Ehrlichia chaffeensis is an obligately intracellular bacterium and the etiologic agent of the emerging life-threatening human zoonosis, human monocytotropic ehrlichiosis (HME) (Paddock and Childs, 2003). E. chaffeensis selectively infects mononuclear phagocytes and resides in endosome-like membrane-bound vacuoles where it replicates and evades innate host defenses (Paddock and Childs, 2003). The mechanisms by which E. chaffeensis enters the host cell, avoids destruction, and establishes persistent infection are not well-understood, but functionally relevant host-pathogen interactions are essential for reprogramming the host cell defense mechanisms. This molecular strategy involves type 1 secreted tandem repeat protein (TRP) effectors (Lina et al., 2016b).

E. chaffeensis TRPs are major immunoreactive proteins that elicit strong host antibody responses during infection. The tandem repeat (TR) domains in TRP120, TRP47, and TRP32 are acidic, serine-rich, and contain protective species-specific epitopes (Doyle et al., 2006; Luo et al., 2008, 2009; Kuriakose et al., 2012). TRP120 and TRP47 are differentially expressed by infectious dense cored cells (DC), while TRP32 is expressed by both DCs and replicating reticulate cells (RC) (Popov et al., 2000; Doyle et al., 2006; Luo et al., 2008). Consistent with type 1 secretion (T1S) signals identified in the C-terminal domains of TRPs, TRPs have been experimentally identified as T1S system substrates through studies using a heterologous T1S apparatus of Escherichia coli (Wakeel et al., 2011).

In order to identify Ehrlichia-host interactions and help define the complex mechanisms by which E. chaffeensis modulates host cells, multiple studies using the yeast two-hybrid (Y2H) approach have been performed to better understand molecular host-pathogen interactions involving TRPs. TRP120, TRP47, and TRP32 have been shown to interact with a diverse network of host proteins involved in many host cellular processes including cell signaling, vesicle trafficking and intracellular transport, transcriptional regulation, metabolism, posttranslational modification and apoptosis, indicating the important roles of TRPs in reprogramming the host cell (Wakeel et al., 2009; Luo et al., 2011; Luo and McBride, 2012).

TRPs are modified by multiple host posttranslational modification pathways, including SUMOylation, ubiquitination and phosphorylation, which appear to mediate functional interactions and extend the number and diversity of interactions with host targets, as well as localization to various subcellular locations, including the nucleus (Wakeel et al., 2010; Dunphy et al., 2014). TRP120 is modified by SUMO at a canonical consensus SUMO conjugation motif located in the C-terminal domain, which has been further confirmed using a high-density microfluidic peptide array (Zhu et al., 2016). TRP120 conjugation with SUMO mediates interactions with host protein targets, and inhibition of the host SUMO pathway significantly decreases interaction between TRP120 and host protein targets, resulting in decreased ehrlichial intracellular survival (Dunphy et al., 2014). TRP120 also interacts with components of the ubiquitin pathways, including the E3 ligases, KLHL12 and FBXW7 as well as ubiquitin (Ub) isoforms UBB and UBC, which suggests TRP120 is a target of Ub conjugation (Luo et al., 2011). TRP47 is phosphorylated and interacts with the Src family tyrosine kinase, Fyn, which may be involved in the tyrosine phosphorylation of TRP47 (Wakeel et al., 2009, 2010). TRPs also contain many additional predicted phosphorylation sites; however, it is not clear which protein kinases are involved and how the phosphorylation affects TRP function or interactions with the host cell.

We have demonstrated the influence of selected TRP120 or TRP32-interacting host proteins on ehrlichial infection by RNA interference (Luo and McBride, 2012; Luo et al., 2016); however, a comprehensive analysis of all TRP-host interactions has not been performed. In this study, we extend the role of TRP-host interactions by investigating the influence of 138 TRP120, TRP47, and TRP32 interacting host target proteins on ehrlichial infection by RNA interference. We directly demonstrate that E. chaffeensis exploits the host cells through complex TRP interactions with a large and diverse array of host targets to promote intracellular survival.

In recent years multiple studies from our laboratory have identified numerous Ehrlichia-host interactions and determined that Ehrlichia TRPs interact with a diverse network of host proteins involved in many host cellular processes including cell signaling, vesicle trafficking and intracellular transport, transcriptional regulation, metabolism, posttranslational modification, and apoptosis (Wakeel et al., 2009; Luo et al., 2011; Luo and McBride, 2012). Previous studies have helped understand the complex mechanisms by which E. chaffeensis modulates host cell processes. This study found that knockdown of host proteins that interact with Ehrlichia TRPs differentially influence intracellular survival. Figure 6 illustrates important host cellular processes that E. chaffeensis TRPs modulate during infection through interactions with host targets.

Notably, E. chaffeensis TRPs interact with numerous host proteins associated with cellular signaling pathways, including 17 proteins with TRP120, 10 proteins with TRP47, and six proteins with TRP32, suggesting that infection and replication of E. chaffeensis involves TRP exploitation of conserved cell signaling pathways, such as Wnt, Notch, mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK), phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin (PI3K/Akt/mTOR), nuclear factor kappa B (NFκB), and cytokine/chemokine-mediated signaling (Table 4). Wnt and Notch are fundamental cell signaling pathways that play major roles in the regulation of gene expression, cell proliferation and differentiation, and embryonic development (Hayward et al., 2008). Five TRP120-interacting proteins including ADAM metallopeptidase domain 17 (ADAM17), annexin A2 (ANXA2), protein phosphatase 3 regulatory subunit B alpha (PPP3R1), transducin like enhancer of split 4 (TLE4), and G protein subunit beta 1 (GNB1) are signaling molecules of Wnt and Notch pathways. This is consistent with recent studies in which we have used different approaches to demonstrate that Wnt and Notch signaling pathways play important roles in ehrlichial infection, and TRPs mediate ehrlichial invasion and survival through activation and modulation of Wnt and Notch pathways (Luo et al., 2016; Lina et al., 2016a). In this study, we further demonstrated that knockdown of nearly all of TRP-interacting proteins involved in host cell signaling, except two TRP47-interacting proteins, calcium binding protein 39 (CAB39) and immunoglobulin lambda like polypeptide 1 (IGLL1), significantly influenced E. chaffeensis survival. These results support the importance of many conserved cellular signaling pathways in ehrlichial survival. For example, G-protein coupled receptor (GPCR) signaling involves a variety of ligands and stimuli and can regulate a highly interconnected network of signal transductions and cellular responses (Roth et al., 2015). We found that six TRP120-interacting proteins including ADAM17, A kinase anchor protein 2 (AKAP2), C-X-C motif chemokine ligand 12 (CXCL12), G protein subunit alpha i2 (GNAI2), G protein pathway suppressor 1 (GPS1), and phosphodiesterase 1B (PDE1B), and one TRP47-interacting protein GNB1 are involved in GPCR signaling. In addition, MAPK signaling is a conserved pathway involved in a variety of fundamental cellular processes (Cargnello and Roux, 2011). It has been reported that p38 MAPK/ERK1/2 are activated at the early stage of E. chaffeensis infection, but the activations are subsequently reduced in infected cells compared with those in uninfected cells (Lin and Rikihisa, 2004). We found that six TRP120-interacting proteins GNAI2, GPS1, immunoglobulin kappa constant (IGKC), interleukin 2 receptor subunit gamma (IL2RG), KRAS proto-oncogene GTPase (KRAS), and PPP3R1, four TRP47-interacting proteins cyclin dependent kinase 1 (CDK1), cyclin dependent kinase 10 (CDK10), FYN, and IGKC, and one TRP32-interacting protein, CD14, are involved in MAPK signaling. Knockdown of all these TRP target proteins significantly influenced infection, indicating that GPCR and MAPK signaling pathways are involved in ehrlichial survival. Based on these studies, Wnt, Notch, and MAPK as well as GPCR signaling pathway appear to be major targets of TRP exploitation. Further investigation is needed to understand the specific role of each TRP target and associated pathway(s) in ehrlichial pathobiology.

An important host cellular process in which many TRP-interacting host proteins are involved is vesicle trafficking and intracellular transport. This process involves 20 TRP120-interacting proteins, seven TRP47-interacting proteins, and three TRP32-interacting proteins. Remarkably, from this group we found a subset of actin-related proteins interacting with Ehrlichia TRPs (particularly TRP120), including eight TRP120-interacting proteins and one TRP47-interacting protein (Table 5). Ehrlichia TRPs interact directly with host cytosolic actin gamma 1 (ACTG1) and eight other actin-binding or related proteins, including actin related protein 2/3 complex subunit 2 (ARPC2), CDC42 small effector 2 (CDC42SE2), integrin subunit alpha M (ITGAM), myosin X (MYO10), spectrin alpha erythrocytic 1 (SPTA1), WAS protein family member 2 (WASF2), WD repeat domain 1 (WDR1), and adenylate cyclase associated protein 1 (CAP1), suggesting that actin cytoskeleton plays an important role in ehrlichial infection. Most of these TRP-interacting proteins have also been found to be involved in phagocytosis or endocytosis. Multiple studies have shown the importance of TRP120 in ehrlichial binding and internalization, and most recently, we demonstrated that TRPs directly activate Wnt signaling to induce ehrlichial phagocytosis (Popov et al., 2000; Kumagai et al., 2010; Luo et al., 2016). In addition, it has been reported that E. chaffeensis is transported through the filopodia of the host cell and inhibition of actin polymerization in infected cells prevents filopodia formation. Moreover, the spreading process of Ehrlichia canis in macrophages is also dependent on actin cytoskeleton (Thomas et al., 2010; Alves et al., 2014). Other studies reported that E. chaffeensis surface protein EtpE binds GPI-anchored protein DNase X to trigger entry by N-WASP-dependent actin polymerization (Mohan Kumar et al., 2013, 2015). Therefore, interaction of TRPs with actin cytoskeleton might facilitate ehrlichial entry/release and necessary intracellular trafficking during Ehrlichia infection. Our RNAi data showed that knockdown of all these Ehrlichia TRP target proteins except WDR1 significantly reduced bacterial load, demonstrating the importance of actin cytoskeleton organization in ehrlichial entry and/or survival.

Another group of TRP-interacting proteins with significant interest is host proteins involved in transcriptional regulation, including 16 TRP120-interacting proteins, seven TRP47-interacting proteins, and two TRP32-interacting proteins (Table 6). These proteins are either transcription factors or transcription regulators (coactivator or corepressor). Both TRP120 and TRP47 interact with PCGF5, a component of the polycomb repressive complex which mediates epigenetic regulation (Gao et al., 2014). Other important TRP120-interacting proteins involved in transcriptional regulation include AT-rich interactive domain 1B (ARID1B), cyclin-dependent kinase 12 (CDK12), interleukin enhancer binding factor 3 (ILF3), IRF2BP2, KDM6B, nuclear receptor binding SET domain protein 1 (NSD1), and tripartite motif containing 24 (TRIM24). Important TRP47-interacting proteins involved in transcriptional regulation include AT-rich interactive domain 2 (ARID2), histone deacetylase 2 (HDAC2), signal transducer and activator of transcription 5A and 6 (STAT5A and STAT6), and transcription factor EC (TFEC). Moreover, TRP32 interacts with DAZ-associated protein 2 (DAZAP2) and hematopoietically expressed homeobox (HHEX). We have previously reported that Ehrlichia TRP120 and TRP32 are nucleomodulins that directly bind host DNA and regulate the expression of numerous genes involved with transcriptional regulation, signal transduction, and cell differentiation and proliferation (Zhu et al., 2011; Farris et al., 2016). In this study, our RNAi data showed that knockdown of all Ehrlichia TRP target proteins involved in transcriptional regulation significantly influenced infection level, therefore, TRPs also play roles in regulation of host gene transcription by direct interactions with transcription factors and regulators in multiple cellular processes including survival, growth, and differentiation.

TRP-interacting targets include some host proteins involved in protein posttranslational modification (PTM). In particular, TRP120 interacts with 10 host proteins that have the main function in posttranslational modification machinery, including phosphorylation and ubiquitination. TRP120 interacts with host kinase and phosphatase involved in protein phosphorylation/dephosphorylation, including BMP2 inducible kinase (BMP2K), CDC-like kinase 1 (CLK1), and protein phosphatase 6 regulatory subunit 1 (PPP6R1). TRP120 also interacts with multiple host proteins involved in protein ubiquitination, including BTB domain containing 6 (BTBD6), a putative ubiquitin ligase adaptor protein (Sobieszczuk et al., 2010); cullin-associated and neddylation dissociated 1 (CAND1), an essential regulator of Cullin-RING ubiquitin ligases (Goldenberg et al., 2004); FBXW7, one of the four subunits of ubiquitin protein ligase complex SCFs (SKP1-cullin-F-box) (Hao et al., 2007); kelch-like family member 12 (KLHL12), a substrate adaptor of the Cullin-3 ubiquitin ligase complex (Jin et al., 2012); OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1), a highly specific ubiquitin iso-peptidase (Edelmann et al., 2009); and an ubiquitin precursor protein ubiquitin C (UBC). Ubiquitin B (UBB), another ubiquitin precursor, is a common interacting target of TRP120 and TRP47. TRP47 also interacts with NEDD4 binding protein 1 (N4BP1), an inhibitor of the E3 ubiquitin ligase (Oberst et al., 2007). Moreover, TRP120 has recently been found to be directly modified by SUMO, and more importantly, SUMO conjugation contributes to interactions with defined host proteins, such as PCGF5, actin and myosin cytoskeleton components, and recruitments of host proteins to the ehrlichial vacuole that influence infection (Dunphy et al., 2014). It remains unclear whether the functional consequences of TRP ubiquitination and phosphorylation are different from those associated with TRP SUMOylation, but it is well-established that many bacterial effectors mimic host proteins involved in the host posttranslational machinery to modify host proteins and signaling (Ribet and Cossart, 2010). Thus, PTMs may contribute to the moonlighting function of Ehrlichia TRPs and facilitate many diverse interactions between TRPs and host proteins. Another intracellular bacterium moonlighting effector is Chlamydia trachomatis CPAF, a protease which targets multiple host and bacterial proteins to maintain vacuole integrity, manipulate signaling pathways and promote virulence (Jorgensen et al., 2011; Zhong, 2011). Our RNAi data demonstrate that knockdown of all TRP target proteins involved in PTM significantly influenced infection, indicating that E. chaffeensis exploits major posttranslational modification machineries of host cells to facilitate a survival strategy.

Host proteins involved in metabolism are also frequent interacting targets of E. chaffeensis TRPs, including 16 TRP120-interacting proteins, six TRP47-interacting proteins, and five TRP32-interacting proteins. This category covers a variety of host metabolism processes, including glycometabolism, lipid metabolism, protein metabolism, nucleic acid metabolism, ion, and other inorganic metabolism. Two proteins, carbonic anhydrase I (CA1) and Charcot-Leyden crystal galectin (CLC) are common interacting targets of TRP120 and TRP47, suggesting their importance during infection. Our RNAi data demonstrated that knockdown of most of these Ehrlichia TRP target proteins significantly influenced bacterial load, indicating that TRPs also play a role in regulating host metabolism to favor the survival.

Interestingly, Y2H studies also discovered that a large number of apoptosis-associated proteins of the host cell were TRP targets, although most of these proteins are classified into other categories of function because their primary functions appear not to be related to apoptosis (Table 7). Two TRP32-interacting proteins glucocorticoid-induced 1 (GLCCI1) and tumor protein p53 inducible protein 11 (TP53I11) have main function in cell apoptosis. Expression of GLCCI1 is induced by glucocorticoids and may be an early marker for glucocorticoid-induced apoptosis (Chapman et al., 1996). TP53I11 is a downstream target of p53 and is involved in the regulation of apoptosis (Wu et al., 2009). Three common interacting proteins of TRPs, eukaryotic elongation factor 1 alpha 1 (EEF1A1), CA1, and CLC, play roles in apoptosis (Ejiri, 2002; Kubach et al., 2007; Zheng et al., 2015). In addition, TRPs interact with a wide variety of immunoglobulin molecules, such as IGKC, immunoglobulin heavy constant alpha 1 (IGHA1) and immunoglobulin lambda like polypeptide 5 (IGLL5), which have also been linked to apoptosis (Yang et al., 2009). TRP47 interacts with other seven host proteins associated with apoptosis, for example, STAT5A and STAT6, both of which are members of the STAT family of transcription factors and have been found to induce the expression of anti-apoptotic protein BCL2L1/BCL-XL (Calò et al., 2003); and CAP1, which has been implicated in promoting apoptosis by functioning as an actin shuttle to mitochondria (Wang et al., 2008). TRP120 interacts with relatively more apoptosis-associated proteins of the host cell. For example, CXCL12 is a negative regulator of intrinsic apoptotic signaling pathway in response to DNA damage, and FBXW7, in contrast, is a positive regulator of oxidative stress-induced intrinsic apoptotic signaling pathway (Hattermann et al., 2010). Previous studies have demonstrated that host cell apoptosis is delayed by stabilization of the mitochondrial membrane potential during E. chaffeensis and E. ewingii infection (Xiong et al., 2008; Liu et al., 2011). Our RNAi data showed that knockdown of all apoptosis-associated TRP target proteins (except for DEAD-box helicase 5 [DDX5]) significantly influenced bacterial load, thus, the interactions of TRPs with different apoptosis-associated proteins may serve an opposite function by inhibiting or promoting apoptosis in the different stages of infection, to facilitate ehrlichial survival and release, respectively.

We classified TRP-interacting host proteins according to their main functions, but many of them are moonlighting proteins which could be included in multiple categories. For example, EEF1A1, a common target protein of TRP120 and TRP32, is the second most abundant protein in eukaryotes after actin. It is an isoform of the alpha subunit of the elongation factor-1 complex, so its main function is the enzymatic delivery of aminoacyl tRNAs to the ribosome during protein translation; however, EEF1A1 is also one of the most important multifunctional eukaryotic proteins and has been reported to be involved in regulation of transcription, cytoskeletal remodeling, cellular response to epidermal growth factor stimulus, regulation of chaperone-mediated autophagy, and apoptosis (Condeelis, 1995; Ejiri, 2002). IGKC, another common target protein of TRP120 and TRP47, is the immunoglobulin kappa constant domain, so its main function is immune response including antigen binding, immunoglobulin receptor binding and complement activation, but it also appears to be involved in other signaling pathways, vesicle-mediated transport, receptor-mediated endocytosis, and phagocytosis (Bentley and Rabbitts, 1980). It is not clear if all or part of the functions of these proteins are involved in Ehrlichia infection, but some of them are very likely moonlighting when interacting with TRPs, since they belong to multiple important common categories of TRP-interacting proteins, such as cell signaling, transcriptional regulation, vesicle trafficking and apoptosis. In addition, some putative host proteins interact with two TRPs, including EF1A1, IGHA1, IGLL5 (interacting with both TRP32 and TRP120), PCGF5, IGKC, CA1, CLC, and UBB (with TRP47 and TRP120), implicating the importance of not only the moonlighting and overlapping host targets but also the crosstalking and converging cellular networks by Ehrlichia effectors.

Our siRNA experiments revealed that knockdown of TRP-interacting host proteins could either increase or decrease E. chaffeensis load. Most siRNAs decreased the bacterial load, indicating that these proteins are necessary for Ehrlichia infection; however, siRNAs of 18 TRP120-interacting proteins and one TRP47-interacting protein (PCGF5) increased the bacterial load, indicating that these proteins inhibit the Ehrlichia infection. For example, Golgi associated gamma adaptin ear containing ARF binding protein 1 (GGA1) is a ubiquitous coat protein that regulates the protein trafficking between the trans-Golgi network and the endosome/lysosome system (Bonifacino, 2004), so E. chaffeensis may inactivate GGA1 to prevent early endosomal maturation into late endosomes and then fusion with lysosome. PCGF5 is a component of the polycomb repressive complex which controls expression of many developmental regulator genes (Gao et al., 2014), thus, E. chaffeensis may inhibit this transcriptional repressor in order to modulate host cell gene expression to favor ehrlichial survival. Uniquely, knockdown of many TRP120 host targets promoted infection, suggesting that TRP120 plays a role in modulating levels of these targets during infection. This result is likely related to our recent finding that TRP120 can function as ubiquitin ligase and degrade host target proteins such as PCGF5 (Zhu and McBride unpublished). In this study, no siRNA of TRP32-interacting proteins was found to increase the bacterial load significantly, probably because only C-terminus of TRP32 was available as the bait of Y2H screening and resulted in the loss of some host proteins interacting with other domains of TRP32. In addition, siRNAs of 14 TRP-interacting proteins could not change bacterial load significantly. This is probably because these proteins are important at the late stage of Ehrlichia infection, or they play a nonessential role in infection. Some of these proteins may be too abundant to be reduced sufficiently, or they are just nonspecific Y2H targets. We did not perform the late time point of infection in our experiments, since some infected cells started to collapse and release Ehrlichia at 3 day p.i.

Our RNAi data also show that changes in Ehrlichia infection status can occur at different stages of infection for different host proteins, suggesting that Ehrlichia TRPs and host proteins interact at different time points during infection. For example, some siRNAs, such as those targeting CDC42SE2, ITGAM, MYO10, unc-13 homolog D (UNC13D) and TLE4, reduced bacterial load significantly at 2 days p.i. but not significantly at day 1, suggesting that these proteins may be not involved in bacterial internalization or early stage of infection. In contrast, some siRNAs, such as those targeting solute carrier family 2 member 3 (SLC2A3), interferon gamma receptor 2 (IFNGR2), PDE1B, ARID1B, and EIF3A, reduced or increased bacterial load significantly at 1 day p.i. but not significantly at day 2, suggesting that these proteins are more important for the bacterial internalization or early stage of infection. Meanwhile, most siRNAs reduced or increased bacterial load significantly at both 1 and 2 days p.i., suggesting that these proteins are important for both early and intermediate stages of infection. Moreover, knockdown of some TRP-interacting proteins had a dramatic effect on infection, suggesting that they play key roles. For example, knockdown of SPTA1, ILF3, CLC, UBB, SEPX1, and PCGF5 reduced or increase the infection very significantly at both 1 and 2 days p.i., suggesting that they are critical factors exploited by Ehrlichia TRPs for bacterial survival; however, we cannot exclude the possibility that siRNAs may have different knockdown efficiency. We also found that the knockdown of any single target protein by RNA interference could not abolish the ehrlichial growth completely. As observed by Western blot, 100% knockdown of a target protein by siRNA is very difficult, thus, a knockout cell line would be necessary to determine if a host protein is essential for E. chaffeensis infection.

The molecular mechanisms by which Ehrlichia enters and survives in the host cell remain unclear. This study documents the extensive effector-host interactions that contribute to survival of this obligately intracellular pathogen, and will help understand the molecular mechanisms utilized by other intracellular pathogens to infect host cells. Further studies will delineate the specific roles of these complex and diverse TRP-host interactions during infection.

TL and PD designed, performed the experiments and analyzed the data. TL wrote the manuscript. JM directed and contributed to the writing of the manuscript.

This work was supported by grants AI106859 and AI126144 from the National Institute of Allergy and Infectious Diseases (NIAID), and by funding from the Clayton Foundation for Research (to JM).