Ehrlichia TRP effectors: moonlighting, mimicry and infection.

Intracellular bacteria have evolved various strategies to evade host defense mechanisms. Remarkably, the obligately intracellular bacterium, Ehrlichia chaffeensis, hijacks host cell processes of the mononuclear phagocyte to evade host defenses through mechanisms executed in part by tandem repeat protein (TRP) effectors secreted by the type 1 secretion system. In the past decade, TRP120 has emerged as a model moonlighting effector, acting as a ligand mimetic, nucleomodulin and ubiquitin ligase. These defined functions illuminate the diverse roles TRP120 plays in exploiting and manipulating host cell processes, including cytoskeletal organization, vesicle trafficking, cell signaling, transcriptional regulation, post-translational modifications, autophagy and apoptosis. This review will focus on TRP effectors and their expanding roles in infection and provide perspective on Ehrlichia chaffeensis as an invaluable model organism for understanding infection strategies of obligately intracellular bacteria.

INTRODUCTION

Nearly 30 years ago, Ehrlichia chaffeensis (E.ch.) was identified as an emerging tick-borne pathogen responsible for the life-threatening zoonosis and human monocytic ehrlichiosis (HME) (Anderson et al. 1992). E.ch. is a Gram-negative, obligatory intracellular bacterium capable of surviving and replicating within mononuclear phagocytes (Paddock and Childs 2003). Previous reviews have provided broad overviews of E.ch. pathogenesis. This review will focus on more recent findings regarding the continually expanding roles of various E.ch. effector proteins and illuminate the versatile and important roles of E.ch. tandem repeat proteins (TRPs) in reprogramming the host cell to promote intracellular infection.

E.ch. exhibits tropism for mononuclear phagocytes which are crucial for innate host defenses, thus it is important to understand the sophisticated immunoevasion strategies that have evolved within the confines of a small genome (∼1.3 Mb). The E.ch. genome encodes just 882 proteins, containing low GC content (∼30%), various tandem repeats (TRs) and long non-coding regions (Andersson and Andersson 1999). The creation and deletion of TRs occur through an unknown mechanism that is compatible with DNA slippage (Dunning Hotopp et al. 2006). DNA TRs are small (12 bp) or large (100–300 bp) and are known to play a critical role in gene expression and phase variation. Further, TRs found in proteins encoded by the genome allow E.ch. adaptation to the host and are phylogenetically distinct from TRs found in other Ehrlichia species (Frutos et al. 2006).

TRs are found in proteins throughout all kingdoms but have been best recognized in multicellular eukaryotes where they have evolved to create functional diversity. The diversity of TRs gives rise to structures capable of interacting with a variety of binding partners (Lin, Hsu and Chang 2012). Well recognized examples in eukaryotes include the C2H2-type zinc finger, WD40 repeats and leucine rich repeat (Björklund, Ekman and Elofsson 2006). They interact with various proteins, small molecules, DNA and RNA to mediate an array of biological outcomes, including cell adhesion, protein folding, signal transduction, immune response, transcription, RNA processing and apoptosis (Lin, Hsu and Chang 2012). The E.ch. genome reveals an array of genes involved in host–pathogen interactions, including genes that encode tandem and ankyrin repeat containing proteins (Wakeel et al. 2011). Although extensive research on TRs has been focused on eukaryotic organisms, there has been a rapid increase in information regarding pathogen utilization of TRs to interface with the host cell. Many pathogenic bacteria are known to possess TRs, and the variation of the repeat domains generates functional and antigenic diversity (Lin, Hsu and Chang 2012). Streptococcus is a well characterized group of pathogens that employ TR surface-associated proteins and secreted effectors for infection. One study identified 52 streptococcal genomes in which 3748 proteins were identified to contain TRs. Highly repeated sequences within bacterial proteins have been associated with increased virulence, implying that Streptococcus tandem domain-containing proteins may be involved in the pathogenesis of streptococci (Scholze and Boch 2011; Lin, Hsu and Chang 2012).

TRP effectors and the type 1 secretion system

During infection, E.ch. utilizes both a type 1 secretion system (T1SS) and a type 4 secretion system (T4SS). E.ch. contains genes which code for VirB and VirD proteins associated with the inner membrane channel and ATPase components of T4SS. Many hypothetical T4SS substrates exist as well as confirmed substrates including Etf-1 and Etf-2, which are involved in apoptosis and endosomal maturation respectively (Rikihisa 2017; Yan et al. 2018). However, perhaps less studied and underappreciated, particularly with respect to intracellular bacteria, is the role of the T1SS during infection. Notably, E.ch. secretes many effector proteins by the type 1 secretion system (T1SS) as recently predicted by bioinformatic analysis (Luo et al. 2020). TRPs contain type 1 secretion signal sequences located in the C-terminal domain and were the first ehrlichial proteins to be identified as T1SS substrates using an E. coli complemented with the hemolysin secretion system (Fig. 1; Wakeel et al. 2011). The T1SS is widely utilized by Gram-negative bacteria and is employed to secrete various exotoxins, adhesins and enzymes (Green and Mecsas 2016). TRPs have many similarities with the repeats-in-toxins (RTX) family comprised of exotoxins, lipases, S-layer proteins and adhesins. The features consistent between TRPs and RTX members are glycine and aspartate rich tandem repeats, a non-cleavable C-terminal T1SS signal, homology with ATP-transporters and acidic pls (Wakeel et al. 2011). Both TRP and RTX family members utilize the T1SS to employ various proteins, which identifies the emerging importance and role of T1SS effectors in promoting intracellular infection.

Figure 1.

E. chaffeensis TRP effectors. Schematic of E. ch. TRPs illustrating TR domains and other important features. TRPs contain molecularly distinct TR domains that vary in sequence, length and number. TRPs are secreted by the T1SS and have predicted type 1 secretion signals located in the terminal amino acids of the C-terminal domain. The TRP effectors are nucleomodulins that bind host cell DNA via TR DNA binding domains to modulate host gene transcription. TRPs are modified by PTMs and sites of ubiquitination, SUMOylation and phosphorylation have been identified. TRP120 is a HECT E3 ubiquitin ligase and contains a conserved catalytic site in the C-terminal domain, allowing it to ubiquitinate and target host proteins for degradation.

The T1SS is an ATP-binding cassette (ABC) transporter that forms a channel for one-step secretion of effector proteins from the bacterial cytoplasm to the extracellular environment. It consists of an ATP-binding cassette protein (ECH0383), a TolC outer membrane protein (ECH1020) and a membrane fusion protein (ECH0970), all of which are located within the cell envelope and are essential components of the secretion nanomachine (Delepelaire 2004). The membrane fusion protein is an adaptor that contains a cytoplasmic domain at the N-terminus, a membrane anchor and a periplasmic domain to connect the outer and inner membrane components of the T1SS, in response to the substrate binding the cytoplasmic side. Additionally, the TolC outer membrane protein is a trimeric protein that is responsible for channel formation throughout the outer membrane and periplasm. Remarkably, the ATP-binding cassette protein is fused to a transmembrane domain and recognizes the substrate's secretion signal, assuring that only specific substrates are recognized. The secretion signal is located at the C-terminal, and although the exact region is not defined, the T1SS substrates typically contain repeat sequences that are enriched in [LDAVTSIF] amino acids and occasionally comprised of [KHPMWC] amino acids within the 50 amino acid C-terminal region of the protein (Delepelaire 2004).

Localization of TRPs has been demonstrated on the surface of ehrlichiae as well as extracellularly. Although the T1SS is traditionally recognized as a one step process, recent investigations describe a two-step process or intermediate step that stalls protein secretion resulting in surface localization (Spitz et al. 2019). A primary example of the intermediate step has been demonstrated in E. coli. The discovery of a retention module (RM) at the N terminus was found to anchor the adhesin to the cell surface to stall further translocation, leaving a stalled plug in the periplasm, most likely wedged in TolC and the translocated E. coli adhesin in the extracellular space. During unfavorable conditions where biofilm formation cannot occur, the RM is removed by proteolysis and LapA is secreted. Therefore, the adhesin–TolC–RM complex occurs as a pseudoperiplasmic intermediate. The RTX adhesin model identifies the secretion of an unfolded substrate with its C terminus facing the OM protein. Ca2+ ions bind the GG repeats to induce folding of the substrate into a β-roll (Spitz et al. 2019). In the case of E. coli LapA and IBA substrates, the N-terminal domain folds before secretion completes to plug the translocon, demonstrating a two-step process (Spitz et al. 2019). The two-step process describes a mechanism in which secreted proteins may act as both a surface protein and an effector protein. The documented surface localization and secreted forms of E.ch. TRP120 suggests it uses this type of multi-mechanistic protein secretion via the T1SS.

TRPs and the pathogen–host interface

E.ch. has emerged as a model organism for understanding the pathobiology of intracellular bacteria, and for investigating the role of TRP effectors (Rogan et al. 2019). TRPs are major immunoreactive proteins that elicit strong host antibody responses and are known to interact with many host cell proteins during infection (Tables 1–3). Using Y2H analyses, an array of pathogen–host interactions involved in diverse cellular processes have been identified. Notably, TRP32, TRP47, TRP75 and TRP120 interact with host proteins associated with cell signaling, cytoskeleton organization, vesicle trafficking and intracellular transport, transcriptional regulation, PTMs and apoptosis (Luo, Dunphy and McBride 2017; Luo, Mitra and McBride 2018).

Table 1.

TRP–host protein interactions that influence transcriptional regulation and PTMs.

	Host protein		Function
TRP	Symbol	Full name	Transcriptional regulation and PTMs
TRP32	DAZAP2	DAZ-associated protein 2	Binds various proteins to regulate transcription
	HHEX	Hematopoietically-expressed homeobox protein	Transcription factor
TRP47	ARID2	AT-rich interactive domain-containing protein 2	SWI/SNF chromatin remodeling complex
	HDAC2	Histone deacetylase 2	Deacetylation of lysine residues
	PIWIL4	Piwi-like protein 4	piRNA metabolic process
	STAT5A	Signal transducer and activator of transcription 5A	Signal transduction and activation of transcription
	STAT6	Signal transducer and activator of transcription 6	Signal transduction and activation of transcription
	STT3B	Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit	Catalytic subunit of the oligosaccharyl transferase complex
	TFEC	Transcription factor EC	Transcriptional regulator: repressor, activator
TRP75	AFF1	AF4/FMR2 family member 1	Gene expression regulation
	CSDE1	Cold shock domain containing E1	RNA-binding protein
	FAM208A	Protein TASOR	Epigenetic repression regulation
	GMEB1	Glucocorticoid modulatory element-binding protein 1	Trans-acting factor
	HERC2	E3 ubiquitin-protein ligase	Ubiquitin-dependent retention regulator
	KDM3B	Lysine-specific demethylase 3B	Histone demethylase
	PIAS1	E3 SUMO-protein ligase PIAS1	E3-type small ubiquitin-like modifier (SUMO) ligase
	PPP1R11	E3 ubiquitin-protein ligase PPP1R11	E3 ubiquitin-protein ligase
	STAT3	Signal transducer and activator of transcription 3.1	Transcription factor
	UBE2I	SUMO-conjugating enzyme	SUMO activity
	USP15	Ubiquitin carboxyl-terminal hydrolase 15	Hydrolase, removes conjugated ubiquitin from target proteins
	USP3	Ubiquitin carboxyl-terminal hydrolase 3	Hydrolase, deubiquitinates monoubiquitinated target proteins
	USP8	Ubiquitin carboxyl-terminal hydrolase 8	Hydrolase, removes conjugated ubiquitin from target proteins
TRP120	ARID1B	AT-rich interactive domain-containing protein 1B	SWI/SNF chromatin remodeling complex, represses Wnt
	ATAD2B	ATPase family AAA domain-containing protein 2B	Chromatin/histone binding
	BAHCC1	BAH and coiled-coil domain-containing protein 1	Chromatin binding
	BMP2K	BMP-2-inducible protein kinase	Phosphatase regulator activity
	BTBD6	BTB/POZ domain-containing protein 6	Adapter protein for the cul3 E3 ubiquitin-protein ligase complex
	CAND1	Cullin-associated NEDD8-dissociated protein 1	Assembly factor of SKP1-CUL1-F-box E3 ubiquitin ligase complexes
	CDK12	Cyclin-dependent kinase 12	Transcription elongation
	CLK1	Dual specificity protein kinase CLK1	Phosphorylates serine/arginine-rich proteins of spliceosomal complex
	DDX5	Probable ATP-dependent RNA helicase	Alternative regulation of pre-mRNA splicing
	FUS	RNA-binding protein FUS	Transcription regulation, RNA splicing, RNA transport, DNA repair
	HNRNPA2B1	Heterogeneous nuclear ribonucleoproteins A2/B1	Packaging pre-mRNAs into hnRNP particles
	ILF3	Interleukin enhancer-binding factor 3	RNA-binding protein
	KDM6B	Lysine-specific demethylase 6B	Histone demethylase
	KLHL12	Kelch-like protein 12	Substrate-specific adapter of BTB-CUL3-RBX1 E3 ubiquitin ligase
	MBNL1	Muscleblind-like protein 1	Pre-mRNA alternative splicing regulation
	NSD1	Histone–lysine N-methyltransferase, H3 lysine-36 specific	Histone methyltransferase
	OTUB1	Ubiquitin thioesterase OTUB1	Hydrolase with regulatory role in protein turnover
	PPP6R1	Serine/threonine-protein phosphatase 6 regulatory subunit 1	Regulatory subunit of protein phosphatase 6
	SFRS2	Serine/arginine-rich splicing factor 2	Splicing of pre-mRNA
	TRIM24	Transcription intermediary factor 1-alpha	Transcriptional coactivator
	UBC	UBC core domain-containing protein	Ubiquitin-protein transferase activity, ATP binding, DNA binding
TRP120/TRP47	PCGF5	Polycomb group RING finger protein 5	Component of Polycomb group multiprotein PRC1-like complex
	UBB	Polyubiquitin-B	Conjugates to target proteins for various functions
TRP120/TRP75	IRF2BP2	Interferon regulatory factor 2-binding protein 2	Transcription corepressor

Table 3.

TRP–host protein interactions to influence apoptosis.

	Host protein		Function
TRP	Symbol	Full name	Apoptosis
TRP32	CD14	Carbonic anhydrase 1	Cell signaling
	GLCCI1	Glucocorticoid-induced transcript 1 protein	Apoptotic function
	TP53I11	Tumor protein p53-inducible protein 11	Negative regulation of cell population proliferation
TRP47	CDK1	Cyclin dependent kinase 1	Cell cycle modulator
	CAP1	Adenylate cyclase associated protein 1	Homeostasis
	GNB1	G protein subunit beta 1	Apoptotic function and cell proliferation
	HDAC2	Histone deacetylase 2	Negative regulator of apoptosis and cell differentiation
	PTPN2	Protein tyrosine posphatase, non-receptor type 2	Homeostasis and positive apoptosis regulation
	STAT5A	Signal transducer and activator of transcription 5A	Cell proliferation regulator
	STAT6	Signal transducer and activator of transcription 6	Cell proliferation regulator
TRP75	PRDX3	Thioredoxin-dependent peroxide reductase, mitochondrial	Homeostasis and cell proliferation
	TPT1	Translationally-controlled tumor protein	Negative regulation of apoptotic process
TRP120	ADAM17	ADAM metallopeptidase domain 17	Cell signaling and regulator of apoptosis and cell proliferation
	CAT	Catalase	Metabolism
	CXCL12	C-C-C motif chemokine ligand 12	Negative regulation of apoptosis
	DDX5	DEAD-box helicase 5	Transcriptional regulation
	ERAL1	Era-like 12S mitachondrial rRNA chaperone 1	Mitochondrial protection
	FBXW7	F-box and WD repeat domain containing 7	Cell signaling, apoptosis regulator and PTM
	ICAM3	Intercellular adhesion molecule 3	Vesicle trafficking and PTM
	IRF2BP	Interferon regulatory factor 2 binding protein 2	Transcriptional corepressor and negative regulator
	KDM6B	Lysine demethylase 6B	Cell fate commitment
	KRAS	KRAS proto-oncogene, GTPase	Homeostasis and negative regulator of apoptosis
	LGALS1	Galectin 1	Regulator of apoptosis, cell proliferation and cell differentiation
	ORAOV1	Oral cancer overexpressed 1	Biogenesis
	PDE1B	Phosphodiesterase 1B	Apoptosis regulation
	PPP3R1	Protein phosphatase 3 regulatory subunit B, alpha	Apoptotic signaling pathway and Wnt signaling
	SEPX1	Selenoprotein X, 1	Innate immune response
	SPTA1	Spectrin alpha and erythrocytic 1	Cell proliferation and cell shape regulator
	TRIM24	Tripartite motif containing 24	Apoptosis regulation. Negative regulation of cell proliferation
TRP120/TRP32	EEF1A1	Eukaryotic translation elongation factor 1 alpha 1	Autophagy and GTPase activity
	IGHA1	Immunoglobulin heavy constant alpha 1	Innate immune response
	IGLL5	Immunoglobulin lambda like polypeptide 5	Innate immune response and phagocytosis
TRP120/TRP47	CLC	Charcot-Leyden crystal galectin	Protein aggregate and cytotoxic
	IGKC	Immunoglobulin kappa constant	Innate immune response

TRP32

Studies indicate that TRP32 has many functional roles in reprogramming host cellular processes through host protein interactions, and that nearly all TRP32 interacting partners promote infection (Luo et al. 2017). Y2H analysis determined that TRP32 has many binding partners with varying functions, including elongation factor 1 alpha (EF1A), immunoglobulin heavy constant alpha 1 (IGHA1), DAZ-associated protein 2 (DAZAP2), p53 inducible protein 11 (TP53I11) and hematopoietically expressed homeobox (HHEX; Tables 1–3; Luo et al. 2011; Luo and McBride 2012; Farris et al. 2016). EF1A1 is one of the most abundant proteins in eukaryotes and has many functional roles including cytoskeletal remodeling, apoptosis, translation and enzyme regulation. Others include IGHA1, involved in antigen binding, transcription factor DAZAP2 that functions during canonical Wnt signaling, TP53I11 which plays a role in inducing apoptosis, and HHEX a homeobox protein involved in hematopoietic cell differentiation (Maruyama et al. 2007; Lukas et al. 2009; Goodings et al. 2015).

TRP47

E.ch. effector TRP47 was the first ehrlichial TRP examined using the Y2H approach to identify pathogen–host interactions (Wakeel et al. 2010). TRP47 was found to interact with an array of host proteins that positively influence infection (Tables 1–3). TRP47 interacting partners included host proteins with functional roles in cell signaling, vesicle trafficking, intracellular transport, metabolism, PTMs and transcription (Luo et al. 2017). The effector also interacts with both actin binding protein (CAP1) and the Src family tyrosine kinase, Fyn (Wakeel et al. 2010). TRP47 interactions with CAP1 at the morula membrane interface modify CAP1 distribution, which may heavily influence host cell homeostasis. Additionally, TRP47 binds cofilin, actin, SH3 domain, adenylyl cyclase and profilin to potentially guide receptor-mediated endocytosis and vesicle trafficking (Kibler et al. 2018).

TRP75

E.ch. TRP75 which has been shown to interact with a variety of host cell targets that regulate cell signaling, vesicle trafficking, intracellular transport, cytoskeleton organization, metabolism, PTMs and cellular homeostasis (Tables 1–3). TRP75 is tyrosine phosphorylated and predicted to be a lipoprotein, based on its lipobox sequence in the N-terminal region (Luo, Mitra and McBride 2018). Y2H analysis identified TRP75 interaction with 13 human proteins, including solute carrier family 4 member 7 (SLC4A7), actin binding and actin related proteins; actin-related protein 2/3 complex subunit 5 (ARPC5), lymphocyte cystolic protein 1 (LCP1), pleckstrin (PLEK), tropomyosin 4 (TPM4) and apoptosis regulators; eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), integrin subunit beta 2 (ITGB2), peroxiredoxin 3 (PRDX3), protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1), proteasome 26S subunit, ATPase 5 (PSMC5), RB1-inducible coiled-coil 1 (RB1CC1), selenoprotein W,1 (SEPW1) and signal transducer and activator of transcription 3 (STAT3; Luo, Mitra and McBride 2018). Although the role of TRP75 has not been fully elucidated, like other TRPs, interactions with various host targets suggests an important role in regulating host cellular processes to promote infection.

TRP120

Y2H studies of E.ch. TRP120 have identified a multitude of molecular interactions between TRP120 and host proteins including a diverse group of eukaryotic proteins involved in multiple cellular processes, including cell signaling, vesicle trafficking, PTMs, transcriptional regulation, apoptosis and homeostasis (Tables 1–3; Luo et al. 2011). Many of these interactions have been investigated in detail confirming the role of TRP120 in modulating host cell processes through interactions with host cell proteins. TRP120 modulates Notch signaling through direct interactions with ADAM17 and FBXW7, exploits host PTM machinery through interactions with NEDD4L and UBC9, and highjacks chromatin remodeling via its interaction with PCGF5 to regulate transcription (Dunphy, Luo and McBride 2014; Mitra et al. 2018; Wang et al. 2020). These novel interactions described in more detail in this review indicate that TRP120 has complex and diverse functions, and further research will elucidate the unique mechanisms underlying these host–pathogen molecular interactions and their importance in ehrlichial pathobiology.

TRP posttranslational modifications

TRP120 effector activity is due in part to its ability to exploit host cell machinery to acquire PTMs such as ubiquitin (Ub) and SUMO (Dunphy, Luo and McBride 2014; Zhu et al. 2017). Notably, studies have revealed that TRP120 is both intrinsically ubiquitinated and ubiquitinated by host HECT ligase activity to enhance interactions between TRP120 and host cell targets to promote infection. TRP120 utilizes intrinsically and extrinsically generated PTMs to promote effector–host interactions (Zhu et al. 2017). Studies have demonstrated that human HECT E3 Ub ligase NEDD4L interacts with and facilitates modification of TRP120 with ubiquitin (Fig. 2; Wang et al. 2020). In addition, TRP120 is selectively conjugated with SUMO2/3 isoforms and ehrlichial inclusions co-localize with SUMO1 and UBC9 (Fig. 2; Dunphy, Luo and McBride 2014). Identified TRP120 interacting host proteins contain SUMO interacting motifs (SIMs) that are short hydrophobic domains decorated with acidic residues essential for SUMO-mediated protein interactions. TRP120 exhibits various interactions with SIM-containing proteins including cytoskeleton component Myo10 (unconventional myosin) and GGA1 (Golgi-localizing, γ-adaptin ear domain homology and Arf-binding protein) recruitment and trafficking regulator, indicating that SUMOylation contributes to the numerous molecular interactions between TRP120 and host proteins (Dunphy, Luo and McBride 2014; Zhu et al. 2017). Initial studies demonstrated that TRP120 is conjugated to SUMO at a carboxyl-terminal canonical consensus SUMO conjugation motif and is specifically SUMOylated at Lys 432 to facilitate interactions with PCGF5 and other host proteins. Inhibition of the SUMO pathway negatively impacts E.ch. infection and prevents PCGF5 interaction, indicating that SUMOylation is critical in this regard (Dunphy, Luo and McBride 2014). Exploitation of the SUMO pathway to mediate the effector–host interactions demonstrates the importance of acquiring such PTMs for pathogen–host interactions that promote intracellular infection.

Figure 2.

TRP120 exploitation of host PTM pathways and host protein interactions. TRP120 is SUMOylated at canonical SUMO motif by host cell PTM machinery (UBC9), which promotes the direct interaction between Myo10 and GGA1. TRP120 auto-ubiquitinates via intrinsic HECT E3 Ub ligase activity and interacts with host NEDD4L to mediate self-ubiquitination. In the nucleus, TRP120 uses Ub ligase activity to target FBW7and PCGF5 for Ub-mediated degradation. TRP120 binds to FBW7 in a trans conformation and ubiquitinates with K48-Ub chains, resulting in the upregulation of Notch genes and oncoproteins for cell survival. SUMOylated TRP120 binds PCGF5 resulting in PCGFs degradation and the upregulation of HOX genes.

Effectors TRP120 and AmpA of Ehrlichia and Anaplasma were the first reported examples of bacterial proteins post translationally modified with SUMO (Dunphy, Luo and McBride 2014; Beyer et al. 2015). Previous studies had shown that other pathogens utilize PTMs to interface with the host cell. They mimic, inhibit and serve as substrates of the SUMOylation and Ub pathways to modulate host cellular functions. Several Gram-negative bacteria directly target SUMOylation as a survival strategy, including the multi-drug resistant bacteria, K. pneumoniae, which prevents SUMOylation of host target proteins to subvert innate immunity of the host cell. K. pneumoniae increases deSUMOylase SENP2 levels in the cytosol through K48 ubiquitylation and degradation by the ubiquitin proteasome (Sá-Pessoa et al. 2020). The direct SUMOylation of host proteins by intracellular bacteria was recently determined in studies where S. Typhimurium mediated the host endocytic vesicular transport pathway (VTP) through SUMOylation of RAB7, a key component of VTP (Mohapatra et al. 2019).

TRP nucleomodulins

Many E.ch. effectors translocate to the host cell nucleus including tandem and ankyrin (Anks) repeat proteins TRP32, TRP120, TRP47 and Ank200 and thus are considered nucleomodulins (Wakeel et al. 2011; Luo, Dunphy and McBride 2017; Rogan et al. 2019). E.ch. Ank200 was the first ehrlichial effector identified as a nucleomodulin, due to its localization in the nucleus and ability to directly bind genomic Alu elements (AT-rich regions) responsible for controlling ATPase activity, transcriptional regulation and cell fate (Zhu et al. 2009). More recently, TRP32, TRP120 and TRP47 have been identified as nucleomodulins (Table 4). TRPs appear to modulate gene expression using various mechanisms, including direct binding through the protein–DNA complexes, interacting with host proteins to modify epigenetics and degrading nuclear host ubiquitin ligases such as FBW7 to upregulate genes associated with cell survival (Kibler et al. 2018; Klema et al. 2018; Mitra et al. 2018; Wang et al. 2020).

Table 4.

Gene regulation of TRP nucleomodulins.

	Host protein		Function
TRP	Symbol	Gene description
TRP32	AKT3	AKT Serine/Threonine Kinase 3	Cell signaling and glycogen synthesis
	ATF4	Activating Transcription Factor 4	Transcriptional regulation
	BTK	Bruton Tyrosine Kinase	Cell signaling
	CALM2	Calmodulin 2	Cell signaling and homeostasis
	FOS	Fos Proto-Oncogene, AP-1 Transcription Factor Subunit	Cell proliferation and differentiation and transformation
	JUN	Jun Proto-Oncogene, AP-1 Transcription Factor Subunit	Cell signaling
	MALAT1	Homo sapiens metastasis associated lung adenocarcinoma transcript 1	Controls cell cycle via B-MYB and mRNA processing
	MED1	Mediator Complex Subunit 1	Transcriptional regulation
	MIR142	Homo sapiens miRNA 142	Homeopoietic cell development and function
	MIR17HG	Homo sapiens miR-17–92 cluster	Cell proliferation and differentiation
	MIR21	Homo sapiens miRNA 21	Oncomir, anti-apoptotic, targets: PTEN, Bcl2 and TGFBRII
	MIR200C	Homo sapiens miRNA 200c	MET progression, TLR, targets: IKBKB, KRAS and MYD88
	MIR505	Homo sapiens miRNA 505	Inhibits cell proliferation and induces apoptosis
	NRAS	NRAS Proto-Oncogene, GTPase	Vesicle trafficking
	RPS23	Ribosomal Protein S23	Protein synthesis
	TNFAIP	TNF Alpha-induced Protein 3	Cell signaling
TRP47	ACTG1	Actin Gamma 1	Cell motility and cytoskeleton organization
	ACTR2	Actin Related Protein 2	Cell motility
	ARTN	Artemin	Gene regulation
	CACNA	Calcium Voltage-Gated Channel Subunit Alpha	Calcium influx
	CACNA1G	Calcium Voltage-Gated Channel Subunit Alpha1 G	Calcium influx
	CAPZB	Capping Actin Protein of Muscle Z-Line Subunit Beta	Actin filament regulation
	CD74	CD74 Molecule	Immune response
	DMTN	Dematin Actin Binding Protein	Cytoskeleton organization
	EZR	Ezrin	Cell adhesion, migration and organization
	GFRA2	GDNF Family Receptor Alpha 2	Cell fate and differentiation
	KCNA10	Potassium Voltage-Gated Channel Subfamily A Member 10	Potassium transport
	KCNA2	Potassium Voltage-Gated Channel Subfamily A Member 2	Potassium transport
	SLC2A1	Solute Carrier Family 2 Member 1	Glucose transporter
	SPTB	Spectrin Beta, Erythrocytic	Cytoskeleton organization
	TIRAP	TIR Domain Containing Adaptor Protein	Cell signaling and immune response
	TNF	Tumor Necrosis Factor	Immune response
TRP120	ADAM17	ADAM Metallopeptidase Domain 17	Notch and homeostasis
	ADRBK1	Adrenergic, beta and receptor kinase 1	Transcriptional regulation and cytoskeletal organization
	CARD9	Caspase recruitment domain family, member 9	Apoptosis
	CD79	CD79 molecule	Adhesion, leukocyte recruitment and activation
	ERN1	Endoplasmic Reticulum to Nucleus Signaling 1	Protein folding
	FOXA2	Forkhead Box A2	Transcriptional regulation
	GTF2H1	General Transcription Factor IIH Subunit 1	Transcriptional regulation
	IKBKB	Inhibitor of kappa light polypeptide gene enhancer in B cells	Cell signaling
	Jak2	Janus kinase 2	Cell signaling
	NOTCH1	Homo sapiens notch1	Notch, homeostasis
	NPM2	Nucleoplasmin 2	Chromatin reprogramming
	LRP5	Low-density lipoprotein receptor-related protein 5	Wnt and homeostasis
	PTK2	Protein tyrosine kinase 2	Cell signaling
	TLR5	Toll-like receptor 5	Innate immune response
	TNFRSF14	Tumor necrosis factor receptor superfamily, member 14	Inflammatory response
	TNFRSF9	Tumor necrosis factor receptor superfamily, member 9	Cell proliferation and inflammatory response
	ZNF670	Zinc finger protein 670	Transcriptional regulation
	ZNF250	Zinc finger protein 250	Transcriptional regulation
	ZNF684	Zinc finger protein 250	Transcriptional regulation
	ZNF282	Zinc finger protein 282	Transcriptional regulation
TRP120/TRP47/TRP32	BMP8B	Bone Morphogenetic Protein 8b	Cell signaling
	CAP1	Cyclase Associated Actin Cytoskeleton Regulatory Protein 1	Cell signaling and cytoskeleton
	CD20	Membrane Spanning 4-Domains A1	Cell differentiation
	CITED4	Cbp/P300-Interacting Transactivator 4	Transcriptional regulation
	CLDN19	Claudin 19	Cell adhesion
	COL9A2	Collagen Type IX Alpha 2 Chain	Collagen structure
	CTPS1	CTP Synthase 1	Biosynthesis
	FOXJ3	Forkhead Box J3	Transcriptional regulation
	GUCA2A	Guanylate Cyclase Activator 2A	Cell signaling
	IRF2BP2	Interferon Regulatory Factor 2 Binding Protein 2	Transcriptional regulation
	KCNQ4	Potassium Voltage-Gated Channel Subfamily Q Member 4	Potassium channel
	LMNA	Lamin A/C	Cell structure
	MFSD2A	Major Facilitator Superfamily Domain Containing 2A	Sodium transportation
	MYCL	MYCL Proto-Oncogene, BHLH Transcription Factor	Transcriptional regulation
	NFYC	Nuclear Transcription Factor Y Subunit Gamma	Transcriptional regulation
	PPT1	Palmitoyl-Protein Thioesterase 1	Lysosomal degradation
	PSMB1	Proteasome 20S Subunit Beta 1	Protein degradation
	RIMS3	Regulating Synaptic Membrane Exocytosis 3	Exocytosis regulation
	RLF	RLF Zinc Finger	Transcriptional regulation
	TRIT1	TRNA Isopentenyltransferase 1	Translation regulation

TRP32 is considered a nucleomodulin because of its ability to control the host cell through direct interactions with host target genes within the nucleus. TRP32 binds G-rich motifs with GGTGGC-like sequence repeats and targets genes that mediate cell signaling, transcription, cell proliferation/differentiation and apoptosis (Table 4; Farris et al. 2018). Further analysis has demonstrated that TRP32 lysine residues are sites of ubiquitination by host ubiquitin machinery. Although TRP32 lacks a PPxY motif, the effector is modified by host protein NEDD4L, similarly to TRP120. NEDD4L-mediated ubiquitination of TRP32 promotes nuclear localization and transcriptional repressor function (Farris et al. 2016). In addition, TRP32 is phosphorylated at Y179, located in the C-terminal tri-tyrosine motif, which plays a role in directing TRP32 nuclear translocation (Farris et al. 2016).

TRP47 has most recently been identified as a nucleomodulin and the gene encoding TRP47 is the most highly expressed ehrlichial gene during infection in mammalian cells (Kuriakose et al. 2011). TRP47 has a TR domain, consisting of seven 19-mer (ASVSEGDAVVNAVSQETPA) TRs within the C-terminal region of the protein (Luo et al. 2010; Fig. 1). The TR region shows homology with various eukaryotic proteins including DNA polymerase III subunit gamma and tau-conserved domain, ribonuclease E and renin receptor/ATP6AP2/CAPER protein (McBride and Walker 2013). TRP47 translocates to the nucleus at least in part by utilizing a MYND-binding domain-dependent mechanism to bind enhancers of host genes (Kibler et al. 2018). Consistent with other TRPs, the tandem repeat domain of TRP47 binds host DNA and primarily targets genes that influence cell signaling, immune responses, cytoskeleton organization and glucose/potassium transport to promote infection (Table 4).

E. ch. TRP120 was the second ehrlichial nucleomodulin identified and shown to directly bind host DNA. Notably, sequence analysis has revealed that TRP120 does not contain DNA-binding domains typical of eukaryotic transcription factors. Instead, TRP120 binds GC-rich DNA in an ordered structure to form a protein–DNA complex (Klema et al. 2018). Studies demonstrate that TRP120 is highly acidic, which allows its interaction with negatively charged DNA to regulate transcription. In addition, TRP120 may recruit host proteins to stabilize repulsive interactions between TRP120 and varying pH regions of DNA, further promoting transcriptional regulation (Klema et al. 2018). As a nucleomodulin, TRP120 binds DNA to regulate multiple functions, including cell signaling, cytoskeletal organization, transcription, translation and apoptosis (Table 4). Additionally, TRP120 interacts with an array of chromatin-modifying proteins, including proteins of the SWI/SNF chromatin remodeling complex and polycomb comb group (PcG) proteins to positively influence infection (Zhu et al. 2017).

Notably, E.ch. nucleomodulins resemble Xanthomonas transcription activator-like (TAL) effectors, which are unique proteins that contain predictable and modifiable sequences, which makes them a DNA-targeting technology (Zhou, Aertsen and Michiels 2014). TALs are secreted via a T3SS into plant cells, where they translocate to the nucleus and directly bind target promoters to induce gene expression favorable for infection (Scholze and Boch 2011). TALs are used in a variety of DNA-specific applications, including DNA probing, mutation, activation, repression and replacement (Zhou, Aertsen and Michiels 2014; Rinaldi et al. 2017). Further understanding the intricate role of bacterial nucleomodulins, including TALs and TRPs, will potentially impact the medical field through the discovery of novel therapeutics.

TRP120 moonlighting portfolio

E.ch. TRP120 is the most characterized TRP and has numerous interactions with host proteins to promote E.ch. survival (Tables 1–3). In the past decade, the various functions of TRP120 as a moonlighting protein have been well defined and demonstrate that E.ch. relies on this effector to reprogram the host cell (Wang et al. 2020). TRP120 is a model moonlighting protein that utilizes intricate molecular strategies to mediate host cell processes. To date, the well documented roles include ligand mimic, nucleomodulin and E3 ubiquitin ligase activity. Further, TRP120 has several other defined functions, including its roles in cell entry, cytoskeletal organization, vesicle trafficking, cell signaling, transcription regulation and apoptosis. In the section below, we will explore the known TRP120 moonlighting functions during infection in more detail.

TRP120 invasin

Several studies have identified mechanisms involved in E.ch. invasion of monocytes including Ca2+ signaling, actin filamentation and Wnt signaling (McBride and Walker 2013; Rogan et al. 2019). Surface protein DNaseX, and potentially other glycophosphatidylinositol (GPI)-anchored proteins associated with caveolae are involved in infectious dense-core cell (DC) ehrlichiae adherence and entry into host cells (Lin and Rikihisa 2003b; Mohan Kumar et al. 2015). Studies have determined that the C-terminus of EtpE (ECH1038) triggers E.ch. entry through its interactions with DNaseX, CD147, N-Wiskott-Aldrich syndrome protein (N-WASP) and hnRNP-K (Mohan Kumar et al. 2013, 2015). However, TRP120 expressed on infectious DC ehrlichiae has also been shown to play a role in ehrlichial host cell entry (Popov et al. 2000). Studies have demonstrated that E.ch. invasion of the host cell requires TRP120 and the stability of TRP120 is regulated by bacterial second messenger, cyclic di-GMP and ehrlichial surface serine protease, HtrA (Kumagai et al. 2010).

Recent studies have demonstrated that E.ch. internalization is dependent on Wnt signaling and TRP–receptor interactions (Rogan et al. 2019). TRP120 serves as an adhesin and interacts with Wnt FZD receptors on the host cell to activate the canonical and non-canonical Wnt signaling pathways to stimulate phagocytosis and entry (Luo et al. 2011; Rogan et al. 2021). TRP-coated microspheres-induced phagocytic uptake in macrophages; however, in the presence of a Wnt signaling small molecule inhibitor, the TRP coated microspheres failed to stimulate uptake phagocytosis (Luo et al. 2016). This data suggests that TRP-induced phagocytosis occurs via non-canonical Wnt signaling, which is further supported through studies demonstrating that Wnt5-FZD5-PI3K signaling induces the uptake of E. coli without negatively impacting infection (Maiti et al. 2012; Luo et al. 2016). Further evidence suggests that E.ch. hijacks the canonical and non-canonical Wnt signaling pathways via the FZD receptor. FZD knockdowns at 1-day p.i. significantly reduce E.ch. infection (Via et al. 2015).

Genes involved in membrane trafficking during E.ch. infection show decreased expression, including synaptosomal associated protein, 23 kDa (SNAP23), membrane of RAS oncogene family (Rab5A) and syntaxin 16 (STX16). Further, it is likely that TRP120 interacts with actins to promote entry, since inhibition of actin polymerization in E.ch. infected cells disrupts filopodia formation (Thomas et al. 2010) and TRP120 is known to interact with host proteins involved in cytoskeletal organization, including actin gamma 1 (ACTG1), actin related protein 2/3 complex (ARPC2) and unc-13 homolog D (UNC13D; Luo et al. 2011). E.ch. TRPs bind genes involved in cytoskeletal rearrangement and vesicle trafficking, such as syntaxins (SNX14, SNX11 and SNX17), clathrin (CTLA), TSNARE1 and caotomer (COPA). Thus, E.ch. TRP120 and other TRPs potentially modulate genes associated with cytoskeletal organization to facilitate ehrlichial entry, vesicular trafficking and exocytosis to promote infection.

TRP120 cellular signaling ligand mimetic

Recent studies have demonstrated that E.ch. exploits host cellular processes to establish a favorable niche through the activation of conserved cell signaling pathways, including the Notch and Wnt signaling pathways (Fig. 3). Other survival strategies employed by E.ch. include the suppression of tyrosine and mitogen activated protein kinase (MAPK) activity and the regulation of Toll-like receptors and transcription factors in monocytes and macrophages (Rogan et al. 2019). The activation of the Notch and Wnt signaling pathways are thought to occur through specific ligand–receptor interactions (e.g. Frizzled (FZD), Notch) to promote E.ch. survival (Guruharsha, Kankel and Artavanis-Tsakonas 2012; Hori, Sen and Artavanis-Tsakonas 2013; Luo et al. 2016; Rogan et al. 2019; Wang et al. 2020). Studies demonstrate that TRP120 likely serves as a ligand mimic to promote intracellular survival, providing a useful model to investigate intracellular bacterial reprograming of the host cell to create a favorable environment for infection.

Figure 3.

TRP-mediated activation of conserved signaling pathways and role in infection. TRPs act as ligand mimetics and interact with Notch and Wnt receptors to activate host cell signaling. On the cell surface, E.ch. TRP120 expressed on the surface of dense-cored ehrlichiae interacts with ADAM17 and possibly Notch receptor to activate Notch signaling. Similarly, TRP120 interacts with FZD receptors to activate canonical and non-canonical Wnt signaling to regulate apoptosis and autophagy. Notably, expression of the FZD5 receptor increases during infection. In addition, TRP32 interactions with Wnt transcription factor DAZAP2 to potentially influence Wnt gene transcription.

Notch signaling

In order to mediate the Notch signaling pathway, TRP120 interacts with a variety of host genes and proteins associated with Notch signaling (Fig. 3). The Notch signaling pathway is a conserved signaling pathway with critical roles in cellular homeostasis, including cell proliferation and differentiation (Hoyne 2003; Palaga et al. 2013; Barth and Köhler 2014; Song et al. 2015). Recent studies have identified Notch signaling as a pathway targeted by numerous intracellular bacteria for infection and survival, including Salmonella, Mycobacterium bovis and Bacillus anthracis, potentially due to Notch activity regulating innate and adaptive immune responses including inflammation, lymphocyte cell development, Toll-like receptor (TLR) expression and apoptosis (Narayana and Balaji 2008; Zhu et al. 2011; Larabee et al. 2013; Larabee and Ballard 2014; Lina et al. 2016). Notably, studies have determined that TRP120 directly activates Notch signaling resulting in the downregulation of innate immune sensing (Lina et al. 2016). Specifically, TRP120 activation of the Notch signaling pathway results in the downregulation of toll-like receptor (TLR) 2/4, which is caused by inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 expression (Hao et al. 2007; Welcker and Clurman 2008; Matsumoto et al. 2011).

Studies using pharmacological inhibitors and small interfering RNAs (siRNAs) against Notch activating proteins demonstrated that Notch signaling is required for ehrlichial survival (Zhu et al. 2011). In subsequent Y2H studies, TRP120 was found to interact with Notch pathway proteins ADAM17, NEDD4L and the host nuclear tumor suppressor F-BOX and WD domain repeating-containing 7 (FBW7; Wang et al. 2020). Further analysis regarding the interaction between FBW7 and TRP120 demonstrated that TRP120 directly interacts with FBW7 FBOX and WD40 domains and ubiquitinates FBW7 for degradation, which regulates Notch signaling and stabilizes oncoproteins involved in cell survival and apoptosis (Wang et al. 2020).

In addition to influencing PRR expression, TRP120 appears to exploit Notch signaling to prevent apoptosis for E.ch. survival (Wang et al. 2020). TRP120 has been specifically associated with proteins involved in the regulation of apoptosis, including eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), and cytochrome c oxidase subunit II (COX2), which indicated a potential role in influencing cell survival (Tsujimoto 1998; Carrington et al. 2017). Further, E.ch. infection promotes the upregulation of apoptotic inhibitors during infection, including NF-κB, IER3, MCL1, BCL-2 and BirC3, and the downregulation of apoptotic inducers, such as Bik and BNIP3L (Luo et al. 2011). Interestingly, the BCL-2 anti-apoptotic protein MCL1 level is increased during E.ch. infection due to Notch activation and degradation of the MCL-1 negative regulator FBW7 via TRP120 ubiquitination (Lin, Hsu and Chang 2012; Rinaldi et al. 2017). Therefore, E.ch. stabilizes and increases MCL1 levels for ehrlichial survival. Based on this information, E.ch. may be exploiting host cell intrinsic apoptosis to promote survival partially through TRP120 ubiquitination, effector–host protein interactions and modulation of host gene transcription.

Wnt signaling

TRP120 plays a major role in mediating Wnt signaling during infection. Initially, studies using Y2H identified interactions between TRP120 and various components and regulators of the Wnt signaling pathway, including positive regulators (PPP3R1 and VPS29) and negative regulators (ARID1B, CEP164, KLHL12, ILF3 and LMO2; Luo et al.2011; Luo and McBride 2012; Rogan et al. 2019). Recent studies determined that TRP120 utilizes ligand mimicry to bind Wnt FZD receptors and activates the Wnt signaling pathway in human monocytes (Fig. 3; Rogan et al. 2021). The Wnt signaling pathway is evolutionarily conserved and critical to eukaryotic development and cell fate (Luo et al. 2016). Notably, Wnt signaling has emerged as a key player in pathogenesis by several bacteria, including E.ch., S. enterica, M. tuberculosis, C. difficile, P. aeruginosa and E. coli. A TRP120 binding motif within the promoter region of Wnt target genes has been identified, which suggests that TRP120 regulates transcription genes associated with Wnt signaling (Luo et al. 2016; Rogan et al. 2019). Additionally, gene silencing of non-canonical and canonical Wnt signaling components was shown to have detrimental effects on E.ch. infection. Specifically, RNA silencing of Wnt components, including β-catenin, CK1, CAMKII, Wnt5a FZD5, FZD9, LRP6 and NFAT resulted in significantly reduced E.ch. infection (Rogan et al. 2019). In contrast, the silencing of Wnt antagonist DKK3 increased infection (Luo et al. 2016).

Wnt signaling is known to regulate autophagy and other innate immune responses. A recent study demonstrated that E.ch. hijacks the Wnt signaling pathway via TRP120 to inhibit autolysosome generation and autophagic destruction (Fig. 3; Lina et al. 2017). E.ch. utilizes TRP120 to exploit both the Wnt and PI3k/AKT pathways to activate mTOR signaling and regulate TFEB nuclear translocation to inhibit lysosomal biogenesis and autolysosomal fusion with E.ch. containing vacuoles (Lina et al. 2017). Increased levels of GSK3-β, a Wnt antagonist, were detected in E.ch. infected cells and were shown to be stimulated by TRP120 (Lina et al. 2017). These effects were abrogated with the treatment of a Wnt-Dvl inhibitor. Therefore, TRP120 is a key player in activating the PI3K/Akt pathway and inhibiting GSK3 activity via phosphorylation to prevent lysosomal fusion.

Molecular mimicry

TRP120 utilizes molecular mimicry and is known as a Wnt ligand mimetic that initiates Wnt signaling through its direct interactions with the FZD family of receptors (Rogan et al. 2021). Recent studies demonstrate the direct binding between TRP120 and multiple FZD receptors to activate the Wnt signaling pathway via a short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands. This study reveals the first example of bacterial mimicry of Wnt signaling ligands. Further investigation is needed to elucidate E.ch. TRP120 interactions with Notch receptors. However, since TRP120 is known as a Wnt ligand mimic and to independently activate Notch signaling, it is likely that TRP120 directly interacts with Notch receptors to activate the Notch signaling pathway via ligand mimicry.

Molecular mimicry is a powerful mechanism utilized by pathogens to exploit host cell functions to promote replication and dissemination. Studies determined that viral mimicry occurs for roughly 30% of motif classes identified in the Eukaryotic Linear Motif (ELM) database. Pathogenic molecular mimicry of host cell components occurs via protein SLiMs, which mimic host SLiMs (Davey, Cyert and Moses 2015; Via et al. 2015). SLiMs were first identified as conserved sequences in evolving regions and have arisen through convergent and co-evolution to enhance ligand binding, protein stability and cell signaling (Via et al. 2015). SLiMs are short stretches of contiguous amino acids that reside within natively disordered protein regions and can be found in accessible loops of folded domains (Davey, Cyert and Moses 2015). A single protein can contain various SLiMs, creating an intricate network of balance and competition between each motif (Davey, Cyert and Moses 2015; Via et al. 2015). Further, bacterial pathogens, including T. gondii, H. pylori, C. trachomatis, A. phagocytophilum, M. tuberculosis and L. monocytogenes, utilize SLiMs that share both composition and function with host SLiMs for molecular mimicry, which poses a threat on the delicate balance of host SLiMs and protein interaction networks due to their ability to titrate the motif-binding partners (Via et al. 2015). Unlike viruses, bacteria produce toxins and secreted proteins, and have larger genomes allowing numerous opportunities for molecular mimicry of various host proteins involved in internalization, transcription, immune response and cell signaling (Lina et al. 2016). Thus, the function of TRP120 SLiMs that mimic endogenous ligands highlights the pathogen's ability to upregulate developmental signaling pathways and makes E.ch. an important model organism for infectious diseases to further understand the pathobiology of bacterial pathogens.

HECT E3 ubiquitin ligase

TRP120 contains a functional HECT E3 ligase domain located at the C-terminal, allowing the effector to target host proteins for degradation. This unique characteristic allows TRP120 to regulate signaling through the ubiquitination of its host binding partners in addition to activating conserved signaling pathways through ligand mimicry. Recent studies identified the interaction between TRP120 and F-BOX and WD domain repeating-containing 7 (FBW7). FBW7 is the F-box protein subunit of the Skp1-cullin-1-FBOX E3 Ub ligase complex (SCF) and is required for substrate recognition to regulate an array of oncoproteins involved in Notch signaling (Elmore 2007). FBW7 negatively regulates oncoproteins (Notch, MCL1, cJun and cMyc) involved in cell survival. Thus, TRP120 not only activates, but regulates Notch signaling and identifies FBW7 as a substrate of the TRP120 HECT E3 Ub ligase to maintain Notch signaling for E.ch. survival (Fig. 2; Wang et al. 2020).

Further, TRP120 interacts with proteins of the SWI/SNF chromatin remodeling complex and polycomb comb group (PcG) proteins (Zhu et al. 2017). Polycomb repressive complexes (PRCs) are multi-subunit complexes divided into two groups (PRC1 and PRC2), important for the regulation of chromatin conformation and transcriptional regulation in eukaryotic cells. PRC1 results in the monoubiquitination of histone 2A at lysine 119, while PRC2 is involved in the trimethylation of histone 3 at lysine 27. PRC histone modifications result in chromatin conformational and transcriptional changes of eukaryotic genes. TRP120 is shown to directly interact with the RING domain of PCGF5, a unit of PRC1 (Mitra et al. 2018). PCGF5 is essential in epigenetics to maintain the transcriptionally repressive state of many host cell genes. TRP120 targets PCGF5 for Ub-mediated degradation within the nucleus, providing evidence that TRP120 mimics mammalian E3 ubiquitin ligase activity (Fig. 2). The interaction between TRP120 and PCGF5 occurs during early infection at the TR domain while the HECT domain remains active, demonstrating that PCGF5 is a substrate of TRP120 ligase activity (Mitra et al. 2018). At 48 h, PCGF is redistributed from the nucleus to the ehrlichial vacuole and PCGF isoforms are shown to undergo proteasomal degradation facilitated by TRP120. The proteasomal degradation of PCGF isoforms causes chromatin conformational changes and altered transcriptional activity of PRC1-associated Hox genes (Wang et al. 2020). The direct interaction between TRP120 and PCGF5 demonstrates a unique strategy whereby E.ch. employs to exploit host epigenetic machinery to modulate gene expression for infection.

Various pathogens have been shown to target host proteins for degradation (Ribet and Cossart 2010). Shigella effector proteins hijack host ubiquitination machinery to promote infection, whereby effectors OspI and OspG interact with host E2 enzymes to disrupt their functions, while NEL family effectors mimic E3 ligases to target host proteins for Ub-mediated degradation (Kim et al. 2005; Sanada et al. 2012; Nishide et al. 2013). Additionally, the Salmonella T3SS substrates SopA and SopB modulate host functions using ubiquitination for SopA E3 ubiquitin activity and SopB relocation from the host cell surface to the Salmonella-containing vacuole (SCV) to recruit Rab5 (Knodler et al. 2009; Herhaus and Dikic Zhang et al. 2006; Patel et al. 2009, 2018).

Temporal interactions

The diverse interactions between TRPs and host proteins is thought to occur in a temporal pattern through different stages of infection. Although the direct temporal interactions have not been fully elucidated, the location of TRPs during infection has been determined at specific timepoints. TRP32 is detected around the morulae at 24 h post-infection (hpi). Within 48 hpi TRP32 is observed in the perinuclear region and by 72 hpi TRP32 is found in the perinuclear region and in the nucleus (Farris et al. 2016). In addition, TRP47 is observed in the morulae at 24 hpi and within the nucleus at 24–72 hpi (Kibler et al. 2018). TRP120 is expressed on the surface of infectious dense-cored ehrlichiae and initiates infection within 3 hpi and is found in the nucleus at 24 hpi.

The diverse functions of TRP120 are thought to be temporally coordinated during infection, beginning within 3 hpi with its interactions with cell surface receptors including the Notch receptor complex and Wnt FZD receptors to activate Notch and Wnt signaling pathways to stimulate phagocytosis and entry, downregulate innate immune recognition receptors and inhibit lysosomal fusion to promote infection (Lina et al. 2016; Wang et al. 2020; Rogan et al. 2021). During bacterial replication, TRP120 interacts with many host proteins involved in an array of cellular functions, including its interactions with NEDD4L and UBC9 to hijack host PTM machinery. Sequentially, TRP120 is found in the nucleus at 24 h interacting with and ubiquitinating FBXW7 and PCGF5 to regulate host cell transcription and cell fate (Dunphy, Luo and McBride 2014; Mitra et al. 2018; Wang et al. 2020). Notably, the elaborate interactions of TRPs throughout infection occur to modulate cell processes in cell signaling, vesicle trafficking and intracellular transport, metabolism, PTMs, transcriptional regulation and apoptosis to avoid host defense systems and permit intracellular replication (Luo et al. 2017).

Summary and perspective

Understanding the strategies that are used by intracellular bacteria to exploit the host cell and survive a hostile environment have been revealed by studying various pathogens. Most notably, E.ch has one of the smallest bacterial genomes, yet has evolved strategies within the confines of its limited genome to reprogram the complex and sophisticated mononuclear phagocyte and circumvent the innate defenses. Pathogen–host interactions that have recently been described using E.ch. are new to science and thus highlighting the value of this organism for understanding how intracellular pathogens cause infection and evade host defense mechanisms. E.ch. secretes TRP effectors via a T1SS to hijacks host cell processes, including cell signaling, cytoskeletal organization, vesicle trafficking, transcriptional regulation, post-translational modifications, autophagy and apoptosis to evade host defenses. Investigations have led to the understanding that many other pathogenic bacteria utilize TR containing proteins for survival. Further studies regarding E.ch. TRP exploitation of host cell processes and the moonlighting mechanisms involved will unravel the role of bacterial effectors during infection and will potentially reveal the breath and complexity of moonlighting effectors in other bacterial species. Understanding the interactions between TRPs and host interacting partners will reveal how pathogens modulate host cell processes for survival and may facilitate the creation of new and innovative broad-based therapeutics for E.ch. and other intracellular bacteria.

Table 2.

TRP–host protein interactions to influence cell signaling.

	Host protein		Function
TRP	Symbol	Full name	Cell signaling
TRP32	CD14	CD14 molecule	MAPK, TLR, IKK/NFκB and LPS
	CD63	CD63 molecule	Integrin and VEGF
	IGHA1	Immunoglobulin heavy constant alpha 1	B cell receptor
	IGHV	Immunoglobulin heavy chain variable region	Notch and B cell receptor
	IGHLL5	Immunoglobulin lambda like polypeptide 5	B cell receptor
	RC3H1	Ring finger and CCCH-type domain 1	T cell receptor and NFκB
TRP47	BPI	Bactericidal/permeability-increasing protein	IL, TNF and TLR
	CAB39	Calcium binding protein 39	PI3K/Akt/mTOR and IGF1R,
	CDK1	Cyclin dependent kinase 1	p53, MAPK and Hedgehog
	CDK10	Cyclin dependent kinase 10	MAPK
	FYN	FYN proto-oncogene, Src family tyrosine kinase	Fcγ receptor, T cell receptor, MAPK, IKK/NFκB, PI3 and C-type lectin receptor
	GNB1	G protein subunit beta 1	GPCR, Ras, Wnt, PI3K/Akt, Hedgehog, CXCR3/4 and cytokine
	IGLL1	Immunoglobulin lambda like polypeptide 1	B cell receptor
	PRTN3	Proteinase 3	Cytokine and IL
	PTPN2	Protein tyrosine phosphatase, non-receptor type 2	ERK1/2, EGF receptor, IFNγ, IL, TNF, IFN and T cell receptor
TRP75	ANXA5	Annexin A5	NFκB, PI3K/Akt/mTOR, ERK and p38 MAPK
	CD84	Cluster of differentiation 84	Cell survival
	CSF1	Colony stimulating factor 1	PI3K/AKT/mTOR
	IFNLR1	Interferon lambda receptor 1	Cytokine ligands IFNL2 and IFNL3 receptor
	ITGB1	Integrin beta-1	Collagen receptor
	ITGB2	Integrin beta-2	ICAMs and ubiquitin-like proteins receptor
	MMP9	Matrix metalloproteinase-9	Cytokine-mediated signaling and peptidase activity
	NPTN	Neuroplastin	FGFR1 signaling
	PI4KA	Phosphatidylinositol 4-kinase alpha	Signal transduction
	PRKAA1	5'-AMP-activated protein kinase catalytic subunit alpha-1	Catalytic subunit of AMP-activated protein kinase (AMPK)
	RAB3GAP1	Rab3 GTPase-activating protein catalytic subunit	GTPase activity
	RAD50	DNA repair protein RAD50	Cellular response to DNA damage
	RB1CC1	RB1-inducible coiled-coil protein 1	Autophagy
	SEPW1	Selenoprotein W	Glutathione (GSH)-dependent antioxidant
	SGSM3	Small G protein signaling modulator 3	GTPase activity
	SH3BP5	SH3 domain-binding protein 5	BTK-related cytoplasmic signaling in B-cells
	SPP1	Secreted Phosphoprotein 1	Hedgehog, PTH and Integrin
TRP120	ADAM17	ADAM metallopeptidase domain 17	Hedgehog, EGFR, TGFβ, GPCR, Notch, TNF and cytokine/chemokine
	AKAP2	A kinase anchor protein 2	GPCR
	ANXA2	Annexin A2	NFκB, IL, EGFR, STAT3, Calcium and Wnt
	CXCL12	C-X-C motif chemokine ligand 12	NFκB, GPCR and chemokine
	GCSAM	Germinal center associated signaling and motility	B cell receptor
	GNAI2	G protein subunit alpha i2	MAPK, GPCR and chemokine
	GPS1	G protein pathway suppressor 1	MAPK, JNK and GPCR
	IFNGR2	Interferon gamma receptor 2	JAK-STAT and IFNγ
	IL2RG	Interleukin 2 receptor subunit gamma	MAPK, PI3K/Akt, IL and FGFR
	KRAS	KRAS proto-oncogene, GTPase	MAPK, NFκB, EGFR, Ras and Rac
	LGALS1	Galectin 1	IKK/NFκB
	PDE1B	Phosphodiesterase 1B	GPCR, PLC, EGFR and FGFR
	PPP3R1	Protein phosphatase 3 regulatory subunit B, alpha	Wnt, MAPK
	TLE4	Transducin like enhancer of split 4	Wnt, Notch
TRP120/TRP32	IGHA1	Immunoglobulin heavy constant alpha 1	B cell receptor
	IGLL5	Immunoglobulin lambda like polypeptide 5	B cell receptor
TRP120/TRP47	IGKC	Immunoglobulin kappa constant	B cell receptor signaling pathway