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
2
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#
5 6
Departments of Pathologya and Microbiology and Immunology,b Center for Biodefense and
7
Emerging Infectious Diseases,c Sealy Center for Vaccine Development,d Institute for Human
8
Infections and Immunity,e University of Texas Medical Branch, Galveston, Texas, USA
9 10
Running title: Wnt signaling during Ehrlichia infection
11 12
# Address correspondence to:
13
Jere W. McBride, Ph.D.
14
Department of Pathology
15
University of Texas Medical Branch
16
Galveston, TX 77555-0609
17
Phone: (409) 747-2498
18
Fax: (409) 747-2455
19
E-mail:
[email protected]
20
1
21
ABSTRACT
22
Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and
23
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
34
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
36
components of canonical and noncanonical (Ca2+ and PCP) Wnt pathways, including IWP-2
37
which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated
38
Wnt signaling as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt
39
pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce
40
ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and
41
noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote
42
intracellular survival.
43
2
44
INTRODUCTION
45
Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the emerging
46
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
49
infection, and avoids host defenses are not completely understood, but occur through
50
functionally relevant host-pathogen interactions involving secreted ehrlichial tandem repeat
51
protein (TRP) effectors that are posttranslationally modified by ubiquitin (Ub) and the small
52
ubiquitin-like modifier (SUMO) (2-5). E. chaffeensis TRPs interact with a diverse group of
53
human proteins associated with major cellular processes, including transcription, translation,
54
protein trafficking, cell signaling, cytoskeleton organization, and apoptosis, indicating that they
55
play a role in manipulating these important cellular processes to facilitate infection (6-8).
56
E. chaffeensis TRPs were first recognized as antigens that elicit strong protective antibody
57
responses during infection directed at continuous species-specific epitopes in tandem repeat
58
regions (9-12) . Subsequently, our understanding of the functional role of TRPs as effectors in
59
pathobiology has been advanced through studies that have defined specific TRP-host protein and
60
DNA interactions (4, 13). Notably, E. chaffeensis TRP120 and TRP32 interact with numerous
61
host proteins and genes associated with the canonical and noncanonical Wnt signaling pathways.
62
One of TRP32-interacting targets, deleted-in-azoospermia associated protein 2 (DAZAP2), is a
63
highly conserved protein that modulates gene transcription driven by Wnt/β-catenin signaling
64
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
66
interactive domain 1B (ARID1B), lysine (K)-specific demethylase 6B (KDM6B), interferon
3
67
regulatory factor 2 binding protein 2 (IRF2BP2), protein phosphatase 3 regulatory subunit B
68
alpha (PPP3R1), and vacuolar protein sorting 29 homolog (S. cerevisiae) (VPS29) are also
69
involved in Wnt pathway signaling (6). Moreover, TRP120 binds host genes associated with
70
Wnt signaling pathways, such as Wnt, Dishevelled (Dvl) and nuclear factor of activated T-cells
71
(NFAT) (15). Thus, E. chaffeensis appears to exploit Wnt pathways through TRP-Wnt signaling
72
proteins and by modulating the expression of Wnt pathway genes via TRP transcriptional
73
modulation.
74
Wnt signaling was initially studied for its role in carcinogenesis, but has more recently
75
been recognized for its central role in embryonic development, differentiation, cell proliferation,
76
cell motility, cell polarity, and adult tissue homeostasis (16, 17).
77
signaling has been demonstrated by mutations that lead to a variety of diseases, including breast
78
and prostate cancer, glioblastoma, diabetes, and others (18, 19). Wnt signaling pathways are
79
highly evolutionarily conserved (20, 21). Thus far, three Wnt pathways have been characterized:
80
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
82
Wnt ligand to a Frizzled (Fzd) receptor (22, 23). In the canonical Wnt/β-catenin pathway,
83
activated Fzd heterodimerizes with lipoprotein receptor-related protein (LRP) to recruit and
84
activate Dishevelled (Dvl), which subsequently recruits the protein complex containing axis
85
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,
88
where it associates with TCF/LEF (lymphoid-enhancing factor) family transcription factors to
89
induce the expression of Wnt target genes (23-25). The noncanonical Wnt/Ca2+ pathway signals
4
The importance of Wnt
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through heterotrimeric G proteins, which further activates phospholipase C (PLC), leading to
91
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),
95
and c-Jun N-terminal kinase (JNK), and regulates cytoskeletal reorganization, cell motility and
96
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
98
be rigorously subdivided according to the pathway they induce (28, 29). However, Wnt3a and
99
Wnt5a are more commonly associated with canonical and noncanonical Wnt signaling,
100
respectively (30).
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Recently, the role of the Wnt pathway in phagocytosis of microorganisms has been
102
demonstrated (31, 32). The Wnt ligand-receptor (Wnt5a-Fzd5) signaling in macrophages was
103
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
105
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,
108
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
110
cytokine activation, e.g. in human macrophages stimulated by Mycobacterium (38-42).
111 112
E.
chaffeensis
binding
and
entry
is
known
to
involve
one
or
more
glycosylphosphatidylinositol (GPI)-anchored proteins associated with caveolae at the cell
5
113
surface, inducing receptor-mediated phagocytosis that triggers Wnt signaling-like events
114
including transglutamination, tyrosine phosphorylation, phospholipase Cγ2 (PLC-γ2) activation,
115
inositol-(1,4,5)-trisphosphate (IP3) production and intracellular calcium release (43, 44). In
116
addition, multiple studies have shown the importance of E. chaffeensis TRP120 in ehrlichial
117
binding and internalization (45, 46); however, the specific cellular pathways exploited to mediate
118
invasion and intracellular survival have not been defined. In this study, we demonstrate that host
119
Wnt signaling pathways are exploited for ehrlichial internalization and infection, and ehrlichial
120
TRPs are directly involved.
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MATERIALS AND METHODS
123 124
Cell culture and cultivation of E. chaffeensis. Human cervical epithelial adenocarcinoma
125
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
130
(Arkansas strain) was cultivated in THP-1 cells as previously described (47).
131 132
Inhibitors, siRNAs and antibodies. Wnt signaling pathway inhibitors included pyrvinium
133
pamoate, IWP-2, BAY11-7082, NSC23766 (Sigma), KN93, SB202190, TBCA, FH535, and
134
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
136
(Calcineurin PPP3CA), PP2B-Aβ (Calcineurin PPP3CB), TCF4, Wnt6, Wnt10a, and control-A
137
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,
139
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
143
mouse anti-human α-tubulin, NFATC1 (Santa Cruz) and β-catenin (Pierce, Rockford, IL) and
144
rabbit anti-human Fzd9 (Pierce), phospho-β-catenin, Dvl2 (Cell Signaling, Beverly, MA) and Jun
145
(Santa Cruz).
146 147
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
149
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
151
genes related to Wnt-mediated signal transduction, including Wnt signaling ligands, receptors
152
and regulators, as well as downstream signaling molecules and target proteins for all three Wnt
153
pathways.
154
classification algorithms to generate the pathway activity score, and determines whether Wnt
155
pathway activity is activated or repressed in experimental samples. The human Wnt signaling
156
targets PCR array profiles the expression of 84 key genes responsive to Wnt signal transduction,
157
including Wnt signaling pathway transcription factors and highly relevant target genes to analyze
158
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
7
159
manufacturer. In brief, uninfected and E. chaffeensis-infected THP-1 cells at different intervals
160
postinfection (p.i.) were collected and total RNA was purified using RNeasy Mini kit (Qiagen).
161
During RNA purification, on-column DNA digestion was performed using the RNase-free
162
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
165
RNA FlashGel (Lonza, Rockland, ME).
166
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)
174
assays on all arrays were between 18 CT and 22 CT. The CT values for all wells were exported
175
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
177
array reproducibility, reverse transcription efficiency control (RTC), human genomic DNA
178
contamination control (HGDC) and PPC.
The concentration and purity were determined by measuring the
Genomic DNA was eliminated and cDNA was
179 180
Western immunoblot. The THP-1 cell lysates were prepared using CytoBuster protein
181
extraction reagent (Novagen/EMD, Gibbstown, NJ), separated by sodium dodecyl sulfate-
8
182
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane.
183
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).
186 187
Immunofluorescence microscopy. Uninfected or E. chaffeensis-infected THP-1 cells at
188
different intervals p.i. were collected, and the indirect immunofluorescent antibody assay was
189
performed as previously described (6), except that anti-β-catenin or NFATC1 antibody (1:100)
190
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 ×
194
104/well) were seeded in a 96-well black plate, and the following day cells were transfected with
195
NFAT reporter plasmid using Lipofectamine 3000 (Invitrogen) and incubated overnight again.
196
The cell-free E. chaffeensis (multiplicity of infection [MOI] = ~50), or uninfected THP-1 control
197
was added to transfected HeLa cells. Firefly and Renilla luciferase activities of each well were
198
measured at different time points using the Dual-Glo luciferase assay system (Promega,
199
Madison, WI) and a Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA).
200
Firefly luciferase activity was normalized with Renilla luciferase.
201 202
RNA interference. THP-1 cells (1 × 105/well on a 96-well plate) were transfected with 5
203
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
9
205
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
207
day and 2 days p.i. for quantitative PCR (qPCR) and Western blot to determine knockdown
208
levels.
209 210
Quantification of E. chaffeensis by qPCR. THP-1 cells were pelleted, washed by PBS,
211
lysed in SideStep lysis and stabilization buffer (Agilent, Santa Clara, CA) for 30 min at room
212
temp, and analyzed for bacterial load using realtime qPCR. Amplification of the integral
213
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
220
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
222
change of dsb copy number relative to the control was normalized to qPCR-detected levels of the
223
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
226
treated with inhibitor or DMSO control 3 h before infection with cell-free E. chaffeensis at a
227
MOI of ~50, or cells were infected and then treated with inhibitor/DMSO 3 h p.i. Percentages of
10
228
infected cells were monitored daily over 3 days by Diff-Quik staining and counting of 100 cells.
229
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
231
higher than the IC50, or Ki as provided by the manufacturer. If the bacteria were inhibited at this
232
concentration, MICs were then determined by using two-fold serial dilutions. The MIC was
233
defined as the minimum inhibitor concentration required for inhibition of the growth of the
234
bacteria compared to that of the control (without the inhibitor) at day 3. An infected culture
235
without the inhibitor served as a positive growth control, and an uninfected cell culture served as
236
a negative control. All experiments were repeated three times to confirm results.
237 238
Phagocytosis of microspheres. Expression and purification of E. chaffeensis tandem
239
repeat proteins (TRP) has been described previously (9, 10, 12).
240
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
243
mixing at 20 rpm. The coated beads were collected by centrifugation, washed twice in MES
244
buffer and resuspended in RPMI medium, then gently sonicated to disperse the beads. The
245
efficiency of bead coating was confirmed by dot blot assay. TRP-coated or control thioredoxin
246
protein-coated beads were added to THP-1 cells at a multiplicity of approximately 50 beads per
247
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
261 262 263
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.
264 265
RESULTS
266 267
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
269
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
274
change in pathway activity observed in E. chaffeensis-infected THP-1 cells at 1 h, 8 h and 24 h
275
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
277
(green) at 72 h, compared to 24 h (Fig. 1A).
278
Among 84 host genes associated with Wnt signaling pathways assessed by PCR array (see
279
Fig S1A top panel for gene table), 18 (21% of total) genes exhibited significant differences in
280
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
295
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]).
300
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
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Wakeel A, Zhu B, Yu XJ, McBride JW. 2010. New insights into molecular Ehrlichia chaffeensishost interactions. Microbes Infect 12:337-345.
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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
*
*
* * *
*
*