection that may be applicable to other intracellular pathogens, in which exploitation of such conserved cellular pathways is required for pathogen survival.

Antibodies and inhibitors.
The polyclonal mouse anti-TRP120 antibody used in this study was previously described (69). A convalescentphase anti-E. chaffeensis dog serum, which was derived from an experimentally infected dog, was described previously (40). Other antibodies that were used in this study include anti-Hes1 ( RT-PCR. Total RNA from E. chaffeensis-infected, TRP120-or thioredoxin-stimulated and control THP-1 cells was isolated using an RNeasy minikit (Qiagen) according to the manufacturer's instructions. On-column DNA digestion was performed using the RNase-free DNase set (Qiagen). cDNA was synthesized from 1 g of total RNA using a qScript cDNA SuperMix kit (Quanta Biosciences). The gene expression level of the target host genes was quantitated by qPCR using Brilliant II SYBR green qPCR master mix (Agilent Technologies) with gene-specific primers and a thermal cycling protocol consisting of an initial denaturation step of 95°C for 10 min and 40 cycles of 95°C for 30 s, 58°C for 1 min, and 72°C for 30 s. Gene expression values were calculated on the basis of the threshold cycle (2 Ϫ⌬⌬CT ) method and normalized with GAPDH.
Human Notch signaling pathway PCR array. The human Notch signaling pathway RT 2 Profiler PCR array (Qiagen) was used according to the manufacturer's protocol to determine the expression of 84 genes, Step 1) E. chaffeensis TRP120 effector interaction with ADAM17 activates the metalloprotease, resulting in cleavage of the substrate Notch1, and subsequent cleavage by ␥-secretase causes (step 2) nuclear translocation of NICD, the transcriptionally active form of which binds with RBPj and MAML proteins. This triprotein complex activates transcription of Notch target genes, which causes inhibition of the ERK1/2 and p38 MAPK pathways (step 3). The downstream transcription factor PU.1 expression is repressed, which causes further inhibition of monocyte TLR2/4 expression (step 4). Inhibition of TLR2/4 expression causes both inhibition of E. chaffeensis recognition and TLRmediated proinflammatory cytokine production needed for the activation of monocytes and clearance of ehrlichiae.
which include genes coding for receptors, and ligands, as well as receptor processing, and transcription factor genes associated with Notch signaling and putative Notch target genes. Briefly, RNA was collected from E. chaffeensis-infected and uninfected cells, cells stimulated with TRP120or thioredoxin-coated FluoroSpheres (sulfate microsphere beads) or TRP120 alone, using the RNeasy minikit (Qiagen). RNA purification, genomic DNA elimination, cDNA synthesis, and the PCR array were performed as previously described (10).
Immunofluorescence microscopy. Uninfected or E. chaffeensisinfected cells or THP-1 cells stimulated with TRP120-or /thioredoxincoated beads and TRP120 in soluble form were cytospun onto glass slides, fixed for 15 min using 3% paraformaldehyde in phosphate-buffered saline (PBS), blocked and permeabilized for 30 min using 0.3% Triton X-100 and 2% bovine serum albumin (BSA) in PBS at room temperature. Cells were then incubated with primary antibodies rabbit anti-TRP120 (1: Slides were mounted with ProLong Gold antifade reagent with DAPI (4=,6-diamidino-2-phenylindole) (Invitrogen) after washing. HeLa cells transfected with the GFP-TRP120 plasmids or GFP control plasmids were fixed in chamber slides, permeabilized, and stained with anti-ADAM17 using the same protocol. Images were obtained using an Olympus BX61 epifluorescence microscope and analyzed using Slidebook software (version 5.0; Intelligent Imaging Innovations, Denver, CO).
Pharmacological inhibitor treatment and determination of bacterial load. THP-1 cells were treated with DAPT, SAHM1, or dimethyl sulfoxide (DMSO), incubated for at least 1 h, and then infected with cell-free E. chaffeensis at a multiplicity of infection (MOI) of 50. According to the 50% inhibitory concentrations (IC 50 s) and previously published concentrations, 5 g/ml of DAPT or 10 M SAHM1 was used to treat the cells unless otherwise stated (38,70). At days 1 and 2 p.i., cells were collected, and infection was determined by either calculating the percentage of infected cells after Diff-Quick staining or by determining the dsb copy number using qPCR as previously described (71). Infected culture without the inhibitors and uninfected cells were used as positive and negative controls, respectively. The absolute E. chaffeensis dsb copy number was determined using a standard curve and was normalized to qPCR-detected levels of the host genomic gapdh gene. To confirm that host cell death did not account for decreased ehrlichial inclusions, differences in cell viability were assessed at days 1, 2, and 3 p.i. using trypan blue staining.
Western immunoblot. THP-1 cells infected with E. chaffeensis in the presence of DMSO or Notch inhibitor SAHM1 and uninfected cells were harvested after 1 and 2 days p.i. and LPS stimulation (100 ng/ml for 1 h). Cell lysates were prepared as previously described (3). Approximately 20 g of total proteins was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane using a semidry transfer apparatus. Mouse anti-ADAM17, rabbit anti-Hes1, mouse anti-␣-tubulin, mouse anti-PU.1, mouse anti-TLR4, mouse anti-TLR2, and mouse anti-GAPDH were used. For the detection, horseradish peroxidase-labeled goat anti-rabbit or mouse IgG (heavy and light chains) conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used. SuperSignal West Dura chemiluminescent substrate (Thermo Scientific) was used for detection of Hes1 protein, and ECL enhanced chemiluminescent Western immunoblot substrate (Thermo Scientific) was used for others.
Bead assay. E. chaffeensis TRP120 protein (thioredoxin fused) was expressed and purified as described previously (6,63,69). Purified proteins were desalted (Zeba Spin desalting column; Thermo Scientific) to change the buffer to 40 mM MES [2-(N-morpholino)ethanesulfonic acid]. FluoroSpheres (sulfate microsphere beads; 1.0 m, yellow-green fluorescent [Invitrogen]) were coated with recombinant purified TRP120 or thioredoxin using the following protocol. Briefly, 10 l of beads (~3.6 ϫ 10 8 beads) was washed two times with 10 volumes of 40 mM MES buffer (5,000 ϫ g for 5 min), resuspended in 10 g of TRP120 desalted protein in 500 l MES buffer, and incubated at room temperature for 2 h in a rotor. After incubation, beads were washed twice with 500 l MES buffer (10,000 ϫ g for 8 min) and resuspended in RPMI medium. Since these beads are light sensitive, they were also protected from exposure to light. TRP120-or thioredoxin-coated beads were used to treat THP-1 cells for different time points, and the cells were incubated at 37°C with 5% CO 2 . After incubation, unbound beads were washed by centrifugation at least 4 times at 400 ϫ g.
Bio-Plex. The levels of total and phosphorylated ERK1/2 and p38 MAPK proteins in THP-1 cells infected with E. chaffeensis in the presence and absence of the Notch inhibitor SAHM1 (10 M) and with or without LPS stimulation (100 ng/ml) were measured using the Luminex array (Millipore, Billerica, MA) according to manufacturer's instructions. Samples were analyzed using Bio-Plex Manager software (Bio-Rad).
Statistics. The results are expressed as the means Ϯ standard deviation (SD) of data obtained from at least three independent experiments done with triplicate sets per experiment, unless otherwise indicated. Differences between means were evaluated by using two-tailed Student's t test. P values of Ͻ0.05 were considered statistically significant.