Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt ...

IAI Accepted Manuscript Posted Online 28 December 2015 Infect. Immun. doi:10.1128/IAI.01289-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways

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to Stimulate Phagocytosis and Promote Intracellular Survival

3 4

Tian Luo,a Paige S. Dunphy,a Taslima T. Lina,a and Jere W. McBridea-e#

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Departments of Pathologya and Microbiology and Immunology,b Center for Biodefense and

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Emerging Infectious Diseases,c Sealy Center for Vaccine Development,d Institute for Human

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Infections and Immunity,e University of Texas Medical Branch, Galveston, Texas, USA

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Running title: Wnt signaling during Ehrlichia infection

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# Address correspondence to:

13

Jere W. McBride, Ph.D.

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Department of Pathology

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University of Texas Medical Branch

16

Galveston, TX 77555-0609

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Phone: (409) 747-2498

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Fax: (409) 747-2455

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E-mail: [email protected]

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ABSTRACT

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Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and

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evading innate defenses, but the mechanisms are not fully defined. Recently we have determined

24

that E. chaffeensis tandem repeat proteins (TRP) are type 1 secreted effectors involved in

25

functionally diverse interactions with host targets, including components of the evolutionarily

26

conserved Wnt signaling pathways. In this study, we demonstrate that induction of host canonical

27

and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and

28

promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt

29

signalings were significantly stimulated during early stages of infection (1-3 h) which coincided

30

with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal

31

transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of

32

~44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-

33

interacting Wnt pathway components/regulators and other critical components, such as Wnt5a

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ligand, Frizzled 5 receptor, β-catenin, NFAT and major signaling molecules, resulted in

35

significant reductions in ehrlichial load. Moreover, small molecule inhibitors specific for

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components of canonical and noncanonical (Ca2+ and PCP) Wnt pathways, including IWP-2

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which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated

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Wnt signaling as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt

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pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce

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ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and

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noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote

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intracellular survival.

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INTRODUCTION

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Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the emerging

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life-threatening human zoonosis, human monocytotropic ehrlichiosis (HME) (1). E. chaffeensis

47

selectively infects mononuclear phagocytes and resides in early-endosome-like membrane-bound

48

vacuoles (1). The mechanisms by which E. chaffeensis enters host cells, establishes persistent

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infection, and avoids host defenses are not completely understood, but occur through

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functionally relevant host-pathogen interactions involving secreted ehrlichial tandem repeat

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protein (TRP) effectors that are posttranslationally modified by ubiquitin (Ub) and the small

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ubiquitin-like modifier (SUMO) (2-5). E. chaffeensis TRPs interact with a diverse group of

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human proteins associated with major cellular processes, including transcription, translation,

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protein trafficking, cell signaling, cytoskeleton organization, and apoptosis, indicating that they

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play a role in manipulating these important cellular processes to facilitate infection (6-8).

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E. chaffeensis TRPs were first recognized as antigens that elicit strong protective antibody

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responses during infection directed at continuous species-specific epitopes in tandem repeat

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regions (9-12) . Subsequently, our understanding of the functional role of TRPs as effectors in

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pathobiology has been advanced through studies that have defined specific TRP-host protein and

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DNA interactions (4, 13). Notably, E. chaffeensis TRP120 and TRP32 interact with numerous

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host proteins and genes associated with the canonical and noncanonical Wnt signaling pathways.

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One of TRP32-interacting targets, deleted-in-azoospermia associated protein 2 (DAZAP2), is a

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highly conserved protein that modulates gene transcription driven by Wnt/β-catenin signaling

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effector T-cell factors (TCF), and knockdown of host DAZAP2 by siRNA reduced E. chaffeensis

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load in infected cells (7, 14). Several other TRP120-interacting host proteins, such as AT rich

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interactive domain 1B (ARID1B), lysine (K)-specific demethylase 6B (KDM6B), interferon

3

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regulatory factor 2 binding protein 2 (IRF2BP2), protein phosphatase 3 regulatory subunit B

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alpha (PPP3R1), and vacuolar protein sorting 29 homolog (S. cerevisiae) (VPS29) are also

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involved in Wnt pathway signaling (6). Moreover, TRP120 binds host genes associated with

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Wnt signaling pathways, such as Wnt, Dishevelled (Dvl) and nuclear factor of activated T-cells

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(NFAT) (15). Thus, E. chaffeensis appears to exploit Wnt pathways through TRP-Wnt signaling

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proteins and by modulating the expression of Wnt pathway genes via TRP transcriptional

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modulation.

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Wnt signaling was initially studied for its role in carcinogenesis, but has more recently

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been recognized for its central role in embryonic development, differentiation, cell proliferation,

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cell motility, cell polarity, and adult tissue homeostasis (16, 17).

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signaling has been demonstrated by mutations that lead to a variety of diseases, including breast

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and prostate cancer, glioblastoma, diabetes, and others (18, 19). Wnt signaling pathways are

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highly evolutionarily conserved (20, 21). Thus far, three Wnt pathways have been characterized:

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the canonical Wnt/β-catenin pathway, and two noncanonical β-catenin-independent pathways

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(Wnt/Ca2+ and Wnt/PCP [planer cell polarity]). Wnt signaling is activated by the binding of a

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Wnt ligand to a Frizzled (Fzd) receptor (22, 23). In the canonical Wnt/β-catenin pathway,

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activated Fzd heterodimerizes with lipoprotein receptor-related protein (LRP) to recruit and

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activate Dishevelled (Dvl), which subsequently recruits the protein complex containing axis

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inhibitor (Axin), adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen

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synthase kinase-3 (GSK-3), leading to the inhibition of phosphorylation of β-catenin by these

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kinases. Unphosphorylated β-catenin accumulates, and subsequently translocates to the nucleus,

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where it associates with TCF/LEF (lymphoid-enhancing factor) family transcription factors to

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induce the expression of Wnt target genes (23-25). The noncanonical Wnt/Ca2+ pathway signals

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The importance of Wnt

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through heterotrimeric G proteins, which further activates phospholipase C (PLC), leading to

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intracellular Ca2+ release and activation of calcium/calmodulin-dependent protein kinase II

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(CaMKII), calcineurin and protein kinase C (PKC) (17). These processes can stimulate NFAT

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and other transcription factors such as cAMP response element-binding protein (CREB) (17, 26).

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The noncanonical Wnt/PCP pathway involves the Rho and Rac GTPases, Rho kinase (ROCK),

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and c-Jun N-terminal kinase (JNK), and regulates cytoskeletal reorganization, cell motility and

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tissue polarity (17, 27). More recent studies have suggested that the activity of Wnt ligands and

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their binding to Fzd receptors depends on the cellular context, thus Wnt and Fzd proteins cannot

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be rigorously subdivided according to the pathway they induce (28, 29). However, Wnt3a and

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Wnt5a are more commonly associated with canonical and noncanonical Wnt signaling,

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respectively (30).

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Recently, the role of the Wnt pathway in phagocytosis of microorganisms has been

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demonstrated (31, 32). The Wnt ligand-receptor (Wnt5a-Fzd5) signaling in macrophages was

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reported to promote phagocytosis of bacteria and enhanced survival through lipid raft with Rac1-

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PI3 kinase (PI3K)-IκB kinase (IKK) activation (31). Others have demonstrated that Salmonella

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activates host β-catenin signaling; moreover, Salmonella type 3 secreted effectors, AvrA and

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SopB, can individually activate Wnt/β-catenin signaling in epithelial cells leading to increase of

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stem cells and proliferative cells and transdifferentiation of primed epithelial cells into M cells,

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respectively, to promote intestinal invasion (33-37). Wnt5a is known to activate canonical and

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noncanonical Wnt pathways, and is linked to cytoskeletal modulation and proinflammatory

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cytokine activation, e.g. in human macrophages stimulated by Mycobacterium (38-42).

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E.

chaffeensis

binding

and

entry

is

known

to

involve

one

or

more

glycosylphosphatidylinositol (GPI)-anchored proteins associated with caveolae at the cell

5

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surface, inducing receptor-mediated phagocytosis that triggers Wnt signaling-like events

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including transglutamination, tyrosine phosphorylation, phospholipase Cγ2 (PLC-γ2) activation,

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inositol-(1,4,5)-trisphosphate (IP3) production and intracellular calcium release (43, 44). In

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addition, multiple studies have shown the importance of E. chaffeensis TRP120 in ehrlichial

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binding and internalization (45, 46); however, the specific cellular pathways exploited to mediate

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invasion and intracellular survival have not been defined. In this study, we demonstrate that host

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Wnt signaling pathways are exploited for ehrlichial internalization and infection, and ehrlichial

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TRPs are directly involved.

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MATERIALS AND METHODS

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Cell culture and cultivation of E. chaffeensis. Human cervical epithelial adenocarcinoma

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cells (HeLa, from ATCC) were propagated in Dulbecco’s modified Eagle’s medium (DMEM;

126

Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT).

127

Human monocytic leukemia cells (THP-1) were propagated in RPMI medium 1640 with L-

128

glutamine and 25 mM HEPES buffer (Invitrogen), supplemented with 1 mM sodium pyruvate,

129

2.5 g/L D-(+)-glucose (Sigma, St. Louis, MO), and 10% fetal bovine serum. E. chaffeensis

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(Arkansas strain) was cultivated in THP-1 cells as previously described (47).

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Inhibitors, siRNAs and antibodies. Wnt signaling pathway inhibitors included pyrvinium

133

pamoate, IWP-2, BAY11-7082, NSC23766 (Sigma), KN93, SB202190, TBCA, FH535, and

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LY294002 (Calbiochem/EMD, Billerica, MA). The lipid raft disrupting agent nystatin was from

135

Sigma. Human DKK3, Dvl2, Fzd9, Jun, NFATC1 (NFAT2), NFATC3 (NFAT4), PP2B-Aα

6

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(Calcineurin PPP3CA), PP2B-Aβ (Calcineurin PPP3CB), TCF4, Wnt6, Wnt10a, and control-A

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siRNAs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Validated siRNAs of

138

human β-catenin, Wnt3a, Wnt5a and LRP6 and esiRNAs of human ARID1B, KDM6B,

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IRF2BP2, PPP3R1 and VPS29 were from Sigma. Human Fzd5, Akt, CKIε, CKII, CaMKII,

140

IKK, PI3K and RhoA siRNAs were obtained from GE Dharmacon (Lafayette, CO). Alexa Fluor

141

488-labeled negative siRNA was from Qiagen (Germantown, MD). Rabbit and mouse anti-

142

TRP32 antibodies have been described previously (12). Other antibodies used in this study were

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mouse anti-human α-tubulin, NFATC1 (Santa Cruz) and β-catenin (Pierce, Rockford, IL) and

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rabbit anti-human Fzd9 (Pierce), phospho-β-catenin, Dvl2 (Cell Signaling, Beverly, MA) and Jun

145

(Santa Cruz).

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PCR array. The RT² Profiler PCR arrays (version 4.0; SABiosciences, Valencia, CA)

148

were used, including human Wnt signaling pathway plus PCR array and human Wnt signaling

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targets PCR array (see Fig. S1 and SABiosciences website for gene list and functional gene

150

grouping). The human Wnt signaling pathway plus PCR array profiles the expression of 84

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genes related to Wnt-mediated signal transduction, including Wnt signaling ligands, receptors

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and regulators, as well as downstream signaling molecules and target proteins for all three Wnt

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pathways.

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classification algorithms to generate the pathway activity score, and determines whether Wnt

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pathway activity is activated or repressed in experimental samples. The human Wnt signaling

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targets PCR array profiles the expression of 84 key genes responsive to Wnt signal transduction,

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including Wnt signaling pathway transcription factors and highly relevant target genes to analyze

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Wnt pathway status. PCR arrays were performed according to the PCR array handbook from the

The array uses experimentally derived signature biomarker genes along with

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manufacturer. In brief, uninfected and E. chaffeensis-infected THP-1 cells at different intervals

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postinfection (p.i.) were collected and total RNA was purified using RNeasy Mini kit (Qiagen).

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During RNA purification, on-column DNA digestion was performed using the RNase-free

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DNase set (Qiagen).

163

absorbance using a Nanodrop 100 spectrophotometer (Thermo Scientific, West Palm Beach, FL),

164

and ribosomal RNA band integrity was verified by running an aliquot of each RNA sample on a

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RNA FlashGel (Lonza, Rockland, ME).

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synthesized from 0.5 μg of total RNA using the RT2 first strand kit (Qiagen). Real-time PCR

167

was performed using RT2 Profiler PCR array in combination with RT2 SYBR Green mastermix

168

(Qiagen) on a Mastercycler EP Realplex2 S (Eppendorf, Germany). Cycling conditions were as

169

follows: 95°C for 10 min and 40 cycles of 95°C for 15 s, 60°C for 1 min. The real-time cycler

170

software RealPlex 1.5 (Eppendorf) was used for PCR and data collection. The baseline was set

171

automatically, the threshold was defined manually, and then the threshold cycle (CT) for each

172

well was calculated by RealPlex. The threshold was set in the proper location and at the same

173

level for all PCR arrays in the same analysis so that the values of the positive PCR control (PPC)

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assays on all arrays were between 18 CT and 22 CT. The CT values for all wells were exported

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for analysis using Web-based PCR array data analysis software (version 3.5; SABiosciences).

176

PCR array quality checks were performed by the software before data analysis, including PCR

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array reproducibility, reverse transcription efficiency control (RTC), human genomic DNA

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contamination control (HGDC) and PPC.

The concentration and purity were determined by measuring the

Genomic DNA was eliminated and cDNA was

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Western immunoblot. The THP-1 cell lysates were prepared using CytoBuster protein

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extraction reagent (Novagen/EMD, Gibbstown, NJ), separated by sodium dodecyl sulfate-

8

182

polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane.

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Western immunoblot was performed with horseradish peroxidase-labeled goat anti-rabbit, or

184

mouse IgG (heavy and light chains) conjugate (Kirkegaard & Perry Laboratories, Gaithersburg,

185

MD) and SuperSignal West Dura chemiluminescent substrate (Thermo Scientific).

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Immunofluorescence microscopy. Uninfected or E. chaffeensis-infected THP-1 cells at

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different intervals p.i. were collected, and the indirect immunofluorescent antibody assay was

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performed as previously described (6), except that anti-β-catenin or NFATC1 antibody (1:100)

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and anti-TRP32 antibody (1:10,000) were used.

191 192

Reporter assay.

Activity of noncanonical Wnt/Ca2+ signal pathway (NFAT-mediated

193

transcription) was monitored using the Cignal NFAT reporter (Qiagen). Briefly, HeLa cells (2 ×

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104/well) were seeded in a 96-well black plate, and the following day cells were transfected with

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NFAT reporter plasmid using Lipofectamine 3000 (Invitrogen) and incubated overnight again.

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The cell-free E. chaffeensis (multiplicity of infection [MOI] = ~50), or uninfected THP-1 control

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was added to transfected HeLa cells. Firefly and Renilla luciferase activities of each well were

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measured at different time points using the Dual-Glo luciferase assay system (Promega,

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Madison, WI) and a Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA).

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Firefly luciferase activity was normalized with Renilla luciferase.

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RNA interference. THP-1 cells (1 × 105/well on a 96-well plate) were transfected with 5

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pmol human siRNA using Lipofectamine 3000 (Invitrogen). A control-A siRNA consisting of a

204

scrambled sequence was used as a negative control, and an Alexa Fluor 488-labeled negative

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siRNA was used as a control to monitor transfection efficiency. At 1 day post transfection, the

206

cells were synchronously infected by cell-free E. chaffeensis at a MOI of ~50, and collected at 1

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day and 2 days p.i. for quantitative PCR (qPCR) and Western blot to determine knockdown

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levels.

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Quantification of E. chaffeensis by qPCR. THP-1 cells were pelleted, washed by PBS,

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lysed in SideStep lysis and stabilization buffer (Agilent, Santa Clara, CA) for 30 min at room

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temp, and analyzed for bacterial load using realtime qPCR. Amplification of the integral

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ehrlichial gene dsb was performed using Brilliant II SYBR Green mastermix (Agilent), 200 nM

214

forward

215

gtttcattagccaagaattccgacact-3'). The qPCR thermal cycling protocol (denaturation at 95°C 10

216

min, then 40 cycles of 95°C 30 s, 58°C 1 min, 72°C 1 min) was performed on the Mastercycler

217

EP Realplex2 S (Eppendorf). A standard plasmid pBAD-dsb was constructed by cloning the

218

ehrlichial dsb gene using the TOPO TA cloning kit (Invitrogen). The plasmid copy number for

219

the standards was calculated using the following formula: plasmid copy/μl = [(plasmid

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concentration g/μl) / (plasmid length in base pairs × 660)] × 6.022 × 1023. The absolute E.

221

chaffeensis dsb copy number in the cells was determined against the standard curve or the fold

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change of dsb copy number relative to the control was normalized to qPCR-detected levels of the

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host genomic glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene.

primer

(5'-gctgctccaccaataaatgtatccct-3')

and

200

nM

reverse

primer

(5'-

224 225

Small molecule inhibitor treatment. THP-1 cells were plated in FBS-free medium and

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treated with inhibitor or DMSO control 3 h before infection with cell-free E. chaffeensis at a

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MOI of ~50, or cells were infected and then treated with inhibitor/DMSO 3 h p.i. Percentages of

10

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infected cells were monitored daily over 3 days by Diff-Quik staining and counting of 100 cells.

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Bright field images of these slides were collected on an Olympus BX61 epifluorescence

230

microscope using a color camera. Initially, inhibitors were tested at the concentrations 5-10 fold

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higher than the IC50, or Ki as provided by the manufacturer. If the bacteria were inhibited at this

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concentration, MICs were then determined by using two-fold serial dilutions. The MIC was

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defined as the minimum inhibitor concentration required for inhibition of the growth of the

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bacteria compared to that of the control (without the inhibitor) at day 3. An infected culture

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without the inhibitor served as a positive growth control, and an uninfected cell culture served as

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a negative control. All experiments were repeated three times to confirm results.

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Phagocytosis of microspheres. Expression and purification of E. chaffeensis tandem

239

repeat proteins (TRP) has been described previously (9, 10, 12).

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microspheres (1.0 µm, yellow-green fluorescent; Invitrogen) (10 μl, ~3.6×108 beads) were

241

washed by 40 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer, pH 6.1, and then

242

incubated with 15 µg of TRP protein in 500 μl MES buffer at room temperature for 1 h with

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mixing at 20 rpm. The coated beads were collected by centrifugation, washed twice in MES

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buffer and resuspended in RPMI medium, then gently sonicated to disperse the beads. The

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efficiency of bead coating was confirmed by dot blot assay. TRP-coated or control thioredoxin

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protein-coated beads were added to THP-1 cells at a multiplicity of approximately 50 beads per

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cell and incubated for 2 h at 37°C with 5% CO2. Unbound beads were removed by washing and

248

low-speed centrifugation for three times, and then cells were observed using an Olympus IX71

249

inverted fluorescence microscope. Fluorescence intensity was measured using a FluoroSkan

11

FluoSpheres® sulfate

250

fluorometer (Thermo Scientific). For estimating inhibition of phagocytosis, designated inhibitors

251

were added 2 h before addition of coated beads.

252 253

Measurement of Ca2+. Intracellular Ca2+ release was measured in THP-1 cells using

254

acetoxymethyl (AM) ester derivative of fluo-3 (fluo-3/AM) (PromoKine, Heidelberg, Germany)

255

as previously described (43).

256

(MOI = ~50), TRP-coated beads (50 beads/cell) or control (cell-free uninfected THP-1 or

257

thioredoxin-coated beads) and mixed briefly. Emission at 525 nm after excitation at 488 nm was

258

recorded every 15 s for 40 min using a FluoroSkan fluorometer (Thermo Scientific).

259

Nonfluorescent polystyrene latex beads (LB-11; Sigma) were coated by TRPs as described

260

above.

Fluo-3-preloaded cells were treated with cell-free E. chaffeensis

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Statistics. The statistical differences between experimental groups were assessed with the two-tailed Student’s t test, and significance was indicated by a P value of < 0.05.

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RESULTS

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Gene activity analysis of Wnt signaling pathway in E. chaffeensis-infected host cells

268

by PCR array. To define the impact of E. chaffeensis on host canonical and noncanonical Wnt

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signaling, the gene activity of the Wnt signaling pathway in E. chaffeensis-infected THP-1 cells

270

at different time points (1 h, 3 h, 8 h, 24 h and 72 h) p.i. was assessed by qPCR array (Table 1).

271

As compared to that in uninfected cells (under basal conditions), Wnt signaling pathway activity

272

in THP-1 cells was stimulated significantly by E. chaffeensis early (at 3 h p.i), whereas the

273

activity was repressed significantly at late infection (72 h p.i). However, there was no significant 12

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change in pathway activity observed in E. chaffeensis-infected THP-1 cells at 1 h, 8 h and 24 h

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p.i relative to uninfected/basal level. Heat maps of gene expression of Wnt signaling pathways at

276

3 h, 24 h and 72 h p.i. also show overall more gene upregulation (red) at 3 h and downregulation

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(green) at 72 h, compared to 24 h (Fig. 1A).

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Among 84 host genes associated with Wnt signaling pathways assessed by PCR array (see

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Fig S1A top panel for gene table), 18 (21% of total) genes exhibited significant differences in

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expression for at least one time point after E. chaffeensis infection, either upregulation (fold

281

regulation/fold change > 2) or downregulation (fold regulation < -2 [fold change < 0.5]),

282

compared to uninfected cells, including Wnt ligands, receptors, inhibitors, a transcription factor

283

and targets of Wnt signaling pathways (Table 2). Figure S1A bottom panel shows the expression

284

levels of seven Wnt component/target genes at different time points p.i., including Wnt ligands

285

Wnt6 and Wnt10a, Frizzled receptors Fzd5 and Fzd9, transcription factor 7 (TCF7), and Wnt

286

targets FOS-like antigen 1 (FOSL1) and MYC. Expression profiles of these important genes of

287

Wnt pathways were captured in the global pathway analysis results which identified significant

288

global upregulation of Wnt pathway at 3 h p.i. and downregulation at 72 h p.i.

289 290

Expression analysis of Wnt signaling target genes in E. chaffeensis-infected host cells

291

by PCR array. We further examined the expression of a large number of genes that are targets

292

of canonical/noncanonical Wnt signaling in order to demonstrate the Wnt pathway regulation

293

and identify specific genes that are most affected with Ehrlichia-induced Wnt signaling. Fig. 1B

294

shows heat maps of gene expression of Wnt signaling targets at 3 h, 24 h and 72 h p.i., and Table

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3 shows functionally categorized target genes with significant changes of expression for at least

296

one time point (3 h, 8 h, 24 h or 72 h p.i ) in infected cells compared to that in uninfected cells.

13

297

Among 84 host genes responsive to Wnt signaling (see Fig S1B top panel for gene table), 37

298

genes (44%) showed significant differences in expression level for at least one time point after

299

infection, either upregulation (fold regulation > 2) or downregulation (fold regulation < -2]).

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Among these 37 genes with significant difference of expression, 19 genes (51% of 37) were

301

upregulated, 13 genes (35%) were downregulated, and 5 genes (14%) were upregulated and

302

downregulated at different time points. The functional categories represented by these 37 Wnt

303

signaling target genes include development and differentiation, calcium binding and signaling,

304

adhesion, migration, cell cycle, proteolysis, signal transduction, and transcription factors.

305

Notably, in the categories of development and differentiation, calcium binding and signaling, and

306

migration, about half (51%, 46% and 52%, respectively) of the genes examined were modulated

307

by E. chaffeensis. Expression levels of six host target genes, including cyclin D1 (CCND1),

308

fibroblast growth factor 9 (FGF9), fibronectin 1 (FN1), MET proto-oncogene (MET), matrix

309

metallopeptidase 2 (MMP2), and secreted frizzled-related protein 2 (SFRP2), are shown at

310

different time points p.i. as determined by Wnt signaling targets PCR array (Figure S1B, bottom

311

panel). Consistent with the observed activities of Wnt pathway, these Wnt signaling target genes

312

had a similar expression pattern with relatively high expression at 3 h and 8 h p.i. and relatively

313

low expression at 72 h p.i. Modulation of many Wnt target genes in the host cell during E.

314

chaffeensis infection supports the regulation of Wnt signaling by E. chaffeensis.

315 316

E. chaffeensis infection suppresses phosphorylation of host β-catenin and mediates

317

nuclear translocation of β-catenin and NFATC1. β-catenin and NFATC1 are important signal

318

transducer/transcription factor involved in Wnt/β-catenin and Wnt/Ca2+-regulated gene

319

transcription, respectively.

Activation of Wnt signaling pathways results in the

14

320

dephosphorylation and translocation of β-catenin and NFATC1 into the nucleus. Therefore, we

321

examined the phosphorylation of β-catenin and the localization of β-catenin and NFATC1 after

322

ehrlichial infection by Western blot and immunofluorescence microscopy. Phosphorylation of β-

323

catenin was reduced at 1 h after infection and almost inhibited completely at 3 h (Fig. 2A).

324

Moreover, a dramatic redistribution of β-catenin and NFATC1 proteins to the nucleus was

325

observed shortly after infection with E. chaffeensis. In uninfected THP-1 cells, β-catenin was

326

diffusely distributed mainly in the cytoplasm and associated with cell membrane, but in E.

327

chaffeensis-infected cells as early as 1 h p.i., β-catenin translocated to the nucleus, exhibiting a

328

punctate distribution.

329

cytoplasm (Fig. 2B). Similarly, NFATC1 was diffusely distributed mainly in the cytoplasm of

330

uninfected THP-1 cells, but translocated to the nucleus as early as 1 h p.i. Notably, there was

331

nearly complete translocation of NFATC1 to the nucleus at 3 h, but by 20 h p.i., NFATC1 was

332

mainly observed in the cytoplasm (Fig. 2B). The inhibition of phosphorylation of β-catenin and

333

translocation of β-catenin and NFATC1 proteins to the nucleus between 1 and 3 h p.i. was

334

consistent with PCR array analysis identifying activation of Wnt component and target genes 1-3

335

h after E. chaffeensis infection.

By 20 h p.i., the majority of the β-catenin was redistributed to the

336 337

E. chaffeensis infection activates NFAT reporter. Wnt signaling PCR arrays analyze the

338

expression of a panel of genes related to all three Wnt pathways, but the pathway activities

339

determined by PCR array are based on well-studied canonical Wnt signaling pathway; therefore,

340

the Cignal NFAT reporter was used to examine the activity of noncanonical Wnt/Ca2+ signal

341

pathway (NFAT-mediated transcription). Compared to the uninfected control, stimulation of

342

reporter signal was not observed at 3 h p.i., but the reporter activity increased at 8 h p.i. and was

15

343

significantly upregulated by 24 h p.i. (Fig. 2C), suggesting that E. chaffeensis activates

344

noncanonical Wnt/Ca2+ signal pathway, but

345

canonical Wnt/β-catenin pathway. In addition, the activation of Wnt/Ca2+ signal pathway at

346

transcriptional and translational levels (8-24 h) occurs later than translocation of NFAT protein

347

into the nucleus (1-3 h).

complete activation occurs later than that of

348 349

Knockdown of Wnt signaling pathway components influences ehrlichial infection of

350

macrophages. We further confirmed the role of host Wnt signaling pathways in ehrlichial

351

infection by RNA interference. In total, 23 siRNAs were used to target important components of

352

Wnt signaling pathways, such as Wnt ligands, receptors and co-receptors, regulators,

353

transcription factors and Wnt targets (Fig. 3A). The siRNAs of some host genes modulated

354

during E. chaffeensis infection, as determined by PCR array, were also included. The decrease

355

of most proteins (17 proteins at 1 day p.i. and 20 proteins at 2 days p.i.) decreased E. chaffeensis

356

infection significantly, indicating that Wnt signaling plays a role in E. chaffeensis infection and

357

survival. An exception was knockdown of DKK3 (at 1 day p.i.), which increased ehrlichial load.

358

Notably, DKK3, Dickkopf homolog 3 (Xenopus laevis), is an antagonist of the canonical Wnt

359

signaling pathway (48), thus further supporting the importance of Wnt signaling in ehrlichial

360

survival. The inhibition of infection by siRNAs of Wnt signaling pathways occurred at 1 and/or

361

2 days p.i. Protein expression of three genes Fzd9, Dvl2, and Jun was reduced in specific

362

siRNA-transfected cells, respectively, compared with the unrelated control siRNA-transfected

363

cells (Fig. 3B).

364

16

365

Small molecule inhibitors of Wnt signaling pathways repress E. chaffeensis infection

366

of host cells. To confirm the important role of host Wnt signaling pathways in E. chaffeensis

367

infection, the effect of various Wnt signaling pathway inhibitors on ehrlichial infection was

368

examined (Table 4). Five inhibitors were found to have significant impact on ehrlichial infection

369

without apparent toxicity to host cells, including CK1α/GSK3 activator (Akt inhibitor)

370

pyrvinium, CaMKII inhibitor KN93, inhibitor of Wnt production II IWP-2, CK1δ/ε inhibitor

371

SB202190, and CK2 inhibitor III TBCA (Fig. 4A-C). Pyrvinium, KN93 and IWP-2 were highly

372

potent inhibitors of ehrlichial infection, and could block the infection almost completely, despite

373

addition of inhibitor 3 h before or after ehrlichial infection. Two inhibitors SB202190 and TBCA

374

reduced ehrlichial infection significantly. Addition of inhibitor SB202190 or TBCA 3 h before

375

or after ehrlichial infection did not make significant difference in reduction of bacterial load,

376

suggesting that inhibitors SB202190 and TBCA function after ehrlichial infection, and thus,

377

CK1δ/ε and CK2 of canonical Wnt pathway are not important for ehrlichial internalization.

378

Notably, three most potent inhibitors pyrvinium, KN93 and IWP-2 target the canonical Wnt

379

pathway, the noncanonical Wnt/Ca2+ pathway and Wnt ligand secretion, respectively, indicating

380

the importance of both canonical and noncanonical Wnt signaling pathways and Wnt ligand

381

secretion for ehrlichial survival. Moreover, MICs of pyrvinium, KN93 and IWP-2 for ehrlichial

382

infection were determined to be 20 nM, 4 µM, and 0.3 µM, respectively, by using two-fold serial

383

dilutions (Table 4). In addition, we found that three other inhibitors, β-catenin/TCF inhibitor

384

FH535, PI3K inhibitor LY294002 and IKK inhibitor BAY11-7082 could influence ehrlichial

385

infection, but exhibited toxicity to host cells after treatment for more than 1 day (data not

386

shown).

387

17

388

E. chaffeensis TRP120 interacts with host targets involved in Wnt signaling that

389

influence infection.

Since E. chaffeensis TRPs have been identified as bacterial effector

390

proteins and interact with multiple host proteins involved in Wnt signaling, we confirmed the

391

role of these host proteins in ehrlichial infection by RNA interference. Similar to our previous

392

result of TRP32-interacting protein DAZAP2, knockdown of five TRP120-interacting host

393

proteins, including ARID1B, KDM6B, IRF2BP2, PPP3R1 and VPS29, influenced E. chaffeensis

394

infection of macrophages significantly (Table 5). The bacterial load in all specific siRNA-

395

transfected cells decreased at both 1 day and 2 days p.i. (fold regulation < -2), except that the

396

bacterial load in ARID1B siRNA-transfected cells increased. ARID1B is an AT-rich DNA

397

interacting domain-containing protein and a component of the SWI/SNF chromatin remodeling

398

complex and has been reported to interact with β-catenin to suppress Wnt signaling (49). The

399

results suggest that TRP120 plays a major role in influencing Wnt pathway activation by

400

interacting with these Wnt pathway regulators during E. chaffeensis infection.

401 402

E. chaffeensis tandem repeat proteins stimulate phagocytosis of macrophages and

403

Wnt pathway inhibitors reduced the stimulation.

404

cytoskeletal changes, cell polarity and phagocytosis (32, 50). To determine if E. chaffeensis

405

TRPs activated Wnt pathway to induce phagocytosis, we coated microspheres with recombinant

406

TRPs and examined their uptake. Phagocytosis of TRP120-coated microspheres by the THP-1

407

cell was significantly increased compared with the control protein (TRP120 N-terminal region;

408

TRP120N)-coated microspheres (Fig. 5A). Similarly, TRP32 or TRP47-coated microspheres

409

were also phagocytosed, but less efficiently than TRP120. Next we examined the role of specific

410

TRP120 domains in inducing Wnt-directed phagocytosis. Truncated TRP120TRC (containing

18

Wnt signaling is known to influence

411

all tandem repeats and C-terminus), TRP120-2R (two tandem repeats) and TRP120C (C-

412

terminus), stimulated phagocytosis, but less efficiently than full length TRP120. The N-terminal

413

region of TRP120 was not phagocytosed suggesting that TR and C-terminal domains both

414

contribute to TRP-induced phagocytosis (Fig. 5B). To examine the role of Wnt pathways

415

associated with TRP-induced phagocytosis, we tested Wnt pathway inhibitors (Table 4).

416

Noncanonical Wnt pathway inhibitors, including IKK inhibitor BAY11-7082 and PI3K inhibitor

417

LY94002, significantly reduced the TRP-coated microsphere uptake, supporting the important

418

role for one or more of these noncanonical Wnt pathways in TRP-induced phagocytosis (Fig. 5A

419

and C). TRP120-induced microsphere internalization was also reduced after treatment with

420

various Wnt pathway inhibitors, including CKIα/GSK3β activator (Akt inhibitor) pyrvinium, β-

421

catenin/TCF inhibitor FH535, CaMKII inhibitor KN93, and Rac1 inhibitor NSC23766,

422

suggesting that all three canonical and noncanonical Wnt pathways are involved in TRP-induced

423

phagocytosis. However, canonical Wnt pathway inhibitors CK1δ/ε inhibitor (SB202190) and

424

CK2 inhibitor III (TBCA) did not reduce TRP microsphere internalization significantly,

425

consistent with our data shown in Figure 4A. Predictably, there was dramatic inhibition in TRP-

426

mediated phagocytosis by the lipid raft disrupting agent nystatin (10 µM) similar to the most

427

potent noncanonical Wnt pathway inhibitors. Inhibitor of Wnt ligand biogenesis and secretion,

428

IWP-2, did not inhibit the phagocytosis mediated by TRP, suggesting that TRP-induced

429

microsphere internalization occurs independent of Wnt ligand secretion (Fig. 5C). These results

430

demonstrate the importance of Wnt signaling pathways in ehrlichial internalization and highlight

431

the roles of PI3K, IKK, Rac1, CK1α/GSK3β/Akt, β-catenin/TCF, and CaMKII as well as lipid

432

rafts in TRP-induced phagocytosis.

433

19

434

E. chaffeensis tandem repeat proteins stimulate intracellular Ca2+ release. Activation

435

of the noncanonical Wnt/Ca2+ pathway leads to increased intracellular Ca2+ release and activation

436

of CaMKII and calcineurin followed by NFAT and other transcription factors. Ca2+ increase was

437

detected in THP-1 cells infected with E. chaffeensis or treated with beads coated by a mixture of

438

TRP32, TRP47 and TRP120. Consistent with a previous study (43), a rapid increase of Ca2+ was

439

detected to about 80 sec after cell-free E. chaffeensis was added. Similarly, TRPs-coated beads

440

also caused a rapid increase of Ca2+ to about 60 sec, although the Ca2+ concentration was lower

441

than that stimulated by Ehrlichia. As controls, uninfected THP-1 cell lysate or control beads did

442

not induce Ca2+ release, indicating that Ehrlichia and TRPs stimulate intracellular Ca2+ release

443

associated with E. chaffeensis entry (Fig. 5D). In addition, Ca2+ release by a single TRP-coated

444

beads was not detected (data not shown), suggesting that all three TRPs are needed for Ca2+

445

release or the TRP mixture caused more Ca2+ release.

446 447

DISCUSSION

448 449

Wnt signaling involves a complex signaling network of ligands, receptors, kinases,

450

transcription factors and other molecules that are associated with three distinct, but

451

interconnected Wnt pathways.

452

proliferation, differentiation and cell polarity, and recent reports have linked microbes and host

453

Wnt signaling. Microbe activation of Wnt pathways has been linked to phagocytosis of bacteria

454

and viruses, differentiation of cells to promote entry, expression of proinflammatory responses to

455

microbial pathogen-associated molecular patterns, and intracellular bacterial survival (31, 32, 36,

456

38).

In the eukaryotic cell, Wnt pathways control cellular

Previously, we have defined interactions between ehrlichial TRP effectors and host

20

457

DNA/proteins, including Wnt pathway components, regulators, and transcriptional regulators of

458

Wnt pathway gene expression (6, 7, 15). This study extends these interactions and demonstrates

459

that canonical and noncanonical Wnt pathway activation occurs during E. chaffeensis infection.

460

In this study, we demonstrate that host Wnt signaling pathways play important roles in ehrlichial

461

internalization and infection, and ehrlichial TRPs mediate the invasion and survival through

462

activation and modulation of Wnt signaling pathways.

463

In order to identify temporal regulation of Wnt pathways, and the specific Wnt signaling

464

components and target genes induced by E. chaffeensis infection, we examined expression of

465

Wnt pathway components and target genes using PCR arrays.

466

demonstrated early activation and late repression of Wnt pathways with significant changes in

467

the expression level of Wnt ligands, receptors, inhibitors, signaling molecules, and transcription

468

factors associated with both canonical and noncanonical Wnt pathways. Gene transcription

469

analysis indicated that Wnt pathway(s) were activated at a very early stage of infection (3 h),

470

suggesting involvement of the pathway in the internalization of bacteria, consistent with a

471

previous report that the Wnt signaling in macrophages promoted phagocytosis of bacteria and

472

enhanced survival (31). Ehrlichial entry occurs within 1 h to 3 h p.i. (45, 51), and the resulting

473

activation of Wnt pathways at 3 h p.i. is consistent with the timeframe of the entry process.

474

Early activation of canonical and noncanonical Wnt pathways was confirmed by the

475

dephosphorylation of β-catenin and nuclear translocation of β-catenin and NFATC1.

476

Translocation of β-catenin and NFATC1 was also detected at 1 h p.i. These findings support that

477

host Wnt signaling pathways are activated during Ehrlichia entry. Conversely, Wnt pathway

478

repression was observed 72 h after infection in which the bacteria have completed a replication

479

cycle and exit the host cell (51), suggesting that the Wnt pathway(s) is downregulated during the

21

Pathway activity scores

480

exit phase. Significant changes in pathway activity at other time points (1 h, 8 h, and 24 h) were

481

not detected, and this finding may be associated with the limitation of signature biomarker genes

482

that our PCR array selects, since different components may be regulated by Ehrlichia at different

483

time points in a different way. In addition, we used the NFAT reporter assay to demonstrate that

484

E. chaffeensis activates the noncanonical Wnt/Ca2+ pathway directly. The inconsistency in time

485

points between NFAT nuclear translocation (1-3 h p.i.) and NFAT reporter expression (8-24 h

486

p.i.) may be due to the later occurrence of gene expression than nuclear translocation of NFAT, or

487

caused by different cell lines and cell context.

488

Genes with significant changes of expression after ehrlichial infection (Table 2) are

489

included in both canonical and noncanonical Wnt pathways. For example, Wnt5b ligand has

490

been found to activate Wnt/PCP and Wnt/Ca2+ pathways and also be associated with β-catenin

491

(52-54); Similarly, receptor Fzd5 is commonly linked to Wnt5a and Wnt/PCP pathway, but

492

prototype noncanonical Wnt5a has been reported to signal to β-catenin in the presence of

493

overexpressed Fzd5 (55). Therefore, it is predicted that these Wnt pathway components/targets

494

with significant changes of expression play important roles during Ehrlichia entry/survival, and

495

all three Wnt signaling pathways are utilized by E. chaffeensis. This is further supported by

496

functionally categorized gene expression changes of Wnt signaling targets after ehrlichial

497

infection, including Wnt targets involved in development and differentiation, calcium binding

498

and signaling and migration, as demonstrated by Wnt targets PCR array (Table 3).

499

pathways are involved in these important cellular processes, suggesting that the regulation of

500

canonical and noncanonical Wnt pathways is important for ehrlichial infection.

Wnt

501

Experiments using small interfering RNAs and small molecule inhibitors support the

502

conclusion that canonical and noncanonical Wnt signaling pathways are activated during

22

503

ehrlichial infection of host cells. Knockdown of some important components of Wnt signaling

504

pathways, including Wnt ligands, receptors and co-receptors, signaling molecules, kinases,

505

transcription factors and targets as well as TRP120-interacting proteins, influenced E. chaffeensis

506

infection significantly, indicating the importance of these proteins and canonical/noncanonical

507

Wnt athway activation during E. chaffeensis infection. Specifically, highly significant reduction

508

of infection was observed at both 1 d and 2 d p.i. after knockdown of ligand Wnt5a or its

509

receptor Fzd5, suggesting that Wnt5a-Fzd5 signaling is very important for Ehrlichia survival

510

after internalization. This is consistent with previous report that Wnt5a-Fzd5 signaling reduced

511

bacterial killing by macrophages (31). However, we found by gene expression analysis that

512

Fzd5, but not Wnt5a, was upregulated by Ehrlichia, suggesting that Ehrlichia regulates the

513

expression of Wnt5a receptor instead of Wnt5a itself, although Wnt5a does appear to be

514

important for bacterial survival after entry. Wnt5a can bind to different classes of receptors and

515

co-receptors, but Wnt5a signals primarily through the noncanonical pathway, where it mediates

516

cell proliferation, adhesion, and movement (56). However, the role of Wnt5a in canonical

517

signaling is still unclear, due to its complex nature. Depending on the receptor availability,

518

Wnt5a can activate or inhibit the canonical Wnt signaling pathway (57). Moreover, Wnt5a has

519

recently emerged as a macrophage effector molecule that triggers inflammation (58, 59).

520

Another protein whose knockdown reduced the infection highly significantly at 1 d p.i. was

521

LRP6 (low density lipoprotein receptor-related protein 6), a co-receptor with Frizzled for Wnt to

522

transmit the canonical Wnt signaling.

523

cytoplasmic tail of LRP were able to bind and internalize sheep red blood cells, and LRP6 was

524

reported to internalize with caveolin and interact with Axin to promote the accumulation of β-

525

catenin (60, 61), so LRP6 may be involved in both Ehrlichia internalization and survival.

Macrophages expressing receptors containing the

23

526

Knockdown of Akt reduced the infection very significantly at both 1 d and 2 d p.i. Akt, also

527

known as protein kinase B (PKB), is a serine/threonine-specific protein kinase that is involved in

528

both canonical and noncanonical Wnt pathways. Akt plays a key role in multiple cellular

529

processes such as cell survival, cell proliferation, cell migration, transcription and apoptosis, and

530

it is also associated with cytoskeleton reorganization and phagocytosis of bacteria by

531

macrophages (62, 63); therefore, Akt is predicted to be involved in both Ehrlichia internalization

532

and survival.

533

consistent with the fact that it is an antagonist of the canonical Wnt pathway.

In our study, the only siRNA which increased ehrlichial load was DKK3,

534

TRP120-interacting host proteins ARID1B, KDM6B, IRF2BP2, PPP3R1 and VPS29 are

535

involved in both canonical/noncanonical Wnt signaling pathways and Wnt ligand secretion and

536

also appear to play important roles during ehrlichial infection. For example, KDM6B is a

537

histone H3 lysine demethylase with an important gene regulatory role in development and

538

physiology. It has been reported to not only interact with β-catenin and contribute to β-catenin-

539

dependent promoter activation, but also upregulate Wnt3 expression and cause activation of Wnt

540

signaling (64, 65).

541

Wnt/Ca2+ pathway with Ca2+/calmodulin binding activity. Therefore, TRP120 interaction with

542

KDM6B and PPP3R1 would be predicted to promote positive regulation of Wnt/β-catenin and

543

Wnt/Ca2+ pathway, respectively, to facilitate ehrlichial survival. VPS29 is a component of a

544

large retromer complex, which is involved in retrograde transport of Wnt carrier protein Wntless

545

(Wls) from endosomes to the trans-Golgi network and is required for Wnt secretion (66).

546

TRP120 may interact with VPS29 to assist the recycling of Wls protein, and thus promote the

547

secretion of Wnt ligands and activation of Wnt pathways during ehrlichial infection.

PPP3R1 is calcineurin subunit B type 1, an important component of

24

548

The siRNA experiments revealed that reduction in E. chaffeensis load occurs at different

549

stages of infection for different host targets, suggesting that the role of these targets varies at

550

different points during infection.

551

siRNAs reduced bacterial load significantly at 2 d p.i. but not significantly at 1 d, suggesting that

552

these components may be not involved in bacterial internalization, or perhaps they play a larger

553

role in Wnt signaling associated with bacterial survival rather than entry. On the contrary, Fzd9

554

and DKK3 siRNAs reduced/increased bacterial load significantly at 1 d p.i. but not significantly

555

at 2 d, suggesting that they are more important for the bacterial entry or early stage. In addition,

556

knockdown of some Wnt pathway components had a dramatic effect on ehrlichial load,

557

suggesting that they play key roles in the Ehrlichia-Wnt axis. For example, knockdown of

558

Wnt5a, Fzd5 or Akt reduced the infection very significantly at both 1 d and 2 d p.i., suggesting

559

that they are critical components of Wnt signaling exploited by Ehrlichia survival; however we

560

cannot exclude the possibility that siRNAs may have different knockdown efficiency. Unlike the

561

effect of some small molecule inhibitors, the reduction of a single target protein by RNA

562

interference could not abolish the ehrlichial growth completely. This may result from the

563

incomplete knockdown of target proteins as was demonstrated by Western blot, or redundancy in

564

Wnt signaling.

For example, Wnt3a, Wnt10a, and calcineurin PPP3CB

565

Several Wnt signaling pathway inhibitors reduced ehrlichial infection significantly, three of

566

which, pyrvinium pamoate, KN93 and IWP-2 were highly effective. Pyrvinium was first found

567

to activate CK1α, but recently reported that it promotes Akt downregulation and GSK3

568

activation rather than activates CK1 (67, 68).

569

mechanism of action, pyrvinium ultimately inhibits Wnt/β-catenin signaling. However, Akt also

570

interacts with Wnt/Ca2+ and Wnt/PCP pathways, so pyrvinium may also influence noncanonical

25

Despite discrepancies in the understood

571

Wnt pathways. KN93 selectively binds to the calmodulin binding site of CaMKII and prevents

572

the association of calmodulin with CaMKII, and thus inhibits Wnt/Ca2+ pathway (69); IWP-2

573

inhibits the cellular Wnt processing and secretion via selective blockage of PORCN-mediated

574

Wnt palmitoylation (70). Two canonical Wnt pathway inhibitors SB202190 and TBCA, which

575

inhibit CK1δ/ε and CK2 respectively, were less effective. These inhibitors act on all three Wnt

576

signaling pathways and Wnt secretion, and thus, reveal the importance of both canonical and

577

noncanonical Wnt signaling pathways, especially CK1α/GSK3/Akt, CaMKII and Wnt ligands in

578

ehrlichial infection.

579

ehrlichial internalization, since addition of inhibitor SB202190 or TBCA before or after

580

ehrlichial infection did not make significant difference for reduction of bacterial load.

581

Pyrvinium, KN93 and IWP-2 could block the infection almost completely, so the roles of

582

CK1α/GSK3/Akt, CaMKII and Wnt ligand secretion in ehrlichial internalization could not be

583

verified by this experiment only.

584

ehrlichiosis treatment, but further study is needed to understand the importance of specific Wnt

585

pathway component in ehrlichial pathobiology.

CK1δ/ε and CK2 of canonical Wnt pathway were not important for

Wnt signaling pathway inhibitors have potential for

586

Consistent with recent reports (31, 44), our experiments indicate that inhibitors of PI3K,

587

IKK and lipid raft reduced TRP-mediated phagocytosis significantly. Activation of Rac1-PI3K-

588

IKK perhaps stimulates the assembly of scavenger receptors at lipid rafts and supports

589

cytoskeletal rearrangements required for phagocytosis (71-73). In addition, inhibitors of Akt,

590

Rac1, β-catenin/TCF and CaMKII, but not CK1δ/ε and CK2, also reduced TRP-induced

591

phagocytosis. Therefore, Wnt pathway components PI3K, IKK, Rac1, CK1α/GSK3β/Akt, β-

592

catenin/TCF, and CaMKII appear to be important in signaling that leads to ehrlichial

593

internalization.

In contrast, CK1δ/ε and CK2 are not involved, consistent with our other

26

594

experiment results. Based on the classification of these components, we may also define TRP-

595

induced phagocytosis primarily as a noncanonical mode of Wnt signaling (most likely Wnt/PCP

596

signaling), similar to Wnt5a-induced phagocytosis, although canonical Wnt signaling may be

597

involved and influence phagocytosis.

598

components/regulators of the Wnt signaling pathway appear to directly activate Wnt signaling.

599

However, in contrast to the recent report demonstrating Wnt5a triggered phagocytosis of a

600

bacteria (31), Wnt production inhibitor IWP-2 could not inhibit the phagocytosis mediated by

601

TRP, suggesting that Wnt ligands, such as Wnt5a, may promote Wnt signaling activation to

602

promote ehrlichial survival after internalization, but are not involved in TRP-induced

603

phagocytosis. Thus, it appears that Ehrlichia internalization is independent of Wnt ligand

604

secretion, although further experimentation is needed to confirm this conclusion.

Interactions between E. chaffeensis TRPs and

605

To date, the molecular mechanisms by which Ehrlichia enters and modulates host cells are

606

not well understood (4, 6-8), but our findings regarding Wnt pathways are consistent with

607

previous report that described some characteristics of cellular signaling upon internalization of

608

ehrlichiae (43, 44). E. chaffeensis enters the monocyte through lipid raft-caveolae-mediated

609

endocytosis, where it can reside within a cytoplasmic vacuole that resembles an early endosome,

610

preventing lysosomal fusion, and protecting it from killing (44). TRP120 is an ehrlichial surface

611

protein preferentially expressed on dense-core cells, and it has been found to interact with host

612

proteins involved in cytoskeleton organization (6). It has also been associated with ehrlichial

613

binding and internalization (45, 46). We found that ehrlichial TRPs stimulate phagocytosis by

614

macrophages and Wnt pathway inhibitors inhibit phagocytosis of TRP-coated microspheres.

615

Thus, ehrlichial TRPs appear to be agonists that directly activate Wnt signaling pathways of the

616

host to induce internalization and facilitate intracellular survival. However, the underlying

27

617

molecular mechanism remains unclear. Our data suggests that TRP120 induces phagocytosis

618

more efficiently than other TRPs, and TR and C-terminal domains of TRP120 mediate TRP-

619

induced phagocytosis. These domains associated with internalization were not examined in other

620

TRPs, but are predicted to be similar. Notably, another study reported that E. chaffeensis uses its

621

surface protein EtpE to bind GPI-anchored protein DNase X to trigger entry via CD147 and

622

hnRNP-K recruitment and actin mobilization (74, 75). The domains in TRPs responsible for

623

internalization are structurally distinct from EtpE and are not predicted to interact with DNase X.

624

Therefore, TRP-induced entry may involve a distinct TRP-interacting Wnt surface protein that

625

remains to be identified, rather than DNase X. Figure 6 is a proposed model showing E.

626

chaffeensis TRP-mediated activation of canonical and noncanonical Wnt signaling pathways.

627

TRP effector proteins bind unidentified cell receptors and activate Dvl protein and other

628

downstream signaling molecules of Wnt pathways identified in this study, resulting in regulation

629

of gene transcription and cytoskeletal reorganization/phagocytosis. Further study is needed to

630

determine the receptor involved, the roles of TRP-interacting proteins, and the relationship

631

between TRP and EtpE in ehrlichial binding and internalization.

632

Our study establishes for the first time an obligately intracellular pathogen-directed Wnt

633

pathway-induced mechanism responsible for invasion and persistence, and will provide insight

634

into mechanisms utilized by intracellular pathogens to invade host cells. Further research will

635

elucidate and define the mechanisms and specific interactions between TRPs and Wnt signaling

636

pathways.

637 638 639

28

640

FUNDING INFORMATION

641 642 643

This work was supported by the National Institutes of Health grants AI105536 and AI106859, and by funding from the Clayton Foundation for Research (to Jere W. McBride).

29

644

References:

645

1.

646 647

Microbiol Rev 16:37-64. 2.

648 649

3.

McBride JW, Walker DH. 2011. Molecular and cellular pathobiology of Ehrlichia infection: targets for new therapeutics and immunomodulation strategies. Expert Rev Mol Med 13:e3.

4.

652 653

Rikihisa Y. 2010. Anaplasma phagocytophilum and Ehrlichia chaffeensis: subversive manipulators of host cells. Nat Rev Microbiol 8:328-339.

650 651

Paddock CD, Childs JE. 2003. Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin

Dunphy PS, Luo T, McBride JW. 2013. Ehrlichia moonlighting effectors and interkingdom interactions with the mononuclear phagocyte. Microbes Infect 15:1005-1016.

5.

Dunphy PS, Luo T, McBride JW. 2014. Ehrlichia chaffeensis Exploits Host SUMOylation Pathways

654

to Mediate Effector-Host Interactions and Promote Intracellular Survival. Infect Immun

655

doi:10.1128/IAI.01984-14.

656

6.

Luo T, Kuriakose JA, Zhu B, Wakeel A, McBride JW. 2011. Ehrlichia chaffeensis TRP120 interacts

657

with a diverse array of eukaryotic proteins involved in transcription, signaling, and cytoskeleton

658

organization. Infect Immun 79:4382-4391.

659

7.

660 661

Luo T, McBride JW. 2012. Ehrlichia chaffeensis TRP32 interacts with host cell targets that influence intracellular survival. Infect Immun 80:2297-2306.

8.

Wakeel A, Kuriakose JA, McBride JW. 2009. An Ehrlichia chaffeensis tandem repeat protein

662

interacts with multiple host targets involved in cell signaling, transcriptional regulation, and

663

vesicle trafficking. Infect Immun 77:1734-1745.

664

9.

Doyle CK, Nethery KA, Popov VL, McBride JW. 2006. Differentially expressed and secreted

665

major immunoreactive protein orthologs of Ehrlichia canis and E. chaffeensis elicit early

666

antibody responses to epitopes on glycosylated tandem repeats. Infect Immun 74:711-720.

30

667

10.

Luo T, Zhang X, McBride JW. 2009. Major species-specific antibody epitopes of the Ehrlichia

668

chaffeensis p120 and E. canis p140 orthologs in surface-exposed tandem repeat regions. Clin

669

Vaccine Immunol 16:982-990.

670

11.

Kuriakose JA, Zhang X, Luo T, McBride JW. 2012. Molecular basis of antibody mediated

671

immunity against Ehrlichia chaffeensis involves species-specific linear epitopes in tandem repeat

672

proteins. Microbes Infect 14:1054-1063.

673

12.

Luo T, Zhang X, Wakeel A, Popov VL, McBride JW. 2008. A variable-length PCR target protein of

674

Ehrlichia chaffeensis contains major species-specific antibody epitopes in acidic serine-rich

675

tandem repeats. Infect Immun 76:1572-1580.

676

13.

677 678

Wakeel A, Zhu B, Yu XJ, McBride JW. 2010. New insights into molecular Ehrlichia chaffeensishost interactions. Microbes Infect 12:337-345.

14.

Lukas J, Mazna P, Valenta T, Doubravska L, Pospichalova V, Vojtechova M, Fafilek B, Ivanek R,

679

Plachy J, Novak J, Korinek V. 2009. Dazap2 modulates transcription driven by the Wnt effector

680

TCF-4. Nucleic Acids Res 37:3007-3020.

681

15.

Zhu B, Kuriakose JA, Luo T, Ballesteros E, Gupta S, Fofanov Y, McBride JW. 2011. Ehrlichia

682

chaffeensis TRP120 binds a G+C-rich motif in host cell DNA and exhibits eukaryotic

683

transcriptional activator function. Infect Immun 79:4370-4381.

684

16.

685

Nusse R, van Ooyen A, Cox D, Fung YK, Varmus H. 1984. Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307:131-136.

686

17.

Komiya Y, Habas R. 2008. Wnt signal transduction pathways. Organogenesis 4:68-75.

687

18.

Logan CY, Nusse R. 2004. The Wnt signaling pathway in development and disease. Annu Rev Cell

688 689 690

Dev Biol 20:781-810. 19.

Klaus A, Birchmeier W. 2008. Wnt signalling and its impact on development and cancer. Nat Rev Cancer 8:387-398.

31

691

20.

Nusse R, Varmus HE. 1992. Wnt genes. Cell 69:1073-1087.

692

21.

Nusse R, Varmus H. 2012. Three decades of Wnts: a personal perspective on how a scientific

693 694

field developed. EMBO J 31:2670-2684. 22.

695 696

4:2. 23.

697 698

24.

25.

26.

Sugimura R, Li L. 2010. Noncanonical Wnt signaling in vertebrate development, stem cells, and diseases. Birth Defects Res C Embryo Today 90:243-256.

27.

705 706

MacDonald BT, Tamai K, He X. 2009. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9-26.

703 704

Staal FJ, Clevers H. 2000. Tcf/Lef transcription factors during T-cell development: unique and overlapping functions. Hematol J 1:3-6.

701 702

Rao TP, Kuhl M. 2010. An updated overview on Wnt signaling pathways: a prelude for more. Circ Res 106:1798-1806.

699 700

Habas R, Dawid IB. 2005. Dishevelled and Wnt signaling: is the nucleus the final frontier? J Biol

Gordon MD, Nusse R. 2006. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 281:22429-22433.

28.

707

Niehrs C. 2012. The complex world of WNT receptor signalling. Nat Rev Mol Cell Biol 13:767779.

708

29.

Willert K, Nusse R. 2012. Wnt proteins. Cold Spring Harb Perspect Biol 4:a007864.

709

30.

Kikuchi A, Yamamoto H, Sato A, Matsumoto S. 2011. New insights into the mechanism of Wnt

710 711

signaling pathway activation. Int Rev Cell Mol Biol 291:21-71. 31.

712 713 714

Maiti G, Naskar D, Sen M. 2012. The Wingless homolog Wnt5a stimulates phagocytosis but not bacterial killing. Proc Natl Acad Sci U S A 109:16600-16605.

32.

Zhu F, Zhang X. 2013. The Wnt signaling pathway is involved in the regulation of phagocytosis of virus in Drosophila. Sci Rep 3:2069.

32

715

33.

716 717

signaling in human epithelia. Am J Physiol Gastrointest Liver Physiol 287:G220-227. 34.

718 719

Sun J, Hobert ME, Rao AS, Neish AS, Madara JL. 2004. Bacterial activation of beta-catenin

Liu X, Lu R, Wu S, Sun J. 2010. Salmonella regulation of intestinal stem cells through the Wnt/beta-catenin pathway. FEBS Lett 584:911-916.

35.

Lu R, Liu X, Wu S, Xia Y, Zhang YG, Petrof EO, Claud EC, Sun J. 2012. Consistent activation of the

720

beta-catenin pathway by Salmonella type-three secretion effector protein AvrA in chronically

721

infected intestine. Am J Physiol Gastrointest Liver Physiol 303:G1113-1125.

722

36.

Tahoun A, Mahajan S, Paxton E, Malterer G, Donaldson DS, Wang D, Tan A, Gillespie TL,

723

O'Shea M, Roe AJ, Shaw DJ, Gally DL, Lengeling A, Mabbott NA, Haas J, Mahajan A. 2012.

724

Salmonella transforms follicle-associated epithelial cells into M cells to promote intestinal

725

invasion. Cell Host Microbe 12:645-656.

726

37.

Lu R, Wu S, Zhang YG, Xia Y, Liu X, Zheng Y, Chen H, Schaefer KL, Zhou Z, Bissonnette M, Li L,

727

Sun J. 2014. Enteric bacterial protein AvrA promotes colonic tumorigenesis and activates colonic

728

beta-catenin signaling pathway. Oncogenesis 3:e105.

729

38.

Blumenthal A, Ehlers S, Lauber J, Buer J, Lange C, Goldmann T, Heine H, Brandt E, Reiling N.

730

2006. The Wingless homolog WNT5A and its receptor Frizzled-5 regulate inflammatory

731

responses of human mononuclear cells induced by microbial stimulation. Blood 108:965-973.

732

39.

733 734

George SJ. 2008. Wnt pathway: a new role in regulation of inflammation. Arterioscler Thromb Vasc Biol 28:400-402.

40.

Pereira C, Schaer DJ, Bachli EB, Kurrer MO, Schoedon G. 2008. Wnt5A/CaMKII signaling

735

contributes to the inflammatory response of macrophages and is a target for the

736

antiinflammatory action of activated protein C and interleukin-10. Arterioscler Thromb Vasc Biol

737

28:504-510.

33

738

41.

739 740

Kim J, Kim J, Kim DW, Ha Y, Ihm MH, Kim H, Song K, Lee I. 2010. Wnt5a induces endothelial inflammation via beta-catenin-independent signaling. J Immunol 185:1274-1282.

42.

Wang Q, Symes AJ, Kane CA, Freeman A, Nariculam J, Munson P, Thrasivoulou C, Masters JR,

741

Ahmed A. 2010. A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell

742

motility in prostate cancer. PLoS One 5:e10456.

743

43.

Lin M, Zhu MX, Rikihisa Y. 2002. Rapid activation of protein tyrosine kinase and phospholipase

744

C-gamma2 and increase in cytosolic free calcium are required by Ehrlichia chaffeensis for

745

internalization and growth in THP-1 cells. Infect Immun 70:889-898.

746

44.

Lin M, Rikihisa Y. 2003. Obligatory intracellular parasitism by Ehrlichia chaffeensis and

747

Anaplasma phagocytophilum involves caveolae and glycosylphosphatidylinositol-anchored

748

proteins. Cell Microbiol 5:809-820.

749

45.

Popov VL, Yu X, Walker DH. 2000. The 120 kDa outer membrane protein of Ehrlichia

750

chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli

751

associated with attachment and entry. Microb Pathog 28:71-80.

752

46.

753 754

Kumagai Y, Matsuo J, Hayakawa Y, Rikihisa Y. 2010. Cyclic di-GMP signaling regulates invasion by Ehrlichia chaffeensis of human monocytes. J Bacteriol 192:4122-4133.

47.

Kuriakose JA, Miyashiro S, Luo T, Zhu B, McBride JW. 2011. Ehrlichia chaffeensis transcriptome

755

in mammalian and arthropod hosts reveals differential gene expression and post transcriptional

756

regulation. PLoS One 6:e24136.

757

48.

758 759

Yin DT, Wu W, Li M, Wang QE, Li H, Wang Y, Tang Y, Xing M. 2013. DKK3 is a potential tumor suppressor gene in papillary thyroid carcinoma. Endocr Relat Cancer 20:507-514.

49.

Vasileiou G, Ekici AB, Uebe S, Zweier C, Hoyer J, Engels H, Behrens J, Reis A, Hadjihannas MV.

760

2015. Chromatin-Remodeling-Factor ARID1B Represses Wnt/beta-Catenin Signaling. Am J Hum

761

Genet 97:445-456.

34

762

50.

763 764

tumorigenesis. Cell Res 19:532-545. 51.

765 766

Lai SL, Chien AJ, Moon RT. 2009. Wnt/Fz signaling and the cytoskeleton: potential roles in

Zhang JZ, Popov VL, Gao S, Walker DH, Yu XJ. 2007. The developmental cycle of Ehrlichia chaffeensis in vertebrate cells. Cell Microbiol 9:610-618.

52.

Fazzi R, Pacini S, Carnicelli V, Trombi L, Montali M, Lazzarini E, Petrini M. 2011. Mesodermal

767

progenitor cells (MPCs) differentiate into mesenchymal stromal cells (MSCs) by activation of

768

Wnt5/calmodulin signalling pathway. PLoS One 6:e25600.

769

53.

Takeshita A, Iwai S, Morita Y, Niki-Yonekawa A, Hamada M, Yura Y. 2014. Wnt5b promotes the

770

cell motility essential for metastasis of oral squamous cell carcinoma through active Cdc42 and

771

RhoA. Int J Oncol 44:59-68.

772

54.

Kim MK, Maeng YI, Sung WJ, Oh HK, Park JB, Yoon GS, Cho CH, Park KK. 2013. The differential

773

expression of TGF-beta1, ILK and wnt signaling inducing epithelial to mesenchymal transition in

774

human renal fibrogenesis: an immunohistochemical study. Int J Clin Exp Pathol 6:1747-1758.

775

55.

776 777

protein family mediating axis induction by Wnt-5A. Science 275:1652-1654. 56.

778 779

57.

Mikels AJ, Nusse R. 2006. Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol 4:e115.

58.

782 783

Bhatt PM, Malgor R. 2014. Wnt5a: a player in the pathogenesis of atherosclerosis and other inflammatory disorders. Atherosclerosis 237:155-162.

780 781

He X, Saint-Jeannet JP, Wang Y, Nathans J, Dawid I, Varmus H. 1997. A member of the Frizzled

Kim J, Chang W, Jung Y, Song K, Lee I. 2012. Wnt5a activates THP-1 monocytic cells via a betacatenin-independent pathway involving JNK and NF-kappaB activation. Cytokine 60:242-248.

59.

Schaale K, Neumann J, Schneider D, Ehlers S, Reiling N. 2011. Wnt signaling in macrophages:

784

augmenting and inhibiting mycobacteria-induced inflammatory responses. Eur J Cell Biol 90:553-

785

559.

35

786

60.

Patel M, Morrow J, Maxfield FR, Strickland DK, Greenberg S, Tabas I. 2003. The cytoplasmic

787

domain of the low density lipoprotein (LDL) receptor-related protein, but not that of the LDL

788

receptor, triggers phagocytosis. J Biol Chem 278:44799-44807.

789

61.

790 791

internalization of LRP6 and accumulation of beta-catenin. Dev Cell 11:213-223. 62.

792 793

Yamamoto H, Komekado H, Kikuchi A. 2006. Caveolin is necessary for Wnt-3a-dependent

Follo MY, Manzoli L, Poli A, McCubrey JA, Cocco L. 2015. PLC and PI3K/Akt/mTOR signalling in disease and cancer. Adv Biol Regul 57:10-16.

63.

Forsberg M, Blomgran R, Lerm M, Sarndahl E, Sebti SM, Hamilton A, Stendahl O, Zheng L.

794

2003. Differential effects of invasion by and phagocytosis of Salmonella typhimurium on

795

apoptosis in human macrophages: potential role of Rho-GTPases and Akt. J Leukoc Biol 74:620-

796

629.

797

64.

Jiang W, Wang J, Zhang Y. 2013. Histone H3K27me3 demethylases KDM6A and KDM6B

798

modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling

799

pathway. Cell Res 23:122-130.

800

65.

Pereira F, Barbachano A, Silva J, Bonilla F, Campbell MJ, Munoz A, Larriba MJ. 2011.

801

KDM6B/JMJD3 histone demethylase is induced by vitamin D and modulates its effects in colon

802

cancer cells. Hum Mol Genet 20:4655-4665.

803

66.

Lorenowicz MJ, Macurkova M, Harterink M, Middelkoop TC, de Groot R, Betist MC, Korswagen

804

HC. 2014. Inhibition of late endosomal maturation restores Wnt secretion in Caenorhabditis

805

elegans vps-29 retromer mutants. Cell Signal 26:19-31.

806

67.

Thorne CA, Hanson AJ, Schneider J, Tahinci E, Orton D, Cselenyi CS, Jernigan KK, Meyers KC,

807

Hang BI, Waterson AG, Kim K, Melancon B, Ghidu VP, Sulikowski GA, LaFleur B, Salic A, Lee LA,

808

Miller DM, 3rd, Lee E. 2010. Small-molecule inhibition of Wnt signaling through activation of

809

casein kinase 1alpha. Nat Chem Biol 6:829-836.

36

810

68.

Venerando A, Girardi C, Ruzzene M, Pinna LA. 2013. Pyrvinium pamoate does not activate

811

protein kinase CK1, but promotes Akt/PKB down-regulation and GSK3 activation. Biochem J

812

452:131-137.

813

69.

Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T, Hidaka H. 1991. The newly

814

synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces

815

dopamine contents in PC12h cells. Biochem Biophys Res Commun 181:968-975.

816

70.

Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, Wei S, Hao W, Kilgore J, Williams NS, Roth

817

MG, Amatruda JF, Chen C, Lum L. 2009. Small molecule-mediated disruption of Wnt-dependent

818

signaling in tissue regeneration and cancer. Nat Chem Biol 5:100-107.

819

71.

820

Yin HL, Janmey PA. 2003. Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol 65:761-789.

821

72.

Lafont F, van der Goot FG. 2005. Bacterial invasion via lipid rafts. Cell Microbiol 7:613-620.

822

73.

Seveau S, Tham TN, Payrastre B, Hoppe AD, Swanson JA, Cossart P. 2007. A FRET analysis to

823

unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling

824

pathway. Cell Microbiol 9:790-803.

825

74.

Mohan Kumar D, Yamaguchi M, Miura K, Lin M, Los M, Coy JF, Rikihisa Y. 2013. Ehrlichia

826

chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger

827

Entry into Mammalian Cells. PLoS Pathog 9:e1003666.

828

75.

Mohan Kumar D, Lin M, Xiong Q, Webber MJ, Kural C, Rikihisa Y. 2015. EtpE Binding to DNase X

829

Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-

830

WASP and Actin. MBio 6:e01541.

831

37

832

FIGURE LEGENDS

833 834

FIG 1 Heat map of gene expression of Wnt signaling pathway in the THP-1 cells at 3, 24, 72 h

835

postinfection of E. chaffeensis by PCR arrays. Each individual well represents an individual

836

gene (see Fig. S1 for the gene tables and line graphs of important gene expression changes). The

837

scale bar shows color-coded differential expression level compared to that in uninfected cells.

838

Red and green indicate upregulation and downregulation, respectively.

839

pathway PCR array. (B) Wnt signaling targets PCR array.

(A) Wnt signaling

840 841

FIG 2 E. chaffeensis upregulates Wnt signaling. (A) Western blots show that phosphorylation of

842

β-catenin is inhibited at 1 h and 3 h postinfection of E. chaffeensis. (B) Wnt signal transducer β-

843

catenin and transcription factor NFATC1 translocate to nucleus in THP-1 cells at 1 h and 3 h

844

postinfection of E. chaffeensis. DAPI staining shows nucleus. Bars, 10 µm. (C) E. chaffeensis

845

infection activates NFAT reporter. The cell-free E. chaffeensis or uninfected THP-1 control was

846

added to NFAT reporter-transfected HeLa cells. Firefly and Renilla luciferase activities of each

847

well were measured at different time points posttreatment. RLU, relative luciferase units of

848

firefly that were normalized against Renilla luciferase activity. The experiment was repeated

849

three times, and the values are means ± standard deviations (*, P < 0.05)

850 851

FIG 3 Knockdown of Wnt signaling pathway component or target influences ehrlichial infection

852

of macrophages. THP-1 cells were transfected with specific or control siRNA and then infected

853

with E. chaffeensis. (A) Bacterial numbers were determined by qPCR at 1 day and 2 days p.i.

854

All experiments were repeated three times, and the values are means ± standard deviations (*, P

38

855

< 0.05). (B) Western blottings confirmed the reduction of Fzd9, Dvl2, and Jun proteins at 2 days

856

p.i.

857 858

FIG 4 Wnt signaling pathway inhibitors reduced ehrlichial infection of host cells.

(A)

859

Percentages of infected cells were determined by Diff-Quik staining and counting of 100 cells at

860

3 days p.i. Inhibitors were added 3 h before or after infection. An infected cell culture with the

861

vehicle (DMSO) only served as a positive control, and an uninfected culture served as a negative

862

control. Results are from three independent experiments, and the values are means ± standard

863

deviations (*, P < 0.05; **, P < 0.01). (B) Trypan blue staining was performed to assess host

864

cell viability graphed as a percentage of total cell count for THP-1 cells exposed to 72 h

865

treatments of vehicle or inhibitor. (C) Bright-field images (magnification, × 40) of Diff-Quik-

866

stained samples collected at 3 days p.i. demonstrate decreased number of infected cells following

867

treatment with Wnt signaling pathway inhibitors Pyrvinium and SB202190, as examples. Bars,

868

10 µm. Concentrations used for inhibitors were as following: 20 nM Pyrvinium, 4 µM KN93,

869

0.3 µM IWP-2, 0.5 µM TBCA, and 1.4 µM SB202190.

870 871

FIG 5 E. chaffeensis tandem repeat proteins stimulate phagocytosis and intracellular Ca2+ release

872

in macrophages, and Wnt pathway inhibitors reduce the stimulation. (A) Compared with control

873

TRP120N-coated beads, TRP120-coated beads were phagocytosed by the THP-1 cell. After Wnt

874

pathway inhibitor Bay11-7082 (10 µM) treatment, the promotion of bead internalization by

875

TRP120 was largely reduced.

876

different tandem repeat proteins and domains. (C) The effect of different Wnt pathway inhibitors

877

on the promotion of bead internalization by TRP120. All experiments were repeated three times,

(B) The efficiency of promotion of bead internalization by

39

878

and the values are means ± standard deviations (*, P < 0.05). (D) E. chaffeensis TRPs stimulate

879

Ca2+ release. Ca2+ release was measured in THP-1 cells using fluo-3/AM. Fluo-3-preloaded cells

880

were treated with cell-free E. chaffeensis (MOI = ~50), TRP-coated beads (50 beads/cell) or

881

control (cell-free uninfected THP-1 or thioredoxin-coated beads) and mixed briefly, and emission

882

at 525 nm after excitation at 488 nm was recorded every 20 seconds.

883 884

FIG 6 Proposed schematic diagram showing E. chaffeensis TRP-mediated activation of

885

canonical and noncanonical Wnt signaling pathways. TRP effector proteins bind unidentified

886

cell receptors and activate Dvl protein, which is the hub molecule of all Wnt pathways. Dvl

887

protein activates other signaling molecules of Wnt pathways in the cytosol, including many

888

kinases and phosphatase, resulting in regulation of gene transcription by transcription factors,

889

such as TCF and NFAT, and cytoskeletal reorganization and phagocytosis. TRPs also interact

890

with direct components or regulators (shown in green) of Wnt pathways to regulate Wnt

891

signaling.

892

40

893

TABLE 1. Activity scores of Wnt signaling pathway in E. chaffeensis-infected host cells as

894

determined by PCR array. Time point (p.i.)

Pathway activity scorea

P value

1h

-0.06

0.42

3h

0.49

0.01

8h

-0.06

0.42

24 h

-0.06

0.42

72 h

-0.37

0.04

Positive activity score > 0.3 or < -0.3 with P value < 0.05 indicates significant stimulation or repression

895

a

896

of pathway activity.

897

41

898

TABLE 2. Functionally categorized Wnt signaling genes with significant changes of expression

899

in E. chaffeensis-infected host cells determined by Wnt pathway PCR array. Fold regulationa Role in Wnt pathway inhibitor

ligand

receptor

900

Gene 1h

3h

8h

24 h

72 h

DKK3

-4.6

-3.0

-8.5

-4.6

-2.8

PRICKLE1

-2.0

-1.6

-1.3

-1.3

-2.2

SFRP4

-1.3

-1.3

-1.7

-5.1

-1.4

WNT10A

3.0

3.5

1.3

-1.1

-1.3

WNT5B

-1.1

1.7

-1.8

-2.2

-1.3

WNT6

3.5

4.4

1.1

1.4

-1.7

WNT7B

1.1

1.0

-1.2

1.6

-2.7

FZD5

1.7

2.0

2.0

1.3

-1.3

FZD7

-1.0

-1.6

-2.1

-1.8

-1.4

FZD9

3.0

3.5

2.8

2.0

1.5

VANGL2

1.8

2.1

-1.5

1.1

2.9

transcription factor

TCF7

-1.1

1.5

1.0

1.0

-2.3

target

FOSL1

2.1

1.7

1.6

1.1

-1.9

JUN

-1.8

-2.1

-3.3

-3.6

-2.3

WISP1

-6.4

-5.7

-6.2

-6.2

-5.4

CHSY1

2.6

-1.0

1.1

1.5

1.1

MYC

-1.5

-1.1

-1.1

-1.2

-3.5

SKP2

-1.6

-1.0

1.3

1.2

-2.2

a

Fold regulation > 2 indicates a significant upregulation, and < -2 indicates a significant downregulation.

42

901

TABLE 3. Functionally categorized Wnt signaling target genes with significant changes of

902

expression in E. chaffeensis-infected host cells determined by Wnt targets PCR array. Fold regulationb 8h

Genea

3h Development & differentiation (45 genes tested) ANTXR1 1.6 BMP4 -1.2 CCND1 2.3 CDH1 2.7 DAB2 -1.2 EFNB1 2.1 EGR1 -6.3 FGF9 1.5 FGF20 9.9 FN1 1.9 FST 6.8 GDNF 6.1 IL6 3.0 MET 1.6 MMP9 2.4 NANOG -1.1 NRP1 2.2 NTRK2 1.2 PDGFRA 2.1 SIX1 1.8 SOX2 -6.9 T (Brachyury) -1.1 VEGFA -1.2 Calcium binding and signaling (13 genes tested) CCND1 2.3 CUBN 4.6 MMP2 2.6 MMP7 -1.6 MMP9 2.4 PTGS2 -1.2 Adhesion (9 genes tested) ABCB1 CDH1

3.1 2.7

Migration (21 genes tested)

43

24 h

72 h

2.1 1.2 2.0 2.6 -1.1 1.4 -9.1 2.3 5.3 2.1 4.5 -1.2 2.8 2.2 2.7 -2.3 1.8 -4.2 1.2 2.1 -6.8 1.3 -1.7

1.4 -1.1 1.4 -8.0 -1.6 1.5 -12.3 2.1 4.3 1.1 3.4 2.9 3.4 2.1 2.0 -1.3 1.1 -4.1 -2.8 2.0 1.0 -3.7 -2.3

1.5 2.3 1.6 1.0 4.0 1.8 -1.7 1.3 7.1 1.4 3.1 -1.5 16.7 -3.7 3.7 1.0 1.8 3.5 1.9 -1.2 -3.2 1.2 1.9

2.0 7.5 2.6 -2.6 2.7 -1.9

1.4 6.6 1.4 -2.5 2.0 -2.6

1.6 9.3 1.9 -1.7 3.7 1.4

2.4 2.6

1.4 -8.0

2.8 1.0

903 904

EFNB1 FGF7 FN1 GDNF IL6 MMP9 NRP1 PDGFRA PPAP2B SIX1 VEGFA

2.1 -1.1 1.9 6.1 3.0 2.4 2.2 2.1 1.0 1.8 -1.2

1.4 -3.3 2.1 -1.2 2.8 2.7 1.8 1.2 -2.3 2.1 -1.7

1.5 1.2 1.1 2.9 3.4 2.0 1.1 -2.8 -4.2 2.0 -2.3

1.8 2.8 1.4 -1.5 16.7 3.7 1.8 1.9 1.4 -1.2 1.9

Cell Cycle (13 genes tested) CCND1 MYC PTGS2 SOX2

2.3 -1.1 -1.2 -6.9

2.0 -1.1 -1.9 -6.8

1.4 -1.2 -2.6 1.0

1.6 -3.5 1.4 -3.2

Proteolysis (5 genes tested) DPP10 MMP2 MMP7 MMP9

-3.3 2.6 -1.6 2.4

-1.7 2.6 -2.6 2.7

-2.0 1.4 -2.5 2.0

-1.1 1.9 -1.7 3.7

Signal transduction (23 genes tested) AXIN2 BMP4 FGF9 FST SFRP2 PPAP2B WISP1

1.8 -1.2 1.5 6.8 2.6 1.0 -5.7

-1.1 1.2 2.3 4.5 3.1 -2.3 -6.2

1.1 -1.1 2.1 3.4 1.4 -4.2 -6.2

-2.2 2.3 1.3 3.1 -1.7 1.4 -5.4

-9.1 -1.1 -2.3 2.1 -6.8 1.3 1.0

-12.3 -1.2 -1.3 2.0 1.0 -3.7 1.0

-1.7 -3.5 1.0 -1.2 -3.2 1.2 -2.3

Transcription factors (22 genes tested) EGR1 -6.3 MYC -1.1 NANOG -1.1 SIX1 1.8 SOX2 -6.9 T (Brachyury) -1.1 TCF7 1.5 a A few genes are classified into different categories. b

Fold regulation > 2 indicates a significant upregulation, and < -2 indicates a significant downregulation.

44

905

TABLE 4. List of small molecule inhibitors of Wnt pathways, their target proteins and

906

concentrations used in this study. Pathway/process

Inhibitor

Target a

Concentration (µM)

Canonical Wnt pathway FH535

Noncanonical Wnt pathways

Wnt ligand secretion 907

a

β-catenin/TCF

10

Pyrvinium pamoate

CK1α or Akt (activate GSK3)

0.02

SB202190

CK1δ/ε

1.4

TBCA

CK2

0.5

BAY11-7082

IKK

10

KN93

CaMKII

4

LY294002

PI3K

10

NSC23766

Rac1

50

IWP-2

PORCN

0.3

All targets are inhibited except CK1α.

45

908

TABLE 5. Fold regulation of E. chaffeensis bacterial load in THP-1 cells transfected with

909

siRNA specific for TRP120-interacting host targets involved in Wnt signaling relative to control

910

siRNA at 1day and 2 days p.i., as determined by qPCRa. Fold regulation siRNA target

Target property/function in Wnt signaling

ARID1B

Interacts with β-catenin to suppress Wnt signaling

KDM6B

β-catenin binding; activate Wnt3 or DKK1 to stimulate or

1 day

2 days

2.3

1.5

-4.3

-4.3

suppress Wnt signaling at different stages IRF2BP2

Interacts with NFATC2 to repress its transcriptional activity

-4.5

-3.9

PPP3R1

Calcineurin regulatory subunit 1: calcium ion and calmodulin

-4.0

-4.0

-9.8

-4.5

binding; calcium-dependent protein phosphatase activity; NFAT protein import into nucleus VPS29

Retrograde transport of proteins from endosomes to the transGolgi network; Wnt ligand biogenesis, secretion and trafficking

911

a

dsb gene copy number normalized to gapdh, average shown, n=2.

46

A

Wnt signaling pathway PCR array

B

Wnt signaling targets PCR array

3h

24 h

72 h

A h p.i.

0

1

3

8

24

48

phosphoβ-catenin

B h p.i. β-catenin

β-catenin α-tubulin

C

4

β-catenin (+ DAPI)

THP-1

THP-1/Ech

*

RLU

3

NFATC1

2 1

NFATC1 (+ DAPI)

0 3h

8h

24h

0

1

3

20

A

* *

* * *

*

* *

*

* *

*

*

* *

* *

* *

*

*

* *

* *

* *

*

*

* * *

* *

*

*

*

B siRNA control

Fzd9 control Fzd9

siRNA control Jun

siRNA control Dvl2

Fzd9

Dvl2

Dvl2

Jun α-tubulin

Jun

infected cells (%)

A 100 80

inhibition before infection inhibition after infection

* *

60 40 20 0

**

**

**

%cell viability

B 100 80 60 40 20 0

C

Pyrvinium

SB202190

Vehicle

A

DIC

fluorescence

merged

B 1.0

TRP120 Fluorescence

0.8

TRP120N

0.6 0.4 0.2 0.0

TRP120 +Bay117082

C

D

1.0

Fluorescence

0.8 0.6 0.4 0.2 0.0

*

*

* * *

*

*