Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival

Abstract

Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, β-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca(2+) and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival.

FIG 1

Heat maps of gene expression of the Wnt signaling pathway in THP-1 cells at 3, 24, and 72 h postinfection with E. chaffeensis determined with PCR arrays. Each individual well represents an individual gene (see Fig. S1 in the supplemental material for gene tables and line graphs of important gene expression changes). The scale bar shows color-coded differential expression levels compared to those in uninfected cells. Red and green indicate upregulation and downregulation, respectively. (A) Wnt signaling pathway PCR array. (B) Wnt signaling target PCR array.

FIG 2

E. chaffeensis upregulates Wnt signaling. (A) Western blots show that phosphorylation of β-catenin is inhibited at 1 h and 3 h postinfection with E. chaffeensis. (B) Wnt signal transducer β-catenin and transcription factor NFATC1 translocate to the nucleus in THP-1 cells at 1 h and 3 h postinfection with E. chaffeensis. DAPI (4′,6′-diamidino-2-phenylindole) staining shows the nucleus. Bars, 10 μm. (C) E. chaffeensis infection activates an NFAT reporter. The cell-free E. chaffeensis or uninfected THP-1 control was added to NFAT reporter-transfected HeLa cells. Firefly and Renilla luciferase activities of each well were measured at different time points posttreatment. RLU, relative luciferase units of firefly that were normalized against Renilla luciferase activity. The experiment was repeated three times, and the values are means ± standard deviations (*, P < 0.05).

FIG 3

Knockdown of a Wnt signaling pathway component or target influences ehrlichial infection of macrophages. THP-1 cells were transfected with a specific or control siRNA and then infected with E. chaffeensis. (A) Bacterial numbers were determined by qPCR at 1 day and 2 days p.i. All experiments were repeated three times, and the values are means ± standard deviations (*, P < 0.05). (B) Western blotting confirmed the reduction of Fzd9, Dvl2, and Jun proteins at 2 days p.i.

FIG 4

Wnt signaling pathway inhibitors reduce ehrlichial infection of host cells. (A) Percentages of infected cells were determined by Diff-Quik staining and counting of 100 cells at 3 days p.i. Inhibitors were added 3 h before or after infection. An infected cell culture with the vehicle (DMSO) only served as a positive control, and an uninfected culture served as a negative control. Results are from three independent experiments, and the values are means ± standard deviations (*, P < 0.05; **, P < 0.01). (B) Trypan blue staining was performed to assess host cell viability and graphed as a percentage of the total cell count for THP-1 cells exposed to 72-h treatments with vehicle or inhibitor. (C) Bright-field images (magnification, ×40) of Diff-Quik-stained samples collected at 3 days p.i. demonstrate decreased numbers of infected cells following treatment with Wnt signaling pathway inhibitors pyrvinium and SB202190, as examples. Bars, 10 μm. Concentrations used for inhibitors were as follows: 20 nM pyrvinium, 4 μM KN93, 0.3 μM IWP-2, 0.5 μM TBCA, and 1.4 μM SB202190.

FIG 5

E. chaffeensis tandem repeat proteins stimulate phagocytosis and intracellular Ca²⁺ release in macrophages, and Wnt pathway inhibitors reduce the stimulation. (A) Compared with control TRP120N-coated beads, TRP120-coated beads were phagocytosed by the THP-1 cells. After treatment with Wnt pathway inhibitor Bay11-7082 (10 μM), the promotion of bead internalization by TRP120 was largely reduced. (B) Efficiency of promotion of bead internalization by different tandem repeat proteins and domains. (C) Effect of different Wnt pathway inhibitors on the promotion of bead internalization by TRP120. All experiments were repeated three times, and the values are means ± standard deviations (*, P < 0.05). (D) E. chaffeensis TRPs stimulate Ca²⁺ release. Ca²⁺ release was measured in THP-1 cells using fluo-3/AM. Fluo-3-preloaded cells were treated with cell-free E. chaffeensis (MOI = ∼50), TRP-coated beads (50 beads/cell), or a control (cell-free uninfected THP-1 or thioredoxin-coated beads) and mixed briefly, and emission at 525 nm after excitation at 488 nm was recorded every 20 s.

FIG 6

Proposed schematic diagram showing E. chaffeensis TRP-mediated activation of canonical and noncanonical Wnt signaling pathways. TRP effector proteins bind unidentified cell receptors and activate Dvl protein, which is the hub molecule of all Wnt pathways. Dvl protein activates other signaling molecules of Wnt pathways in the cytosol, including many kinases and phosphatases, resulting in regulation of gene transcription by transcription factors, such as TCF and NFAT, and cytoskeletal reorganization and phagocytosis. TRPs also interact with direct components or regulators (shown in green) of Wnt pathways to regulate Wnt signaling.