WO2016191695A2 - Treatments for obligately intracellular infections - Google Patents

Abstract

Provided are compositions and methods for treating bacterial infections, such as infections by obligately intracellular bacteria. In some embodiments, a Wnt inhibitor may be administered to a mammalian subject to treat ehrlichiosis.

Description

DESCRIPTION

TREATMENTS FOR OBLIGATELY INTRACELLULAR INFECTIONS BACKGROUND OF THE INVENTION [0001] This application claims the benefit of United States Provisional Patent Application No. 62/167,669, filed May 28, 2015, the entirety of which is incorporated herein by reference. [0002] The invention was made with government support under Grant No. AI105536 and AI106859 awarded by the National Institutes of Health. The government has certain rights in the invention. 1. Field of the Invention

[0003] The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns methods of treating bacterial infections. 2. Description of Related Art

[0004] Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the emerging life-threatening human zoonosis, human monocytotropic ehrlichiosis (HME) (Paddock and Childs, 2003). E. chaffeensis selectively infects mononuclear phagocytes and resides in early-endosome-like membrane-bound vacuoles (Paddock and Childs, 2003). The mechanisms by which E. chaffeensis enters host cells, establishes persistent infection, and avoids host defenses are not completely understood, but occur through functionally relevant host-pathogen interactions involving secreted ehrlichial effector proteins (Rikihisa, 2010; McBride and Walker, 2011; Dunphy et al., 2013). Recently a family of tandem repeat proteins (TRP) was characterized that are type 1 secretion system substrates and interact with diverse human proteins associated with major cellular processes, including transcription, translation, protein trafficking, cell signaling, cytoskeleton organization, and apoptosis, indicating that E. chaffeensis TRPs play a role in manipulating these important cellular processes to facilitate infection (Luo et al., 2011; Luo and McBride, 2012; Wakeel et al., 2011; Wakeel et al., 2009; Wakeel et al., 2010). [0005] Recent findings of ehrlichial effector TRPs highlight the importance and complexity of interactions between bacterial effectors and host processes; however, the molecular mechanisms by which Ehrlichia modulates host cells are still not well understood (Dunphy et al., 2013; Luo et al., 2011; Luo and McBride, 2012; Wakeel et al., 2009). Phagocytosis is a specific form of endocytosis and a major mechanism used to remove pathogens in the immune system. Usually the pathogen becomes trapped in a phagosome which then fuses with a lysosome to form a phagolysosome, but E. chaffeensis enters the monocyte through lipid raft-caveolae-mediated endocytosis and then it can reside within a cytoplasmic vacuole that resembles an early endosome and does not fuse with lysosomes, protecting it from killing (Lin and Rikihisa, 2003). However, the underlying molecular mechanism remains unclear. [0006] While significant improvements have been made in understanding various ehrlichial proteins, much about these bacteria remain not well understood. For example, E. chaffeensis TRPs were initially identified as major immunoreactive proteins that elicit strong antibody responses during infection (Luo and McBride, 2012; Doyle et al., 2006; Luo et al., 2009), and antibodies directed at continuous species-specific epitopes in tandem repeat regions are protective against infection (Kuriakose et al., 2012). Subsequently, understanding of the functional role of TRPs as effectors in ehrlichial pathobiology has significantly advanced through studies that have defined specific TRP-host protein and DNA interactions (Dunphy et al., 2013). Despite these advances treatment with antibiotics remains the primary therapeutic approach for treating ehrlichiosis, and in many instances. Ehrlichiosis has been emerging as a significant problem in many parts of the world, and the possible emergence of antibiotic resistance may further complicate treatment options for infection by Ehrlichia. Clearly, there exists a need for additional therapies for the treatment of obligately intracellular bacteria such as Ehrlichia.

SUMMARY OF THE INVENTION [0008] The present invention is based, in part, on the discovery that Ehrlichia bacteria utilize canonical and noncanonical host Wnt signaling pathways to stimulate phagocytosis and promote intracellular survival, and inhibition of Wnt signaling may be used to treat or inhibit bacterial infection by obligately intracellular bacteria such as Ehrlichia canis or Ehrlichia chaffeensis. Additionally, it has also been observed as described herein that Notch signaling can promote ehrlichial survival, and a Notch inhibitor may be used to treat infection by an obligately intracellular bacteria such as Ehrlichia canis or Ehrlichia chaffeensis. In some embodiments and as shown in the examples, RNAi or small molecule inhibitors may be used to inhibit canonical and/or non-canonical Wnt signaling to decrease bacterial load or treat ehrlichiosis. In various aspects, a Wnt inhibitor may be used to treat (e.g., inhibit phagocytosis and replication) of an obligately intracellular bacteria, an obligately intracellular protozoa, or an obligately intracellular fungi in a host. [0009] As shown in the below examples, induction of host Wnt signaling pathways by E. chaffeensis, directed by TRP effectors, can stimulate phagocytosis and reduces ehrlichial killing. After E. chaffeensis infection, canonical and noncanonical Wnt signaling pathways were observed to be significantly stimulated at the very early stage (1-3 h) of infection as determined by β-catenin and NFATC1 nuclear translocation, but repressed at the later stage (72 h) of infection. Similarly, expression levels of approximately 46% of Wnt component and signaling target genes were observed to be modulated during E. chaffeensis infection, indicating early activation and late repression of the pathway. Knockdown of critical components of Wnt signaling pathways by RNAi, including Wnt5a ligand, receptor and co- receptor, regulator, other signaling molecules, transcription factor and target, resulted in significant reductions in ehrlichial load. Similarly, small molecule inhibitors of the canonical and noncanonical (Ca²⁺ and PCP) Wnt signaling pathways significantly decreased ehrlichial infection in vitro, including casein kinase Iα/GSK3β activator pyrvinium pamoate, calmodulin kinase II inhibitor KN93 and inhibitor of Wnt secretion, IWP-2, which dramatically reduced bacterial and TRP coated latex bead internalization. Latex beads coated with E. chaffeensis TRP120 and TRP32 were observed to be phagocytized through Wnt5a-directed mechanisms, demonstrating a key role of these effectors in Wnt5a-mediated phagocytosis. Without wishing to be bound by any theory, these findings demonstrate that E. chaffeensis exploits canonical and noncanonical Wnt pathways to induce phagocytosis and promote intracellular survival. [0010] Data is also presented herein showing that E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, including the Notch receptor cleaving metalloprotease ADAM17. In the below examples, it is shown that E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. Nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) was observed to occur in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. Notch pathway activation was linked specifically to the TRP120 effector, which was observed to directly interact with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and siRNAs against γ-secretase enzyme, Notch transcription factor complex, Notch1 and ADAM17, Notch signaling was demonstrated o be required for ehrlichial survival. The inventors studied the downstream effects and found that E. chaffeensis and TRP120-mediated activation of Notch pathway causes inhibition of ERK1/2 and p38 MAPK pathway required for PU.1 and subsequent TLR2/4 expression. These experiments reveals a mechanism whereby E. chaffeensis exploits Notch pathway to evade the host innate immune response for intracellular survival. Thus, inhibiting Notch signaling may be used to treat ehrlichiosis or an infection by E. canis or E. chaffeensis in a subject. [0011] An aspect of the present invention relates to a method of treating a bacterial infection in a mammalian subject, comprising administering a therapeutically effective amount of a Wnt inhibitor to said subject, wherein said bacterial infection comprises an obligately intracellular bacteria. The obligately intracellular bacteria may be Ehrlichia, Chlamydia, Rickettsia, Coxiella, Orientia, or Mycobacteria. In some embodiments, the obligately intracellular bacteria is Ehrlichia chaffeensis. In some embodiments, the obligately intracellular bacteria is Ehrlichia canis. The subject may be a human. The subject may be a dog. In some embodiments, the Wnt inhibitor is a small interfering RNA (RNAi), an antibody, or a small molecule Wnt inhibitor. The Wnt inhibitor may be administered orally, intravenously, or parenterally. The Wnt inhibitor may be an inhibitor of a canonical Wnt pathway. In some embodiments, the Wnt inhibitor selectively inhibits PI3K, CKII, CK1ε, or β-catenin/TCF/LEF, or the Wnt inhibitor selectively activates casein kinase Iα/GSK3β. In some embodiments, the Wnt inhibitor selectively activates casein kinase Iα/GSK3β. In some embodiments, the Wnt inhibitor is pyrvinium pamoate, TBCA, SB202190, LY294002, or FH535. In some embodiments, the Wnt inhibitor is pyrvinium pamoate. The Wnt inhibitor may be an inhibitor of a non-canonical Wnt pathway. In some embodiments, the Wnt inhibitor is an inhibitor of a Wnt/Ca²⁺ pathway. The Wnt inhibitor may selectively inhibit calmodulin kinase II (CaMKII) or IKK. In some embodiments, the Wnt inhibitor selectively inhibits calmodulin kinase II. In some embodiments, the Wnt inhibitor is KN93. The Wnt inhibitor may be an inhibitor of a Wnt/PCP pathway. In some embodiments, the Wnt inhibitor selectively inhibits PI3K, Akt, or IKK. In some embodiments, the Wnt inhibitor selectively inhibits Akt. In some embodiments, the Wnt inhibitor is pyrvinium pamoate, LY294002, or BAY 11-7082. In some embodiments, the Wnt inhibitor is pyrvinium pamoate. The Wnt inhibitor may selectively inhibit Wnt secretion. In some embodiments, the Wnt inhibitor selectively inhibits PORCN. In some embodiments, the Wnt inhibitor is IWP-2. In some embodiments, the Wnt inhibitor is comprised in a pharmaceutical preparation. The pharmaceutical preparation may be formulated, e.g., for oral, intravenous, topical, or parenteral administration. [0012] Another aspect of the present invention relates to a method of treating a bacterial infection in a mammalian subject, comprising administering a therapeutically effective amount of a Notch inhibitor to said subject, wherein said bacterial infection comprises an obligately intracellular bacteria. The obligately intracellular bacteria may be Ehrlichia, Chlamydia, Rickettsia, Coxiella, Orientia, or Mycobacteria. In some embodiments, the obligately intracellular bacteria is Ehrlichia chaffeensis. In some embodiments, the obligately intracellular bacteria is Ehrlichia canis. The subject may be a human or a dog. The Notch inhibitor may be a small interfering RNA (RNAi), an antibody, or a small molecule Notch inhibitor. In some embodiments, the Notch inhibitor is administered orally, intravenously, or parenterally. In some embodiments, the Notch inhibitor selectively inhibits γ-secretase, Notch transcription factor complex, Notch1, ADAM17, or ADAM10. In some embodiments, the Notch inhibitor selectively inhibits γ-secretase. In some embodiments, the Notch inhibitor is DAPT, BMS-906024, MK0752, PF-03084014, MRK0003, or RO4929097. In some embodiments, the Notch inhibitor is an antibody, an antibody fragment, or a Notch blocking peptide. For example, the Notch inhibitor may be a blocking peptide (e.g., MAM peptide agonist SAHM1), an antibody fragment or a neutralizing antibody (e.g., OMP-59R5 (anti- Notch 2/3 mAb), NRR1 anti-Notch1 mAB, NRR2 anti-Notch2 mAb, NRR3 anti-Notch3 mAb, OMP-21M18 anti-DLL4 mAb, a DLL1-Fc Fc anti-delta like 1 chimeric mAb, a JAG1 Fc or jagged 1 Fc chimeric mAb, or A5622A anti-nicastrin mAb), a monoclonal antibody (mAb), or a Notch decoy (e.g., a soluble Notch1, Dll1, jagged1). The Notch inhibitor may be comprised in a pharmaceutical preparation. In some embodiments, the pharmaceutical preparation is formulated for oral, intravenous, topical, or parenteral administration. In some embodiments, the bacterial infection is not Salmonella typhimurium, Mycobacterium bovis (M. bovis) BSG, Bacillus anthracis, or Clostridium difficile. In some embodiments, both the Wnt inhibitor and the Notch inhibitor are administered to the subject to treat the bacterial infection. [0013] As used herein the specification,“a” or“an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word“comprising”, the words“a” or “an” may mean one or more than one. [0014] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” As used herein “another” may mean at least a second or more. [0015] Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0016] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0017] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0018] FIG. 1: Expression levels of seven host genes at different time points postinfection of E. chaffeensis by Wnt signaling pathway PCR array. Fold regulation > 1 indicates an upregulation, and <‐1 indicates a downregulation, compared to that in uninfected cells. [0019] FIG 2: Expression levels of six target genes of Wnt signaling at different time points postinfection of E. chaffeensis by Wnt signaling target PCR array. Fold regulation > 1 indicates an upregulation, and <‐1 indicates a downregulation, compared to that in uninfected cells. [0020] FIGS. 3A-B: Knockdown of Wnt signaling pathway component or target influences ehrlichial infection of macrophages. THP‐1 cells were transfected with specific or control siRNA and then infected with E. chaffeensis. (FIG. 3A) 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). (FIG. 3B) Western blottings confirmed the reduction of FZD9, DVL2, GSK3β and JUN proteins at 2 days p.i. [0021] FIGS.4A-B: Wnt signaling pathway inhibitors reduced ehrlichial infection of host cells. (FIG.4A) Percentages of infected cells were determined by Diff‐Quik staining and counting of 100 cells at 3 days p.i. An infected cell culture without the inhibitor 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). (FIG. 4B) Bright‐field images (magnification, × 40) of Diff‐Quik‐stained samples collected at 3 days p.i. demonstrate decreased number of infected cells following treatment with Wnt signaling pathway inhibitors Pyrvinium and SB202190, as examples. Concentrations used for inhibitors were as following: 20 nM Pyrvinium, 4 μM KN93, 0.3 μM IWP‐2, 0.5 μM TBCA, and 1.4 μM SB202190. [0022] FIGS. 5A-B: E. chaffeensis upregulatesWnt signaling. (FIG. 5A) β‐catenin translocates to nucleus in THP‐1 cells at 3 h postinfection of E. chaffeensis. (FIG. 5B) Wnt transcription factor NFATC1 translocates to nucleus in THP‐1 cells at 1 h and 3 h postinfection of E. chaffeensis. DAPI staining shows nucleus. [0023] FIGS. 6A-C: Wnt pathway inhibitor pyrvinium abolished the stimulation of phagocytosis by the ehrlichial tandem repeat protein TRP120 in macrophages. (FIG.6A and FIG. 6B) Compared with control TRP120N-coated beads, TRP120‐coated beads were phagocytosed by the THP‐1 cell dramatically. (FIG. 6C) After Wnt pathway inhibitor pyrvinium (30 nM) treatment, TRP120 could not promote the internalization of beads by the THP‐1 cell. [0024] FIG. 7: Illustration of canonical and noncanonical Wnt signaling pathways, potent inhibitors and involved TRPinteracting proteins. Wnt pathways regulate cellular processes including gene transcription, phagocytosis, and cytoskeletal reorganization. Small molecule inhibitors disrupt components of Wnt pathways. Ehrlichial TRPs interact with proteins that regulate Wnt signaling. [0025] FIGS.8A-C: E. chaffeensis activates Notch signaling pathway in THP-1 cells. (FIG. 8A) Nuclear translocation of NICD was analyzed at 2 h p.i. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and probed with anti-NICD antibody (Alexa Fluor 568, red), and DNA (DAPI, blue) then visualized by immunofluorescence microscopy (40x) (Bars, 10μm). (FIG.8B) Expression levels of Notch signaling components in THP-1 cells were analyzed using real time RT-PCR in uninfected and E. chaffeensis infected THP-1 cells, 2 h (open bar) and 72 h p.i. (closed bar). mRNA level of notch1, hes1, hes5 and hey2 was normalized to GAPDH and compared with the level of uninfected cells (Student’s t test: ** p< 0.01, ***p < 0.001, n=3). (FIG. 8C) Induction of Hes1, ADAM17 and α-tubulin protein expression was analyzed by Western immunoblot in THP-1 cells at 48 h p.i. Representative data (n=4). [0026] FIGS. 9A-C: Expression array analysis of Notch signaling pathway genes during E. chaffeensis infection. (FIG. 9A) Heat map showing relative expression levels of Notch signaling pathway components and its downstream target genes at 12, 24, 48 and 72 h p.i. Each individual well in the heat map represents individual gene, and the scale bar shows differential expression from the mean gene expression level of uninfected cells. The levels of induction/repression are shown. (FIG. 9B) Scatter plot showing the Notch PCR array data. Genes displaying upregulation, no significant change of expression, and downregulation are shown. (FIG. 9C) List of genes with their fold-change which showed differential expression (up and down-regulation) at 24 h p.i. Notch signaling pathway target genes, Notch pathway components and other genes involved in signaling pathway that cross talks with the Notch signaling pathway are grouped as shown. [0027] FIGS. 10A-D: Inhibition of Notch pathway decreases E. chaffeensis load. THP-1 cells were treated with pharmacological inhibitors against γ-secretase enzyme (DAPT) and RBPjκ transcription complex (SAHM1). Cells were infected with E. chaffeensis after 1 h post treatment. Bacterial loads were determined at 24 and 48 h p.i. either by (FIG. 10A) calculating the percentage of infected cells by counting 100 Diff-Quik-stained cells or (FIG. 10B) using qPCR measurement of dsb copy number. (FIG.10C) THP-1 cells were transfected with specific or control siRNA to knockdown Notch1/ADAM17/RBPjκ and then infected with E. chaffeensis (1 day post transfection). Ehrlichial loads were determined using qPCR measurement of dsb copy number at 24 and 48 h p.i. Data represented as means ± SD, *p < 0.05, ** p < 0.01, ***p < 0.001, n=3). (FIG.10D) Western blots confirmed the reduction of Notch1, ADAM17 and RBPjκ proteins at day 2 p.i. [0028] FIG. 11: ADAM17 and Notch1 interact with TRP120 expressing E. chaffeensis. E. chaffeensis-infected or uninfected THP-1 cells (48 h) were fixed, permeabilized and probed with (A) anti-TRP120 (green), anti-ADAM17 (red) (B) anti-TRP120 (green), anti- Notch1 (red). ADAM17 and Notch1 protein co-localization with TRP120 was observed. (C) HeLa cells were transfected with GFP-TRP120 WT or GFP-control plasmids and probed with anti-ADAM17 (red) antibody (24 h post transfection). Direct ADAM17 and TRP120 interaction through co-localization was observed. Cells were visualized by immunofluoresence microscopy (40x; Bars, 10μm). DAPI shows DNA, blue. Representative data (n=4). [0029] FIGS. 12A-E: Activation of Notch signaling pathway by E. chaffeensis TRP120. (FIG. 12A) THP-1 cells were treated with TRP120 coated beads, fixed, permeabilized, and probed with anti-NICD (red) and DNA (DAPI, blue) then visualized by immunofluoresence microscopy (40x). NICD nuclear translocation after 15 min stimulation with TRP120-coated beads was observed, (Bars, 10μm). (FIG. 12B) Expression levels of notch1, hes1 and hes5 in THP-1 cells were analyzed using real time RT-PCR. RNA was isolated from cells stimulated with TRP120 or thioredoxin coated beads (2 h). mRNA level was normalized to GAPDH and compared with the level of control cells (student’s t test: *p < 0.05, ** p< 0.01, n=3). Notch pathway PCR array was performed to analyze gene expression level in THP-1 cells stimulated with TRP120 coated beads compared to thioredoxin control (24 h). (FIG. 12C) Heat map showing the expression level of Notch signaling genes after TRP120 stimulation. For each gene, the scale bar shows differential expression from the mean gene expression level of thioredoxin stimulated cells. The level of induction (red) or repression (green) are shown. (FIG. 12D) Scatter plot showing the Notch gene expression. Increased gene expression, no significant change of expression, and decreased gene expression compared to control cells are shown. The cutoff was 2 fold. (FIG. 12E) List of genes with their fold change which showed differential expression (up and down-regulation) at 24 h post stimulation with TRP120 coated beads. [0030] FIGS. 13A-D: Notch signaling regulates ERK1/2 and p38 MAPK signaling during ehrlichial infection. THP-1 cells were infected with E. chaffeensis (MOI 100, 1 h post treatment) in the presence or absence of Notch inhibitor SAHM1 (10 μM). Medium or LPS (100 ng/ml) was added at the indicated time and cells were incubated for 30 min. Cells were lysed after incubation and cell lysates were analyzed for (FIG.13A) phosphorylated ERK1/2, (FIG. 13B) total ERK1/2, (FIG. 13C) phosphorylated p38 MAPK and (FIG. 13D) total p38 MAPK using Luminex bead arrays. Results represents as the mean ± SD (n=4), *p < 0.05, ** p< 0.01. [0031] FIGS. 14A-D E. chaffeensis mediated downregulation of PU.1 depends on Notch signaling pathway. THP-1 cells were transfected with either control siRNA or RBPjκ siRNA, incubated for 24 h and then infected with E. chaffeensis. After 24 h p.i. cells were stimulated with LPS (100 ng/ml) for 1 h and expression of PU.1 was determined (FIG. 14A) by probing with anti-PU.1 (red), anti-TRP120 (green) and visualized by immunofluorescence microscopy (Bars, 10μm) or using (FIG.14B) Western blot confirmed the reduction of RBPjκ protein. (FIG.14C) Western blot of whole cell lysates of uninfected and E. chaffeensis infected cells (48 h) in the presence of vehicle (DMSO) or Notch inhibitor SAHM1. Representative data (n=4). (FIG. 14D) Quantitative analysis of the Western blot data using image J software (Student’s t test: *p < 0.05, ** p< 0.01, ***p < 0.001, n=4). [0032] FIGS. 15A-D Notch signaling pathway plays critical role in inhibition of TLR2/4 expression during E. chaffeensis infection. THP-1 cells were treated with either vehicle (DMSO), or with Notch inhibitor DAPT and SAHM1 for 1 h then infected with E. chaffeensis. Expression of TLR2 and 4 was measured using RT-PCR (24 h p.i.) and Western blot (48 h p.i.) after 1 h stimulation with LPS (100 ng/ml). (FIG.15A) TLR2 and (FIG. 15B) TLR4 mRNA level was normalized to GAPDH and compared with the level of untreated cells (*p < 0.05, ** p < 0.01, n=3). (FIG. 15C) Immunoblot analysis was done using anti-TLR4 and anti-TLR2 antibody. Representative data (n=4). (FIG. 15D) Relative band intensities of TLR2/4 have been normalized to the loading control GAPDH and was determined using Image J software (Student’s t test: *p < 0.05, ** p< 0.01, ***p < 0.001, n=4). [0033] FIGS. 16A-D: TRP120 mediated downregulation of PU.1, TLR2 and TLR4 expression. THP-1 cells were treated with thioredoxin (control) or TRP120 coated latex beads or TRP120 in suspension (1 μg/ml) for 24 h and stimulated with LPS for 1 h. IFA analysis was done to measure (FIG.16A) PU.1 (FIG.16B) TLR2 and (FIG.16C) TLR4 expression. (FIG. 16D) Western blot showing reduced expression of PU.1, TLR2 and TLR4 with TRP120 stimulation. Representative data (n=4). [0034] FIG. 17 Proposed model for E. chaffeensis TRP120 mediated activation of canonical Notch signaling pathway and inhibition of TLR2/4 expression. (1) E. chaffeensis TRP120 effector interaction with ADAM17 activates the metalloprotease resulting in cleavage of the substrate Notch1 and subsequent cleavage by γ-secretase causes (2) nuclear translocation of NICD, the transcriptionally active form which binds with RBPjκ and MAML proteins. (3) This tri-protein complex activates transcription of Notch target genes which causes inhibition of ERK1/2 and p38 MAPK pathway (4). The downstream transcription factor PU.1 expression is repressed, which causes further inhibition of monocyte TLR2/4 expression. Inhibition of TLR2/4 expression causes both inhibition of E. chaffeensis recognition and TLR mediated proinflammatory cytokine production needed for the activation of monocytes and clearance of ehrlichiae.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0035] The present invention provides, in some aspects, methods for treating an infection by an obligately intracellular pathogen, such as an obligately intracellular bacteria, in a subject by administering a pharmaceutically effective amount of a Wnt inhibitor or a Notch inhibitor to the subject. In some aspects and as shown in the below examples, it has been observed that inhibition of Wnt signaling in cells by modulating canonical or non-canonical Wnt signaling at a variety of stages in the Wnt signaling pathway can decrease infection of cells by obligately intracellular bacteria such as Ehrlichia (e.g., E. chaffeensis or E. canis). In some embodiments, the bacteria is tuberculosis or chlamydia. [0036] In some aspects, the present invention provides methods for treating an obligately intracellular bacteria by administering a pharmacologically effective dose of a Notch inhibitor to the subject. In some embodiments, the method may comprise administering both a Wnt inhibitor and a Notch inhibitor to treat an obligately intracellular bacteria such as, e.g., E. chaffeensis or E. canis. As shown in the below examples, a host pathogen interaction was observed whereby E. chaffeensis exploits the Notch signaling pathway to subvert innate host defenses. The Notch pathway was activated by E. chaffeensis and linked specifically to the TRP120 effector. The inventors further analyzed the underlying survival mechanism and showed that E. chaffeensis and TRP120-mediated activation of the Notch pathway causes inhibition of ERK1/2 and p38 MAPK signaling pathways and expression of transcription factor PU.1, which represses TLR2/4 expression. To the knowledge of the inventors, this investigation is the first to demonstrate pathogen exploitation of Notch signaling to modulate PRR expression and to promote intracellular survival. I. Wnt Signaling and Wnt Inhibitors

[0037] Wnt signaling was first identified for its role in carcinogenesis, but has since been recognized for its central role in embryonic development, differentiation, cell proliferation, cell motility, cell polarity, and adult tissue homeostasis (Klaus and Birchmeier, 2008; Nusse et al., 1984). The importance of Wnt signaling has been demonstrated by mutations that lead to a variety of diseases, including breast and prostate cancer, glioblastoma, type II diabetes, and others (Komiya and Habas, 2008; Logan and Nusse, 2004). Wnt signaling pathways are highly evolutionarily conserved (Nusse and Varmus, 2012; Nusse and Varmus, 1992). At least three Wnt signaling pathways have been characterized: a canonical Wnt/β- catenin pathway and two noncanonical β-catenin-independent pathways, the Wnt/PCP (planar cell polarity) pathway, and the Wnt/Ca²⁺ pathway. All three Wnt signaling pathways are activated by the binding of a Wnt ligand to a transmembrane receptor Frizzled (FZD), which activates the protein Dishevelled (DVL) inside the cell (Habas and Dawid, 2005; Rao and Kuhl, 2010). In the canonical Wnt/β-catenin pathway, Dishevelled recruits the protein complex containing axis inhibitor (Axin), adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase-3 (GSK-3), leading to the inhibition of phosphorylation of β-catenin by these kinases. Unphosphorylated β-catenin accumulates and subsequently translocates to the nucleus, where it associates with TCF/LEF (lymphoid-enhancing factor) family transcription factors to induce the expression of Wnt target genes (Rao and Kuhl, 2010; MacDonald et al., 2009; Staal and clevers, 2000). The noncanonical Wnt/PCP pathway involves the Rho and Rac GTPases, Rho kinase (ROCK), MAP kinases and c-Jun N-terminal kinase (JNK), and regulates cell motility and tissue polarity (Komiya and Habas, 2008; Gordon and Nusse, 2006). The noncanonical Wnt/Ca²⁺ pathway involves stimulation of heterotrimeric G-proteins, which further activates phospholipase C (PLC), leading to increased intracellular Ca²⁺ release and activation of calcium/calmodulin-dependent protein kinase II (CaMKII), calcineurin and protein kinase C (PKC) (Komiya and Habas, 2008). These processes can stimulate nuclear factor of activated T-cells (NFAT) and other transcription factors like cAMP response element-binding protein (CREB) (Komiya and Habas, 2008; Sugimura and Li, 2010). [0038] E. chaffeensis binding and entry appear to involve one or more glycosylphosphatidylinositol (GPI)-anchored proteins associated with caveolae at the cell surface, inducing receptor-mediated endocytosis that triggers Wnt signaling-like events including transglutamination, tyrosine phosphorylation, phospholipase Cγ2 (PLC-γ2) activation, inositol-(1,4,5)-trisphosphate (IP₃) production and intracellular calcium release (Lin and Rikihisa, 2003; Lin et al., 2002). Moreover, multiple studies have shown the importance of E. chaffeensis TRP120 in ehrlichial binding and internalization (Kumagai et al., 2010; Popov et al., 2000). Recently, E. chaffeensis outer membrane protein, EtpE, was also shown to play a role in this process by binding the cellular GPI-anchored protein, DNaseX (Mohan et al., 2013); however, the specific cellular pathways exploited to mediate invasion have not been defined. As shown herein and in the below examples, host Wnt signaling pathways play important roles in the ehrlichial internalization and infection, and ehrlichial TRPs play key roles in this process. [0039] A simplified diagram showing the canonical and non-canonical Wnt pathways is shown in FIG.7. As would be appreciated by one of skill, various methods may be used to inhibit Wnt signaling. For example and as shown in the below examples, small interfering RNA (RNAi), antibodies, and/or small molecule Wnt inhibitors may be used to inhibit Wnt signaling. In some embodiments, the Wnt inhibitor does not selectively inhibit Wnt5a. It is anticipated that additional Wnt inhibitors that may be subsequently discovered may be used in embodiments of the present invention. A. Canonical Wnt Inhibitors

[0040] A variety of Wnt inhibtors may be used to inhibit canonical Wnt signaling to treat infection by an obligately intracellular bacteria such as an Ehrlichia. For example, the Wnt inhibitor may selectively inhibit phosphoinositide 3-kinase (PI3K), Casein kinase 2 (CKII), Casein kinase 1 epsilon (CK1ε), β-catenin/TCF/LEF, or Protein kinase B (AKT), or the Wnt inhibitor may selectively activate casein kinase Iα/GSK3β. [0041] In some embodiments, pyrvinium pamoate (2-[(E)-2-(2,5-Dimethyl-1- phenylpyrrol-3-yl)ethenyl]-N,N,1-trimethylquinolin-1-ium-6-amine), TBCA ( (E)-3-(2,3,4,5- Tetrabromophenyl)acrylic acid), SB202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4- pyridyl)1H-imidazole, FHPI), LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4- one), or FH535 (N-(2-Methyl-4-nitro)-2,4-dichlorosulfonamide) may be used to inhibit canonical Wnt signaling. [0042] In some embodiments, about 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x of the IC50 amount of the Wnt inhibitor may be administered to treat infection by an obligately intracellular bacteria such as an Ehrlichia. B. Non-Canonical Wnt Inhibitors

[0043] In some embodiments a Wnt inhibitor may be used to selectively inhibit non- canonical Wnt signaling to treat infection by an obligately intracellular bacteria such as an Ehrlichia. [0044] In some embodiments, an Wnt inhibitor may be used to selectively inhibit the Wnt/Ca²⁺ pathway. For example, in some embodiments, the Wnt inhibitor selectively inhibits calmodulin kinase II (CaMKII), IκB kinase (IKK), or Protein kinase B (AKT). In some embodiments, the Wnt inhibitor is KN93 (2-[N-(2-hydroxyethyl)]-N-(4- methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)). [0045] In some embodiments, an Wnt inhibitor may be used to selectively inhibit the Wnt/PCP pathway. For example, in some embodiments, the Wnt inhibitor selectively inhibits Phosphoinositide 3-kinase (PI3K), Protein kinase B (Akt), or IκB kinase (IKK). In some embodiments, pyrvinium pamoate (2-[(E)-2-(2,5-Dimethyl-1-phenylpyrrol-3-yl)ethenyl]- N,N,1-trimethylquinolin-1-ium-6-amine), LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1- benzopyran-4-one), or BAY 11-7082 ( (E)3-[(4-Methylphenyl)sulfonyl]-2-propenenitrile) is used to inhibit Wnt signaling. C. Inhibitors of Wnt Secretion

[0046] In some embodiments a Wnt inhibitor may be used to selectively inhibit Wnt secretion. For example, a Wnt inhibitor may inhibit Wnt secretion by selectively inhibiting X- chromosomal porcupine homolog (PORCN). In some embodiments, the Wnt inhibitor is IWP- 2 (N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2- d]pyrimidin-2-yl)thio]-acetamide). II. Notch inhibitors

[0047] The Notch signaling pathway is evolutionarily conserved in eukaryotes and plays important roles in cell proliferation, differentiation, and apoptosis, thereby influencing cell fate (Fortini 2012; Artavanis-Tsakonas et al., 1999; Hoyne 2003, Radtke et al., 2013). Three proteolytic cleavage steps are essential for the production of fully functional Notch receptor signaling. The first occurs at site 1 (S1) by furin in the trans-golgi (Sasamura et al., 2003; Shi and Stanley 2003), resulting in translocation of the heterodimer to the cell surface. The canonical Notch pathway is activated when the extracellular domain of Notch receptor (NECD) binds to the ligand (DLL1, 3, 4 and Jagged 1, 2) expressed on the membrane of neighboring cells. This receptor-ligand interaction results in the exposure of site 2 (S2) in Notch for cleavage by ADAM metalloproteases (Brou et al., 2000), resulting in NECD shedding and subsequent cleavage of intracellular domain (NICD) by γ-secretase enzyme (S3 cleavage) (Wolfe and Kopan 2004). NICD translocates to the nucleus where it forms a tri- protein complex with DNA-binding transcription factor RBPjκ (CSL) and transcriptional coactivator Mastermind (MAM), activating Notch target gene transcription (Barrick and Kopan 2006; Kovall 2007). It has been previously observed that TRP120 interacts with the ADAM17 metalloprotease (Luo et al., 2011), and also acts as a nucleomodulin, binding target genes associated with the Notch signaling pathway, including notch1 (Zhu et al., 2011). [0048] As shown in the below examples, during E. chaffeensis infection, nuclear localization of transcriptionally active NICD and induction of a panel of Notch genes expression in monocytes was observed. Comprehensive molecular modulation of Notch signaling pathway genes and downstream target genes was observed at different times after infection. This supports the idea that activation of canonical Notch signaling pathway occurs at both early and late phase of ehrlichial infection. Induction of TRP120 interacting protein ADAM17 was also observed during ehrlichial infection. ADAM17, also known as tumor necrosis factor converting enzyme or TACE is an important regulator of Notch signaling and plays critical role in regulation of different cellular events including proliferation and migration. ADAM17 has been implicated in different human diseases including cancer and is a promising target for treatment (Gooz 2010). Without wishing to be bound by any theory, the data supports a mechanism of action of Ehrlichia modulating the Notch pathway as shown in FIG.17. [0049] A variety of Notch inhibitors may be used to treat an obligately intracellular pathogen such as, e.g., Ehrlichia, Chlamydia trachomatis, or Mycobacterium tuberculosis. In some embodiments, the Notch inhibitor selectively inhibits γ-secretase, Notch transcription factor complex, Notch1, ADAM17, or ADAM10. In some embodiments, the Notch inhibitor is a γ-secretase inhibitor, such as:

inhibitor is a blocking peptide (e.g., MAM peptide agonist SAHM1), an antibody fragment or a neutralizing antibody (e.g., OMP-59R5 (anti-Notch 2/3 mAb), NRR1 anti-Notch1 mAB, NRR2 anti-Notch2 mAb, NRR3 anti-Notch3 mAb, OMP-21M18 anti-DLL4 mAb, DLL1-Fc and JAG1 Fc anti-delta like 1 and jagged 1 Fc chimeric mAbs, A5622A anti-nicastrin mAb), or a Notch decoy (e.g., a soluble Notch1, Dll1, jagged1). Additional Notch inhibitors are also described in Espinoza and Miele (2013) that may be used in various aspects of the present invention, e.g., to treat an infection by an obligately intracellular bacteria. III. Obligately Intracellular Pathogens

[0050] Obligately intracellular pathogens are capable of growing and reproducing inside the cells of a host. Obligately intracellular pathogens include bacteria, protozoa, and fungi. Generally, obligately intracellular pathogens cannot reproduce outside their host cell, and the reproduction is primarily or entirely reliant on intracellular host resources. Obligately intracellular bacteria include Ehrlichia bacteria, such as Ehrlichia chaffeensis and Ehrlichia canis. [0051] Ehrlichiosis is a bacterial illness resulting from infection of a human or dog host by Ehrlichia chaffeensis or Ehrlichia canis, respectively. Ehrlichiosis is transmitted by ticks, and typically results in flu-like symptoms. The signs and symptoms of ehrlichiosis range from mild body aches to severe fever and usually appear within a week or two of a tick bite. Ehrlichiosis is typically treated with antibiotics. [0052] Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the life-threatening tick transmitted zoonosis, human monocytotropic ehrlichiosis. E. chaffeensis invades and survives in mononuclear phagocytes by modulating cell processes and evading host defenses, but the mechanisms are not fully defined. Recently it has been observed that E. chaffeensis tandem repeat proteins (TRP) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved canonical and non-canonical Wnt signaling pathways. [0053] Other obligately intracellular bacteria that may be treated with a Wnt inhibitor in various embodiments include, e.g., Chlamydia, Rickettsia, Coxiella, certain Mycobacterium such as Mycobacterium leprae, Mycobacterium tuberculosis. [0054] In some aspects, it is anticipated that a Wnt inhibitor may be used to treat an obligately intracellular protozoa. Obligately intracellular protozoa include, e.g., Apicomplexans (e.g., Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum), Trypanosomatids (e.g., Leishmania spp., Trypanosoma cruzi). [0055] In some aspects, it is anticipated that a Wnt inhibitor may be used to treat an obligately intracellular fungi. Obligately intracellular fungi include, e.g., Pneumocystis jirovecii, and Histoplasma capsulatum. IV. Pharmaceutical Preparations

[0056] Pharmaceutical compositions or pharmaceutical preparations of the present invention comprise an effective amount of one or more compounds of the present invention, e.g., a Wnt inhibitor, or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier or excipient. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one compound or Wnt inhibitor or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21^st Ed. Lippincott et al., 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. [0057] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. [0058] The compound, Wnt inhibitor, or Notch inhibitor of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18^th Ed. Mack Printing Company, 1990, incorporated herein by reference). [0059] The compound, Wnt inhibitor, or Notch inhibitor of the present invention may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like. [0060] Further in accordance with the present invention, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid (e.g., pastes) or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof. [0061] In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art. [0062] In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, e.g., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc. [0063] In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include a compound or Wnt inhibitor of the present invention, one or more lipids, and an aqueous solvent. As used herein, the term“lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention. [0064] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the compound or Wnt inhibitor of the present invention may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes. [0065] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. [0066] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. [0067] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above. In some embodiments, about 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x of the IC₅₀ amount of the Wnt inhibitor or Notch inhibitor may be administered to treat infection by an obligately intracellular bacteria such as an Ehrlichia. A. Alimentary Compositions and Formulations

[0068] In some embodiments, a Wnt inhibitor or Notch inhibitor can be formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. [0069] In some embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. [0070] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth. [0071] Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%. B. Parenteral Compositions and Formulations

[0072] In further embodiments, a compound, Wnt inhibitor, or Notch inhibitor of the present invention may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).. [0073] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0074] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035- 1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should preferably meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards. [0075] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent. C. Miscellaneous Pharmaceutical Compositions and Formulations

[0076] In other preferred embodiments of the invention, the active compound, Wnt inhibitor, or Notch inhibitor may be formulated for administration via various miscellaneous routes, for example, topical or transdermal administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation. [0077] Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time. [0078] In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos.5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No.5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety). [0079] The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosols that may be used can include a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject’s age, weight and the severity and response of the symptoms. V. Examples

[0080] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1

MATERIALS AND METHODS [0081] Cell culture and cultivation of E. chaffeensis. Human cervical epithelial adenocarcinoma cells (HeLa) were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT). Human monocytic leukemia cells (THP-1) were propagated in RPMI medium 1640 with L-glutamine and 25 mM HEPES buffer (Invitrogen), supplemented with 1 mM sodium pyruvate (Sigma, St. Louis, MO), 2.5 g/L D-(+)-glucose (Sigma), and 10% fetal bovine serum (HyClone). E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described. [0082] Inhibitors, siRNAs and antibodies. InhibitorSelect Wnt signaling pathway inhibitor panel was purchased from Calbiochem/EMD (Billerica, MA). This panel contains 15 potent and selective inhibitors for the study of the Wnt signaling pathways: casein kinase I inhibitor D4476, casein kinase II inhibitor III TBCA, β-catenin/TCF inhibitor FH535, GSK-3 inhibitor IX BIO, protein kinase G Iα inhibitor DT3, protein kinase A inhibitor H89 dihydrochloride, JNK inhibitor II SP600125, protein kinase inhibitor K252a Nocardiopsis sp., CaM kinase II inhibitor KN93, PI3 kinase inhibitor LY294002, TAK1 inhibitor (5Z)-7- oxozeaenol Curvularia sp., Src family kinase inhibitor PP2, mTOR inhibitor rapamycin, casein kinase Iδ/ε and p38 MAP kinase inhibitor SB202190, and MEK1/2 inhibitor U0126. Other Wnt signaling inhibitors included casein kinase Iα/GSK3 activator (Akt/PKB inhibitor) pyrvinium pamoate salt hydrate and inhibitor of Wnt production II IWP-2 (Sigma). The lipid raft disrupting agents methyl-β–cyclodextrin (MCD) and n-octyl-β–D-glucopyranoside (OGP) and the IκB kinase inhibitor BAY11-7082 were from Sigma. Rabbit or mouse anti-TRP32 antibodies have been described previously. Other antibodies used in this study were mouse anti-human α-tubulin, NFATC1 (Santa Cruz Biotechnology, Santa Cruz, CA) and β-catenin (Pierce, Rockford, IL) and rabbit anti-human FZD9 (Pierce), JUN (Santa Cruz), DVL2 and GSK3β (Cell signaling, Beverly, MA). siRNAs of human DKK3, DVL2, FZD9, JUN, NFATC1 (NFAT2), NFATC3 (NFAT4), PP2B-Aα (Calcineurin PPP3CA), PP2B-Aβ (Calcineurin PPP3CB), TCF4, WNT10A, WNT6, and control-A were purchased from Santa Cruz. Validated siRNAs of human CTNNB1 (β-catenin), GSK3β, WNT3A, WNT5A and LRP6 and esiRNAs of human ARID1B, KDM6B, IRF2BP2, PPP3R1 and VPS29 were from Sigma. Alexa Fluor 488-labeled negative siRNA was from Qiagen (Germantown, MD). [0083] PCR array. The RT² Profiler PCR arrays (version 4.0; SABiosciences, Valencia, CA) were used, including human Wnt signaling pathway plus PCR array and human Wnt signaling targets PCR array (see SABiosciences website for gene list and functional gene grouping). The human Wnt signaling pathway plus PCR array profiles the expression of 84 genes related to Wnt-mediated signal transduction, including Wnt signaling ligands, receptors and regulators as well as downstream signaling molecules and target proteins for all three Wnt pathways, and uses experimentally derived signature biomarker genes along with classification algorithms to generate the pathway activity score and determines whether Wnt pathway activity is activated or repressed in experimental samples. The human Wnt signaling targets PCR array profiles the expression of 84 key genes responsive to Wnt signal transduction, including Wnt signaling pathway transcription factors and highly relevant target genes identified by multiple studies, and can be used to analyze activation or inhibition of Wnt signaling. PCR arrays were performed according to the PCR array handbook from the manufacturer. In brief, uninfected and E. chaffeensis-infected THP-1 cells at different time point postinfection (p.i.) were collected and total RNA was purified using RNeasy Mini kit (Qiagen). During RNA purification, on-column DNA digestion was performed using the RNase-free DNase set (Qiagen). All RNA samples demonstrated consistent good quality. The concentration and purity were determined by measuring the absorbance in a Nanodrop 100 spectrophotometer (Thermo Scientific, West Palm Beach, FL), and ribosomal RNA band integrity was verified by running an aliquot of each RNA sample on a RNA FlashGel (Lonza, Rockland, ME). Then, genomic DNA was eliminated and cDNA was synthesized from 0.5 μg of total RNA using the RT² first strand kit (Qiagen). Real-time PCR was performed using RT² Profiler PCR array in combination with RT² SYBR Green mastermix (Qiagen) on a Mastercycler EP Realplex² S (Eppendorf, Germany). Cycling conditions were as follows: 95°C for 10 min and 40 cycles of 95°C for 15 s, 60°C for 1 min. The real-time cycler software RealPlex 1.5 (Eppendorf) was used for PCR and data collection. The baseline was set automatically, the threshold was defined manually, and then the threshold cycle (CT) for each well was calculated by RealPlex. The threshold was set in the proper location and at the same level for all PCR arrays in the same analysis so that the values of the positive PCR control (PPC) assays on all arrays were between 18 C_T and 22 C_T. The C_T values for all wells were exported for analysis using Web- based PCR array data analysis software (version 3.5; SABiosciences). PCR array quality checks were performed by the software before data analysis, including PCR array reproducibility, reverse transcription efficiency control, genomic DNA contamination control and positive PCR control. [0084] RNA interference. THP-1 cells (1 × 10⁵/well on a 96-well plate) were transfected with 5 pmol human siRNA using Lipofectamine 2000 (Invitrogen). A control-A siRNA consisting of a scrambled sequence was used as a negative control, and an Alexa Fluor 488-labeled negative siRNA was used as a control to monitor transfection efficiency. At 1 day posttransfection, the cells were infected by cell-free E. chaffeensis at a multiplicity of infection (MOI) of ~50. Then, the cells were collected at 1 day and 2 days p.i., and subjected to quantitative PCR (qPCR) and Western blot. [0085] Quantification of E. chaffeensis by qPCR. Treated THP-1 cells were pelleted, washed by PBS, lysed in SideStep lysis and stabilization buffer (Agilent, Santa Clara, CA) for 30 min at room temp, and analyzed for bacterial load using realtime qPCR. Amplification of the integral ehrlichial gene dsb was performed using Brilliant II SYBR Green mastermix (Agilent), 200 nM forward primer (5'-gctgctccaccaataaatgtatccct-3') and 200 nM reverse primer (5'-gtttcattagccaagaattccgacact-3'). The qPCR thermal cycling protocol (denaturation at 95°C 10 min, then 40 cycles of 95°C 30 s, 58°C 1 min, 72°C 1 min) was performed on the Mastercycler EP Realplex² S (Eppendorf). A standard plasmid pBAD-dsb was constructed by cloning the ehrlichial dsb gene using the TOPO TA cloning kit (Invitrogen). The plasmid copy number for the standards was calculated using the following formula: plasmid copy/μl = [(plasmid concentration g/μl) / (plasmid length in base pairs × 660)] × 6.022 × 10²³. The absolute E. chaffeensis dsb copy number in the cells was determined against the standard curve or the fold change of dsb copy number relative to the control was normalized to qPCR-detected levels of the host genomic glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene. [0086] Western immunoblot. The THP-1 cell lysates were prepared using CytoBuster protein extraction reagent (Novagen/EMD, Gibbstown, NJ), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. Then, Western immunoblot was performed, and horseradish peroxidase-labeled goat anti- rabbit or mouse IgG (heavy and light chains) conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and SuperSignal West Dura chemiluminescent substrate (Thermo Scientific) were used. [0087] Small molecule inhibitor treatment. THP-1 cells were plated in FBS-free medium and treated with different concentrations of inhibitor or DMSO control, and then infected with cell-free E. chaffeensis at a multiplicity of infection (MOI) of ~50. Percentages of infected cells were monitored daily over 3 days by Diff-Quik staining and counting of 100 cells. Bright field images of these slides were collected on an Olympus BX61 epifluorescence microscope using a color camera. Initially, inhibitors were tested at the concentration of 5-10 folds of IC50 or Ki as provided by the manufacturer. If the bacteria were inhibited at this concentration, MICs were then determined by using two-fold serial dilutions. The MIC was defined as the minimum inhibitor concentration required for inhibition of the growth of the bacteria compared to that of the control (without the inhibitor) at day 3. An infected culture without the inhibitor served as a positive growth control, and an uninfected cell culture served as a negative control. All experiments were repeated three times to confirm results. [0088] Immunofluorescence microscopy. Uninfected or E. chaffeensis-infected THP-1 cells were collected, and the indirect immunofluorescent antibody assay was performed as previously described, except that anti-β-catenin or NFATC1 antibody (1:100) and anti- TRP32 antibody (1:10,000) were used. [0089] Phagocytosis of microspheres. Expression and purification of E. chaffeensis tandem repeat proteins (TRP) has been described previously (Doyle et al., 2006; Luo et al., 2009; Klaus and Birchmeier, 2008). FluoSpheres® sulfate microspheres (1.0 µm, yellow- green fluorescent; Invitrogen) (10 μl, ~3.6×10⁸ beads) were washed by 40 mM 2-(N- morpholino)ethanesulfonic acid (MES) buffer, pH 6.1, and then incubated with 15 µg of TRP protein in 500 μl MES buffer at room temperature for 1 h with mixing at 20 rpm. The coated beads were collected by centrifugation, washed twice in MES buffer and resuspended in RPMI medium, then gently sonicated to disperse the beads. Protein coating of the beads were confirmed by dot blot assay. TRP-coated or control protein-coated beads were added to THP- 1 cells at a multiplicity of approximately 50 beads per cell and incubated for 2 h at 37°C with 5% CO2. Unbound beads were removed by washing and low-speed centrifugation for three times, and then cells were collected by cytospin onto the slide and fixed with 3% paraformaldehyde to observe internalized beads. For estimating inhibition of phagocytosis, designated inhibitors were added 2 h before addition of coated beads. Specific concentrations of inhibitors were used as following: 20 nM pyrvinium, 4 µM KN93, 0.3 µM IWP-2, 10 µM LY294002, 10 µM BAY11-7082, 5 mM MCD, and 2.5 mM OGP. [0090] 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. Example 2

Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways to

Stimulate Phagocytosis and Promote Intracellular Survival [0091] Activity analysis of Wnt signaling pathway in E. chaffeensis-infected host cells by PCR array. To define the impact of E. chaffeensis on host Wnt signaling, the activity of Wnt signaling pathways in E. chaffeensis-infected THP-1 cells at different time points (1 h, 3 h, 8 h, 24 h and 72 h) p.i. was assessed by PCR array (Table 1). As compared to that in uninfected cells, the activities of Wnt signaling pathway in E. chaffeensis-infected THP-1 cells at 1 h, 8 h and 24 h p.i. showed no significant change, but Wnt pathway activity in THP-1 cells was stimulated significantly by E. chaffeensis at 3 h p.i., whereas the activity was repressed significantly at 72 h p.i. TABLE 1 Activity scores of Wnt signaling pathway in E. chaffeensis-infected host cells by PCR arra as com ared to that in uninfected cells

[0092] Among 84 host genes related to Wnt signaling pathways assessed by PCR array, 21 (25% of total) genes’ expression showed significant differences for at least one time point p.i. of E. chaffeensis, either upregulation (fold regulation/fold change > 2) or downregulation (fold regulation < -2 [fold change < 0.5]), comparing to those in uninfected cells (see Table S1). FIG.1 shows the expression levels of seven important host genes at different time points p.i. by Wnt signaling pathway PCR array. Gene expressions of two Wnt ligands (WNT6 and WNT10a) and two Frizzled receptors (FZD5 and FZD9) were upregulated at most time points, and exhibited the similar expression pattern with the highest expression at 3 h p.i. and relatively the lowest expression at 72 h p.i. Similarly, gene expression of the transcription factor 7 (TCF7) was upregulated at 3 h but significantly downregulated at 72 h p.i. Gene expression of a Wnt signaling target protein FOS-like antigen 1 (FOSL1) was upregulated at most time points but downregulated at 72 h p.i. Gene expression of another Wnt signaling target protein MYC was downregulated at all time points, significantly at 72 h p.i. of E. chaffeensis. Expression profiles of these important components and targets of Wnt signaling pathways supported the analysis results of pathway activity. [0093] Expression analysis of Wnt signaling target genes in E. chaffeensis-infected host cells by PCR array. The gene expression of Wnt signaling targets in E. chaffeensis- infected THP-1 cells at different time points (3 h, 8 h, 24 h and 72 h) p.i. was also analyzed by PCR array. Table S2 shows functionally categorized gene expression changes of Wnt signaling target proteins in infected cells compared to that in uninfected cells. Among all 84 important host genes responsive to Wnt signal transduction assessed by PCR array, 39 genes (46%) showed significant difference of expression level for at least one time point p.i. of E. chaffeensis, either upregulation (fold regulation/fold change > 2) or downregulation (fold regulation < -2 [fold change < 0.5]). Among these 39 genes with significant difference of expression, 20 genes (51% of 39) were upregulated, 14 genes (36%) were downregulated, and 5 genes (13%) were upregulated and downregulated at different time points. The functional categories of Wnt signaling target proteins include development and differentiation, calcium binding and signaling, adhesion, migration, cell cycle, proteolysis, signal transduction, and transcription factors, which are associated with major biological processes of host cells. Notably, in the categories of development and differentiation, calcium binding and signaling and migration, more than half (51%, 62% and 52%, respectively) of the genes tested were regulated by E. chaffeensis. FIG. 2 shows the expression levels of six host target genes at different time points p.i. by Wnt signaling target PCR array, including cyclin D1 (CCND1), fibroblast growth factor 9 (FGF9), fibronectin 1 (FN1), MET proto-oncogene (MET), matrix metallopeptidase 2 (MMP2), and secreted frizzled-related protein 2 (SFRP2). Consistent with the activity of Wnt signaling, gene expressions of these target genes exhibited the similar expression pattern with relatively high expression at 3 h and 8 h p.i. and relatively low expression at 72 h p.i. Modulation of many Wnt target genes in the host cell during infection indicates the regulation of Wnt signaling pathways by E. chaffeensis.

TABLE 2 Fold regulations of E. chaffeensis bacterial load in THP-1 cells transfected with specific siRNA of TRP120-interacting host targets involved in Wnt signaling relative to control siRNA at 1day and 2 days p.i., as determined by qPCR^a.

[0094] Knockdown of Wnt signaling pathway components influences ehrlichial infection of macrophages. The role of host Wnt signaling pathways in ehrlichial infection was further confirmed using RNA interference. In total, 16 siRNAs were used, including those of some important components of Wnt signaling pathways, such as Wnt ligand, receptor and co-receptor, regulator, transcription factor and target (FIG. 3A). The siRNAs of some host genes modulated during E. chaffeensis infection, as determined by PCR array, were also included. The decrease of most target proteins (12 proteins at 1 day p.i. and 11 proteins at 2 days p.i.) influenced E. chaffeensis infection significantly, indicating that those host components of Wnt signaling pathways play a role in E. chaffeensis infection. Among these siRNAs with significant influence on ehrlichial infection, all but DKK3 siRNA (at 1 day p.i.) inhibited the infection. DKK3, Dickkopf homolog 3 (Xenopus laevis), is an antagonist of the canonical Wnt signaling pathway. The inhibition of infection by siRNAs of Wnt signaling pathways could occur at 1 day and/or 2 days p.i. FIG. 3B shows examples that protein expression of four target genes FZD9, DVL2, GSK3β and JUN was reduced in specific siRNA- transfected cells, respectively, compared with the unrelated control siRNA-transfected cells. [0095] Small molecule inhibitors of Wnt signaling pathways repress E. chaffeensis infection of host cells. To confirm the role of host Wnt signaling pathways in E. chaffeensis infection, a large panel of 17 Wnt signaling pathway inhibitors was examined on ehrlichial infection of host cells, and five inhibitors were found to have significant impact on ehrlichial infection but without apparent toxicity to host cells, including casein kinase Iα/GSK3 activator (Akt/PKB inhibitor) pyrvinium pamoate, CaM kinase II inhibitor KN93, inhibitor of Wnt production II IWP-2, casein kinase II inhibitor III TBCA, and casein kinase Iδ/ε inhibitor SB202190 (FIG.4A and FIG.4B). Pyrvinium, KN93 and IWP-2 were highly potent inhibitors of ehrlichial infection, and could block the infection almost completely. Two inhibitors TBCA and SB202190 reduced ehrlichial infection significantly. In addition, it was observed that two other inhibitors, β-catenin/TCF inhibitor FH535 and PI3 kinase inhibitor LY294002 could influence ehrlichial infection but showed some toxicity to host cells. Notably, three most potent inhibitors pyrvinium, KN93 and IWP-2 target the canonical Wnt pathway, the noncanonical Wnt/Ca²⁺ pathway and Wnt production, respectively, indicating that the importance of both canonical and noncanonical Wnt signaling pathways in ehrlichial infection. Moreover, MICs of pyrvinium, KN93 and IWP-2 for ehrlichial infection were determined to be 20 nM, 4 µM, and 0.3 µM, respectively, by using two-fold serial dilutions. [0096] Host β-catenin and NFATC1 proteins translocate to the nucleus after E. chaffeensis infection. β-catenin and NFATC1 are important nuclear factors involved in Wnt/β-catenin and Wnt/Ca²⁺ pathways, respectively; thus, the localization of these two proteins after infection was examined using immunofluorescence microscopy. Remarkable redistribution of β-catenin and NFATC1 proteins in E. chaffeensis-infected cells compared to their distribution in uninfected cells was observed. In uninfected THP-1 cells, β-catenin was diffusely distributed mainly in the cytoplasm and associated with cell membrane, but in E. chaffeensis-infected cells at 3 h p.i., β-catenin accumulated and was punctately distributed mainly in the nucleus (FIG.5A). Similarly, NFATC1 was diffusely distributed mainly in the cytoplasm of uninfected THP-1 cells, but translocated to the nucleus as early as 1 h p.i. of E. chaffeensis, and notably, almost all of NFATC1 proteins localized in the nucleus of THP-1 cells at 3 h p.i. (FIG. 5B). Translocation of β-catenin and NFATC1 proteins to the nucleus supports the activation of host Wnt signaling at about 3 h after E. chaffeensis infection. [0097] E. chaffeensis TRP120 interacts with host targets involved in Wnt signaling that influence infection. Since E. chaffeensis TRPs have been identified as bacterial effector proteins and interact with multiple host proteins involved in Wnt signaling, the role of these host proteins in ehrlichial infection was confirmed using RNA interference. Similar to the previous result of TRP32-interacting protein DAZAP2, knockdown of five TRP120-interacting host proteins, including ARID1B, KDM6B, IRF2BP2, PPP3R1 and VPS29, influenced ehrlichial infection of macrophages significantly (Table 2). The bacterial load in all specific siRNA-transfected cells decreased at both 1 day and 2 days p.i. (fold regulation < -2), except that the bacterial load in ARID1B siRNA-transfected cells increased at 1 day p.i. ARID1B is an AT-rich DNA interacting domain-containing protein and a component of the SWI/SNF chromatin remodeling complex and has been reported to interact with β-catenin to suppress Wnt signaling. The result indicates the importance of these TRP120 interactions during E. chaffeensis infection. [0098] E. chaffeensis tandem repeat proteins stimulate phagocytosis of macrophages and Wnt pathway inhibitors abolished the stimulation. Recently Wnt signaling pathway has been demonstrated to stimulate phagocytosis but not bacterial killing. Ehrlichial tandem repeat proteins were observed to stimulate the internalization of latex beads by macrophages. FIG. 6A and FIG. 6B show that TRP120-coated latex beads were phagocytosed by the THP-1 cell dramatically compared with the control protein TRP120 N- terminal region only (TRP120N)-coated latex beads. Other ehrlichial tandem repeat proteins, TRP32 and TRP47 can stimulate the internalization of latex beads in THP-1 cells as well. However, some Wnt pathway inhibitors can abolish the phagocytosis in macrophages promoted by the ehrlichial TRP. FIG. 6C shows that TRP120 could not promote the internalization of latex beads by the THP-1 cell after treatment with CKIα/GSK3β activator (Akt/protein kinase B inhibitor) pyrvinium (30 nM). Some other Wnt pathway inhibitors, including CaMKII inhibitor KN93 and PI3K inhibitor LY94002, also blocked TRP-stimulated phagocytosis. Surprisingly, IWP-2 did not inhibit the phagocytosis mediated by TRP. In addition, there was complete inhibition in TRP-mediated phagocytosis by the lipid raft disrupting agents MCD and OGP and the IκB kinase inhibitor BAY11-7082. These results suggest the importance of Wnt signaling pathways in ehrlichial internalization and highlight the roles of CKIα/GSK3β (Akt/PKB), CaMKII, PI3 kinase and IκB kinase, as well as lipid rafts in TRP-induced enhancement in phagocytosis. [0099] Discussion. Recent findings of ehrlichial effector TRPs highlight the importance and complexity of interactions between bacterial effectors and host processes; however, the molecular mechanisms by which Ehrlichia modulates host cells are still not well understood. Some interactions have been identified between TRPs and signaling pathways. The above studies demonstrate that host Wnt signaling pathways play important roles in ehrlichial internalization and infection and ehrlichial TRPs mediate the invasion and survival. [00100] The above data from Wnt signaling pathway PCR array shows that E. chaffeensis activates Wnt signaling pathways in macrophages, remarkably, at a very early stage of infection (3 h) and inhibits the pathway activity at the late stage of infection (72 h). In mammalian cells, E. chaffeensis replication occurs in a 72 h life cycle. The developmental cycle starts with the dense-cored cells (DC), which attaches to and enters into the host cell (Zhang et al., 2007) . Inside a vacuole in the host cell, the DC rapidly transforms into a larger reticulate cell (RC), which multiplies by binary fission for approximately 48 h and then matures into DC at 72 h after infection (Zhang et al., 2007). Then the mature DCs are released and start a new cycle of infection. The general entry time of DC into the host cell is about 1 h to 3 h (Popov et al., 2000; Zhang et al., 2007), but without synchronization of bacteria and cells, the internalization may take longer. So these time point results indicates that host Wnt signaling pathways may be utilized by Ehrlichia for its entry, transformation, and exit. Ehrlichia may activate host Wnt signaling for the entry and transformation, and suppress the Wnt signaling for the exit. Comparison of pathway activity scores and P values of 3h and 72 h suggests that regulation of Wnt signaling at 3 h p.i. is more significant than that at 72 h. [00101] The pathway activity score determined by PCR array was calculated by a data analysis software and represents an overall activity of the pathway, which may reflect a balance of regulations of multiple components. Different components may be regulated by Ehrlichia at different time points in a different way. In addition, the pathway activities determined by PCR array are based on canonical Wnt signaling pathway due to its good documentation, but Wnt signaling pathway PCR array does include many components from other two noncanonical Wnt pathways. Genes with significant changes of expression after infection include ligand, receptor, inhibitor, signaling molecule, transcription factor and target of Wnt signaling pathways and are involved in both canonical and noncanonical Wnt pathways, e.g., ligands WNT6 and WNT7b are also included in the Wnt/Ca²⁺ pathway (Schmidt et al., 2007; Zhang et al., 2007), and receptor FZD5 and target VANGL2 (Vang-like 2) are involved in the regulation of planar cell polarity (Croce et al., 2006; Shafer et al., 2011) (Table S1). Therefore, without wishing to be bound by any theory, the inventors anticipate that these components can play important roles when Ehrlichia exploits host Wnt signaling pathways and all three Wnt signaling pathways can be utilized by E. chaffeensis, which is further supported by functionally categorized gene expression changes of Wnt signaling target proteins after ehrlichial infection by Wnt target PCR array. [00102] Experiments using small interfering RNAs, small molecule inhibitors and immunofluorescent antibodies substantially demonstrate that Wnt signaling pathways are involved in the ehrlichial infection of host cells. Knockdown of some important components of Wnt signaling pathways, including Wnt ligand, receptor and co-receptor, signaling molecule, transcription factor and target as well as TRP120-interacting proteins, influenced E. chaffeensis infection significantly, indicating the importance of these proteins and Wnt signaling in the E. chaffeensis infection. Moreover, specific Wnt signaling pathway inhibitors reduce ehrlichial infection significantly, three of which, pyrvinium pamoate, KN93 and IWP- 2 were highly potent. Pyrvinium was first found to activate casein kinase 1α, but recently reported that it does not activate protein kinase CK1, but promotes Akt/PKB downregulation and GSK3 activation, despite that pyrvinium ultimately inhibits Wnt signaling (Thorne et al., 2010; Venerando et al., 2013). KN93 selectively binds to the CaM binding site of CaM kinase II and prevents the association of CaM with CaM kinase II (Sumi et al., 1991); IWP-2 inhibits the cellular Wnt processing and secretion via selective blockage of Porcn-mediated Wnt palmitoylation (Chen et al., 2009). Thus, three most potent inhibitors pyrvinium, KN93 and IWP-2 revealed the importance of both canonical and noncanonical Wnt signaling pathways as well as the Wnt ligand in ehrlichial infection, respectively. FIG. 7 illustrates canonical and noncanonical Wnt signaling pathways, potent inhibitors and involved TRP-interacting proteins. [00103] The RNA interference experiments indicated that the inhibition can occur at different stages of infection with different efficiency for different targets, suggesting that different targets play different roles at different stages of infection, or the different siRNAs have different knockdown efficiency. Unlike the effect of small molecule inhibitors, the reduction of a single target protein by RNA interference was observed to not abolish the ehrlichial growth completely. Without wishing to be bound by any theory, this may result from the incomplete knockdown of target proteins, which was shown by Western blot. [00104] Phagocytosis is a specific form of endocytosis and a major mechanism used to remove pathogens in the immune system. Usually the pathogen becomes trapped in a phagosome which then fuses with a lysosome to form a phagolysosome, but E. chaffeensis enters the monocyte through lipid raft-caveolae-mediated endocytosis and then it can reside within a cytoplasmic vacuole that resembles an early endosome and does not fuse with lysosomes, protecting it from killing. However, the underlying molecular mechanism remains unclear. Recently Wnt signaling ligand WNT5a has been found to stimulate phagocytosis through Rac1-PI3 kinase-IκB kinase and lipid raft-dependent processes but not bacterial killing (Maiti et al., 2012). Ehrlichial TRPs were observed to stimulate phagocytosis of macrophages and Wnt pathway inhibitors can abolish the stimulation. Without wishing to be bound by any theory, ehrlichial TRPs appear to be ligands that activate Wnt signaling pathways of the host to induce phagocytosis and facilitate intracellular survival. Consistent with this idea, inhibitor experiments suggest that PI3 kinase and IκB kinase as well as lipid rafts play important roles in TRP-induced phagocytosis and infection. Activation of PI3 kinase and IκB kinase might stimulate the assembly of scavenger receptors through lipid raft and supports cytoskeletal rearrangements for enhanced internalization (Lafont et al., 2005; Seveau et al., 2007; Yin and Janmey, 2003). In addition, Akt/protein kinase B and CaM kinase II are also involved. Based on the classification of these components, TRP-induced phagocytosis might occur through lipid raft as a noncanonical mode of Wnt signaling, similar to Wnt5a-induced phagocytosis. However, Wnt production inhibitor IWP-2 could not inhibit the phagocytosis mediated by TRP, suggesting that Wnt ligand secretion/recycling may be involved in the ehrlichial infection but not in TRP-induced phagocytosis, or the phagocytosis is either Wnt ligand-independent or IWP-2-resistant Wnt ligand-dependent, such as WNT3a, since IWP-2 was found to have a much more significant effect on Wnt5a secretion than on Wnt3a secretion (Maiti et al., 2012). There are about 20 Wnt ligands, which act as ligands to the Frizzled family of cell surface receptors, and the Fz family receptors comprise about 10 members (Schulte and Bryia, 2007; Willert and Nusse, 2012), therefore, the phagocytosis mechanism of different bacteria and host cells may have some diversity. [00105] These results establish an obligately intracellular pathogen-directed Wnt pathway-induced mechanism responsible for invasion and persistence, and these mechanisms may apply to the pathobiology of other intracellular microbes. It is envisioned that one or more of the above experiments may be performed for other obligately intracellular bacteria such as, e.g., Chlamydia, Rickettsia, Orientia, and Mycobacteria, and it is envisioned that similar effects including, e.g., inhibition of phagocytosis and/or reduction of bacterial load, may be observed regarding these and other obligately intracellular bacteria. Example 3

Ehrlichia chaffeensis TRP120 Inhibits TLR2/4 Expression and Promotes Intracellular

Survival by Exploiting the Canonical Notch Signaling Pathway MATERIALS AND METHODS

[00106] Cell culture and cultivation of E. chaffeensis. Human monocytic leukemia cells (THP-1) were propagated in RPMI medium 1640 with L-glutamine and 25 mM HEPES buffer (Invitrogen), supplemented with 1 mM sodium pyruvate (Sigma, St. Louis, MO), 2.5 g/L D-(+)-glucose (Sigma), and 10% fetal bovine serum at 37˚C in a 5% CO2 atmosphere. E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described (8). [00107] Antibodies and inhibitors. Polyclonal mouse anti-TRP120 antibody used in this study was previously described (Luo et al., 2009). A convalescent-phase anti-E. chaffeensis dog serum which was derived from an experimentally infected dog was previously described (Kuriakose et al., 2012). Other antibodies that were used in this study include anti- Hes1 (ab71559) (Abcam, Cambridge, MA), anti-Notch1 (C-20), anti-cleaved Notch1 (m1711), anti-α tubulin (B7), anti-TLR2 (TL2.1), anti-TLR4 (15), anti-PU.1 (A-7) (Santa Cruz Biotechnology, NY), anti-ADAM17 (TACE, D22H4), anti-Notch1 (D1E11), anti-RBPSUH (D10A4) (Cell signaling Technology, Inc) and anti-GAPDH (clone 6C5, EMD Millipore, CA). For inhibition of Notch signaling pathway the following inhibitors were used: γ-secretase inhibitor IX named DAPT or N-[N-(3, 5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t- Butyl Ester (Calbiochem, Canada), and Notch transcription factor inhibitor SAHM1 (Calbiochem, Canada). [00108] siRNAs and transfection. To knockdown Notch signaling components, THP-1 cells (1×10⁵/well on a 96-well plate) were transfected with siRNA for TACE (ADAM17), RBP-jκ or Notch1 (Santa Cruz Biotechnology, NY) using the lipofectamine 2000 reagent (Life Technologies, CA) according to the manufacturer’s instructions with a cocktail of 5 picomole siRNA or a negative control siRNA (Santa Cruz Biotechnology, NY). [00109] RT-PCR. Total RNA from E. chaffeensis infected, TRP 120 or thioredoxin stimulated, and control THP-1 cells, was isolated using an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. On-column DNA digestion was performed using the RNase-free DNase set (Qiagen). cDNA was synthesized from 1 µg of total RNA using a qScript cDNA SuperMix kit (Quanta Biosciences). Gene expression level of target host genes were quantitated by qPCR using brilliant II SYBR green qPCR master mix (Agilent Technologies) with gene-specific primers and a thermal cycling protocol consisting of an initial denaturation step of 95°C for 10 min and 40 cycles of 95°C for 30 s, 58°C for 1:00 m, and 72°C for 30 s. Gene expression values were calculated on the basis of the 2^-ΔΔCT method and normalized with GAPDH. [00110] Human Notch Signaling Pathway PCR array. The human Notch signaling pathway RT² profiler PCR array (Qiagen) was used according to the manufacturer’s protocol to determine the expression of 84 genes which contains receptors, ligands, receptor processing and transcription factor genes associated with Notch signaling and putative Notch target genes. Briefly, RNA was collected from E. chaffeensis infected and uninfected cells; cells stimulated with TRP120 or thioredoxin-coated FluoroSpheres sulfate microsphere beads or TRP120 alone, using RNeasy mini kit (Qiagen). RNA purification, Genomic DNA elimination, cDNA synthesis and PCR array was performed as previously described (10). [00111] Immunofluorescence microscopy. Uninfected, E. chaffeensis infected, or THP-1 cells stimulated with TRP120/thioredoxin-coated beads and TRP120 in soluble form were cytospinned on to glass slides, fixed for 15 min using 3% paraformaldehyde in PBS, blocked and permeabilized for 30 min using 0.3% Triton X-100 and 2% BSA in PBS at RT. Cells were then incubated with primary antibodies Rabbit anti-TRP120 (1:1000), dog anti-E. chaffeensis serum (1:100), goat anti-NICD (1:50), mouse anti-ADAM17 (1:50), goat anti- Notch1 (1:50), rabbit anti-Hes1 (1:50), mouse anti-TLR2 (1:50, mouse anti-TLR4 (1:50) and mouse anti-PU.1 (1:50) for 1 h, washed and incubated with Alexa Fluor 488 IgG (H+L) and Alexa Fluor 568 IgG (H+L) secondary antibodies (1:100, Molecular probes) for 30 min. Slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) after washing. HeLa cells transfected with the GFP-TRP120 plasmids or GFP-control plasmids were fixed in chamber slides, permeabilized and stained with anti-ADAM17 using the same protocol. Images were obtained using Olympus BX61 epifluorescence microscope and analyzed using Slidebook software (version 5.0; Intelligent Imaging Innovations, Denver, CO). [00112] Pharmacological inhibitor treatment and determination of bacterial load. THP-1 cells were treated with DAPT, SAHM1 or DMSO and incubated for at least 1 h and then infected with cell free E. chaffeensis at a multiplicity of infection (MOI) of 50. According to the IC₅₀ values and previously published concentrations 5 µg/ml of DAPT or 10 µM SAHM1 was used to treat the cells unless otherwise stated (Moellering et al., 2009; Oliver et al., 2006). At day 1, and 2 p.i. cells were collected and infection was determined by either calculating the percent of infected cells after Diff-Quick staining, or by determining the dsb copy number using qPCR as previously described (Dunphy et al., 2014). Infected culture without the inhibitors, and uninfected cells were used as positive and negative control, respectively. The absolute E. chaffeensis dsb copy number was determined using a standard curve and was normalized to qPCR-detected levels of the host genomic gapdh gene. To confirm host cell death did not account for decreased ehrlichial inclusions, differences in cell viability were assessed at day 1, 2 and 3 p.i. using trypan blue staining. [00113] Western immunoblot. THP-1 cells infected with E. chaffeensis in the presence of DMSO or Notch inhibitor SAHM1, and uninfected cells were harvested after 1 and 2 days p.i. and LPS stimulation (100 ng/ml for 1 h). Cell lysates were prepared as previously described (Wakeel et al., 2011). Approximately 20 µg of total proteins was separated by dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane using a semidry transfer apparatus. Mouse anti-ADAM17, rabbit anti- Hes1, mouse anti-α-tubulin, mouse anti-PU.1, mouse anti-TLR4, mouse anti-TLR2 and mouse anti-GAPDH were used. For the detection, horseradish peroxidase-labeled goat anti-rabbit, or mouse IgG (heavy and light chains) conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used. SuperSignal West Dura chemiluminescent substrate (Thermo Scientific) was used for detection of ADAM17 and Hes1 protein, and ECL Western immunoblot substrate (Thermo Scientific) for others. [00114] Bead assay. E. chaffeensis TRP120 protein (thioredoxin-fused) was expressed and purified as described previously (Doyle et al., 2011; Luo et al., 2008; Luo et al., 2009). Purified proteins were desalted (Zeba Spin desalting column, Thermo Scientific) to change the buffer to 40 mM MES [2-(N-morpholino) ethanesulfonic acid]. Recombinant purified TRP120 or thioredoxin were coated on FluoroSpheres sulfate microsphere beads (1.0 µm, yellow-green fluorescent; Invitrogen) using the following protocol. Briefly, 10 µl (~3.6×10⁸ beads) of beads were washed two times with 10 volumes of 40 mM MES buffer (5000g for 5 min), re-suspended in 10 µg of TRP120 desalted protein in 500 µl MES buffer and incubated at RT for 2 h in a rotor. After incubation beads were washed twice with 500 µl MES buffer (10,000g for 8 min) and re-suspended in RPMI media. Since these beads are light sensitive, they were also protected from exposure to light. TRP120 or thioredoxin-coated beads were used to treat THP-1 cells for different time points, and the cells were incubated at 37ºC with 5% CO₂. After incubation unbound beads were washed by centrifuging at least 4 times at 400g. [00115] Bio-Plex. The level of total and phosphorylated ERK1/2 and p38MAPK proteins in THP-1 cells infected with E. chaffeensis in the presence and absence of Notch inhibitor, SAHM1 (10 µM) and with or without LPS stimulation (100 ng/ml) were measured using Luminex array (Millipore, Beillerica, MA) according to manufacturer’s instructions. Samples were analyzed using Bioplex manager software (Bio-Rad). [00116] Statistics. The results were expressed as the means ±SD of data obtained from at least three independent experiments done with triplicate sets per experiment, unless otherwise indicated. Differences between means were evaluated by using two tailed Student t test. The p values, 0.05 were considered statistically significant. RESULTS [00117] E. chaffeensis activates canonical Notch pathway during infection. Using Y2H and ChIPSeq the inventors previously observed that E. chaffeensis TRP120 interacts with many different cellular targets at different times during infection, including interaction with ADAM17 and binding to the promoter region of notch1 (Zhu et al., 2011; Luo et al., 2011). Since E. chaffeensis interacts with a component of the Notch signaling receptor complex the inventors sought to investigate whether E. chaffeensis might somehow alter the function of this pathway. Activation of Notch receptor following interaction with its ligand and proteolytic cleavage by the ADAM17 and γ-secretase enzyme involves nuclear translocation of NICD (Brou et al., 2000; Wolfe and Kopan 2004). Immunofluorescence microscopy was used to measure NICD expression and localization in uninfected and E. chaffeensis-infected cells. NICD translocation to the nucleus was observed within 2 h of E. chaffeensis infection (FIG. 8A). Since nuclear translocation of NICD results in activation of specific Notch target genes (Barrick and Kopan 2006; Kovall 2007), next the expression of different Notch signaling components and target genes were examined in E. chaffeensis infected cells. The most important and well characterized Notch target genes are the families of basic helix-loop-helix proteins, hairy and enhancer of split (Hes) and hairy and enhancer of split with YRPW motif (Hey) (Fisher and Gessler 2007). These DNA binding proteins function as transcriptional repressors and are the primary effectors of Notch signaling. RT-PCR data showed notch1, hes1, hes5 and hey2 mRNA expression was significantly increased as early as 2 h p.i., reaching a maximum at 72 h p.i. (FIG. 8B). Consistent with RT-PCR data, increased expression of Hes1 and ADAM17 protein was also observed by Western immunoblot after 2 days p.i. The housekeeping protein α-tubulin was unchanged (FIG. 8C). Collectively, these results demonstrate that E. chaffeensis activates canonical Notch signaling pathway during infection. [00118] Analysis of Notch signaling pathway gene expression during E. chaffeensis infection. To understand the global effect of E. chaffeensis on Notch signaling pathway and to examine ehrlichial activation of this pathway, a transcriptional analysis was performed to examine Notch regulated gene expression. A human Notch signaling PCR array consisting of 84 genes, including Notch binding and receptor processing genes, the putative Notch target genes, genes from Sonic Hedgehog and Wnt Receptor Signaling Pathways that cross-talk with the Notch signaling pathway was used. Heat maps were constructed depicting the differential expression of the Notch signaling pathway genes in the E. chaffeensis infected and uninfected cells (FIG.9A). The intensities of the red and green in the heat map represents the level of induction and repression, respectively. PCR array data identified activation of canonical Notch signaling pathway by E. chaffeensis at 12 h, 24 h, 48 h and 72 h p.i. The expression patterns of genes that were consistently upregulated throughout all different time points included Notch target genes: hes1, hey2, NFκB1, NFκB2, nr4a2, pax5, fosl1, chuk and ccne1; Notch pathway component genes, e.g. notch1 (receptor), dll4 (ligand) and maml2 (transcription complex protein). The transcription factor rbpjκ and E3 ubiquitin ligase dtx1, which play important roles in Notch pathway activation and regulation were upregulated at 48 and 72 h p.i. Only a small percentage of genes were downregulated during the infection, including genes for the Notch pathway components dll1 and mmp7 (FIG.9A). In FIG.9B, the scatter plot shows the comparison of the normalized expression of all genes in the Notch PCR array between infected and uninfected cells. The central line indicates unchanged gene expression (2-fold regulation cut-off), the red dots represent the genes which were upregulated, the green dots represent genes that were downregulated, and the black dots represent genes with no significant difference in expression level. Though the gene expression pattern was similar throughout the different time points, maximum changes in Notch gene expression occurred at 24 h p.i. When the differential expression of these individual genes was analyzed, 38 genes showed significant differential expression (p<0.05), including 28 (33.33 %) that were up-regulated and 10 (11.90 %) were down-regulated (FIG. 9C). These results support canonical Notch signaling pathway activation during early and late phase of E. chaffeensis infection. [00119] Notch signaling pathway is required for E. chaffeensis survival. Notch signaling is required not only for the cell growth and proliferation, but also plays important role in determining the fate of mature immune cells (Hoyne 2003; Radtke et al., 2013). Since Notch pathway regulates both innate and adaptive immune responses, and this pathway is activated during E. chaffeensis infection, the role of this pathway in ehrlichial survival was examined. To that end, cells were treated with Notch signaling transcription complex inhibitor SAHM1 and γ-secretase inhibitor DAPT. SAHM1 is a cell permeable small peptide that targets critical protein-protein interaction in Notch transcription complex and prevents their assembly (Moellering et al., 2009). Gamma-secretase inhibitor DAPT is a dipeptide and targets the C terminal fragment of presenilin that is a component of γ-secretase protein (Morohasi et al., 2006 ). THP-1 cells were treated with different concentrations of SAHM1 (1 µM, 5 µM and 10 µM) and DAPT (0.5 µg, 1 µg and 5 µg/ml) and cells were infected with cell free E. chaffeensis (at MOI 50). A dose dependent effect of both of inhibitors on bacterial load was observed as percent of ehrlichiae infected cells was determined using Diff- Quik staining. A significant decrease in bacteria load in cells treated with 10 µM SAHM1 and 5 µg/ml of DAPT was observed; therefore this concentration was used in additional experiments. There was >50% decrease in percentage of infected cells after inhibition of Notch signaling pathway at day 1 p.i. and >80% inhibition at day 2 p.i (FIG.10A), and real time qPCR detected a ~90% decrease in bacterial load in the presence of Notch inhibitors (FIG.10B). No apparent cell death or toxicity was observed with inhibitor treatment within the experimental window, as cell viability measured by trypan blue. To obtain additional evidence supporting the role of Notch pathway in E. chaffeensis survival, siRNAs were used to knock-down expression of the receptor Notch1, metalloprotease ADAM17 and transcription factor RBPjκ in THP-1 cells, then infected them with E. chaffeensis. These components were selected since they play critical role in canonical Notch signal transduction. Bacterial load was measured using real time qPCR by amplification of the integral ehrlichial gene dsb. Consistent with Notch pathway inhibitor experiments, significant reduction of bacterial load was also found in cells in which Notch component genes were knocked down (FIG.10C). Protein expression of Notch1, ADAM17 and RBPjκ was reduced in siRNA-transfected cells compared with the control siRNA-transfected cells (FIG. 10D). Overall, results obtained from pharmacological inhibitors and siRNA experiments support the importance of Notch signaling in E. chaffeensis survival. [00120] E. chaffeensis TRP120 protein interacts with Notch receptor complex. E. chaffeensis activates canonical Notch signaling, which is requisite for survival. However, the mechanism of Ehrlichia-induced Notch activation remains undefined. E. chaffeensis TRP effectors are the major immunoprotective proteins, and contains species specific epitopes (Gooz 2010). TRP120 functions as an adhesin, facilitating ehrlichial entry and is a nucleomodulin (Popov et al., 2000). Moreover, the Y2H data showed TRP120 interacts with ADAM17 and binds to the promoter region of notch1 (Zhu et al., 2011; Luo et al., 2011). The inventors hypothesized that TRP120 interaction with the Notch receptor complex components is required for the activation of Notch pathway. In order to further examine the distribution and colocalization of ADAM17 with the TRP120 expressing ehrlichial inclusions, cells were stained with anti-ADAM17 and anti-TRP120 antibody. Immunofluorescence microscopy showed diffused cytoplasmic localization of ADAM17 in uninfected THP-1 cells; however consistent with previous Y2H data, co-localization of ADAM17 with morulae expressing TRP120 was observed (FIG. 11A). Since Notch1 and ADAM17 are components of the receptor complex, colocalization of Notch1 with E. chaffeensis morulae was also examined. In contrast to the diffused cytoplasmic localization of Notch1 in uninfected cells colocalization of Notch1 with TRP120 expressing morulae was observed. (FIG. 11B). This data was further validated by transfecting HeLa cells with GFP- tagged TRP120 or GFP-control plasmids. Colocalization of ADAM17 and TRP120 was observed (FIG. 11C), demonstrating that Notch receptor components are associated with ehrlichial vacuoles and confirms previous TRP120-ADAM17 interaction data. [00121] TRP120 activates canonical Notch signaling pathway. To further examine the role of TRP120 in Notch pathway activation, TRP120 coated FluoroSphere sulfate microspheres was used to stimulate the THP-1 cells. Notch activation involves proteolytic release of NICD from Notch by the furin, ADAM17 and γ-secretase enzymes and translocation of NICD to the nucleus. Thus, the expression of NICD was used to monitor activation of Notch pathway. Since human monocytes constitutively express Notch receptors and ligands at basal levels, NICD basal level expression was confirmed in the cytoplasm of untreated cells. However, within 5 min of stimulation condensed expression of NICD was observed near the nucleus which then translocated to nucleus within 15 min of stimulation (Fig. 12A). Since, thioredoxin tag was used as the TRP120 fusion protein; cells were treated with thioredoxin as control. Nuclear translocation of NICD was not observed in untreated cells, or in cells treated with thioredoxin. [00122] To further delineate the role of TRP120 modulating Notch pathway gene expression levels, cells were stimulated with TRP120-coated beads for 2 h and the gene expression level of notch1 and the Notch pathway target genes hes1 and hes5 was examined. A significant increase in expression of all the selected genes in TRP120 treated compared to thioredoxin treated cells was detected (FIG. 12B). To further establish the role of TRP120 in induction of Notch signaling pathway, the expression pattern of genes involved in Notch pathway was globally analyzed using human Notch signaling PCR array. The heat map in FIG. 12C shows the graphical representation of gene expression pattern of all the 84 genes involved in Notch signaling, in cells stimulated with TRP120, and normalized to control cells treated with thioredoxin. Examination of individual gene expression identified 33 (39.3%) genes were differentially expressed (p<0.05). The scatter plot in Fig 5D shows differential expression of Notch pathway genes during TRP120 stimulation compared to the thioredoxin control, where the up and downregulation of genes are represented as red and green dots (2-fold cutoff), respectively. FIG. 12E, shows the list of genes and the fold change during the TRP120 stimulation. TRP120 stimulated cells showed increased expression of 16 Notch pathway components (19%) including receptors (notch1, notch3); ligands (dll 1, 3, 4), transcription factor complex proteins (maml2, rbpjκ); γ-secretase protein (psen1) and the TRP120 interacting enzyme adam17. Significant induction of 12 target genes (14%) including hes1, hes5, hey2, NFκB 1, NFκB 2, il2ra and lor, and downregulation of 4 genes (5%) (lmo2, id1, fosl1 and lrp5) was also observed. Previously, it has been observed that ehrlichial TRPs including TRP120 are secreted during infection (Wakeel et al., 2011); therefore, the inventors were also interested in exploring whether soluble TRP120 could potentially modulate Notch signaling in neighboring cells. Approximately 65% of the Notch signaling pathway component and target gene exhibited significant differential expression when stimulated with soluble TRP120. Together these data demonstrate that TRP120 independently and efficiently activates canonical Notch signaling pathway. Moreover, these findings also suggest that ehrlichial infection not only manipulates the host Notch signaling pathway through direct interactions between ehrlichiae and host cells, but potentially involves uninfected neighboring cells through the release of soluble TRP120 during the exit phase. [00123] E. chaffeensis represses ERK1/2 and p38 MAPK pathway through Notch signaling. Previous studies reported down-regulation of PU.1 and TLR2/4 expression during E. chaffeensis infection, and demonstrated that host cells become progressively less responsive to LPS mediated stimulation. Moreover, the underlying mechanism involved inhibition of ERK1/2 and p38 MAPK pathway (Lin and Rikihisa 2004). Since recent studies linked Notch signaling pathway with inhibition of TLR triggered inflammation and inhibition of ERK1/2 (Zhang et al., 2012), the inventors sought to determine the role of E. chaffeensis mediated activation of Notch signaling in inhibition of ERK1/2 and p38 MAPK pathways. Therefore, the phosphorylated and total level of ERK1/2 and p38 MAPK protein was examined in response to E. chaffeensis infection and LPS stimulation in the presence and absence of Notch transcription factor inhibitor SAHM1. Decreased levels of phosphorylated ERK1/2 in response to LPS was detected within 3 h of ehrlichial infection. In contrast, inhibition of ERK1/2 phosphorylation was blocked in the absence of Notch signaling (FIG.13A). However, the total level of ERK1/2 remained unchanged (FIG.13B), and the level of phosphorylated p38 MAPK decreased beginning at 3 h of p.i. and was significantly down regulated at 1 d.p.i, resulting in a decreased responsiveness of p38 MAPK to LPS. However, phosopho-p38 MAPK levels were not decreased when the cells were pre-treated with SAHM1 (FIG. 13C) and the total level of p38 MAPK remained unchanged (FIG.13D). These results support the idea that activation of Notch signaling plays a key role in downregulation of ERK1/2 and p38 MAPK pathway during E. chaffeensis infection. [00124] E. chaffeensis mediated PU.1 inhibition and TLR2/4 down- regulation depends on canonical Notch signaling pathway. To investigate the role of Notch signaling in regulation of PU.1 expression, RBPjκ (Notch transcription factor) expression was silenced in THP-1 cells with specific siRNA. Control siRNA and RBPjκ siRNA transfected cells were infected with E. chaffeensis one day post transfection, and stimulated with LPS (100 ng/ml) for 1 h after 1 d.p.i. Using immunofluorescent microscopy, high levels and predominant localization of PU.1 in the nucleus of uninfected and control siRNA treated THP-1 cells was observed, but there was a reduction of expression in E. chaffeensis infected cells. However, the inventors observed reconstitution of PU.1 expression level in the nucleus of THP-1 cells which were treated with RBPjκ siRNA to inhibit Notch signaling before infection (FIG.14A). Protein expression of RBPjκ was reduced in specific siRNA-transfected cells compared to control siRNA treated cells (FIG. 14B). To independently confirm the results seen in immunofluorescence microscopy, PU.1 protein levels during E. chaffeensis infection in whole cell lysates in the presence or absence of the Notch transcription factor inhibitor SAHM1 were determined by Western blot. As shown in FIG. 14C, the level of PU.1 was reduced in E. chaffeensis infected cells compared to controls. However, no inhibition was seen in E. chaffeensis infected cells which were treated with Notch inhibitor SAHM1. Densitometry data generated by the software image J and normalized with the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed significant differences in the level of PU.1 in E. chaffeensis infected cells compared to control and inhibitor treated cells (FIG.7D). Thus, the Western blot and IFA data were consistent and indicated that the Notch signaling was responsible for E. chaffeensis mediated inhibition of PU.1. [00125] To demonstrate that the Notch induced inhibition of PU.1 expression resulted in down-regulation of TLR2/4 expression, THP-1 cells were infected with E. chaffeensis in the presence or absence of Notch inhibitors e.g. DAPT and SAHM1. After 24 h p.i., cells were treated with LPS (100 ng/mL) for 1 h. As shown in FIG.15A and FIG.15B, E. chaffeensis infection caused significant decrease in TLR2 and 4 expressions compared to uninfected cells even after LPS stimulation. However, E. chaffeensis was unable to downregulate TLR2/4 expression when Notch signaling was blocked. Western blot analysis of THP-1 cells treated under the same conditions provided an independent approach to validate differential expression of these PRRs in the whole cell lysate of E. chaffeensis infected cells (2 day p.i) in the presence or absence of Notch signaling. Immunoblot data correlated with gene expression results, showing reduced expression of TLR2/4 proteins in E. chaffeensis-infected cells compared to uninfected cells. Whereas, in the presence of Notch signaling inhibitor, differences in TLR expression compared to uninfected control were not observed (FIG.15C). In FIG.15D, densitometry data shows quantitative comparison of the Western blot where the levels of TLR2/4 were normalized with the housekeeping protein GAPDH. Collectively, these data support a key role of Notch signaling in TLR2/4 down-regulation during E. chaffeensis infection. [00126] TRP120 effector protein plays crucial role in inhibition of TLR2/4 response. The inventors observed that E. chaffeensis activates canonical Notch signaling pathway, and this pathway suppresses TLR2/4 expression during infection. Since TRP120 plays critical role in activation of Notch components and target gene expression, the inventors sought to evaluate their effect on PU.1 inhibition and TLR2/4 expression. To test this, THP-1 cells were treated with either 1 µg/ml of TRP120 or thioredoxin (control) in soluble form, or TRP120 coated on latex beads and incubated at 37ºC for 24 h. TRP120 treated and control cells were then stimulated with LPS (100 ng/ml) for 1 h and expression of PU.1, TLR2 and TLR4 was determined using immunofluorescence microscopy. FIG.16A shows, strong PU.1 expression in the nucleus of thioredoxin treated cells in response to LPS stimulation. However, after LPS treatment, reduction in the expression level of PU.1 in response to TRP120 stimulation (both bead bound and in suspension) was observed. Since PU.1 transcription factor regulates TLR2/4 expression, the effect of TRP120 in TLR expression was also determined using the same method. Thioredoxin treated cells showed strong TLR2 expression in both cytoplasm and nucleus of the cells in response to LPS; however cells stimulated with TRP120 showed reduced expression of TLR2 (FIG. 16B). Similar results were found when TLR4 expression was measured in response to TRP120 stimulation (FIG. 16C). Western blot was done to analyze the protein expression of PU.1, TLR2 and TLR4 in control and TRP120 stimulated (bead bound and soluble) THP-1 cells in response to LPS. Decreased expression of all three proteins were observed, which further validated the IFA data (FIG. 16D). These studies demonstrate that TRP120 effector protein plays a direct role in modulating in TLR expression during E. chaffeensis infection. [00127] Notch plays critical role in regulating the maturation and function of different immune cells including monocytes and macrophages. Without wishing to be bound by any theory, data presented herein supports the idea that Notch signaling can mediate changes in the properties of these cells and may also affect the bacterial growth. Pharmacological inhibitors against Notch transcription factor protein (SAHM1), γ-secretase enzyme (DAPT) and siRNAs against Notch1, ADAM17 and RBPjκ confirmed that canonical Notch signaling pathway is required for ehrlichial survival. Differences in ehrlichial load observed between different siRNA treatments (at day 1 p.i.) might be because the role of these targets varies at different stages of infection. Also, the siRNAs might vary in their efficacy, and it is possible that the ADAM17 and RBPjκ siRNAs required more time for efficient knockdown. Together, the gene expression studies correlated strongly with inhibitor data supporting the conclusion that canonical Notch signaling is required for ehrlichial survival and may therapeutically inhibited, e.g., to treat ehrlichiosis. [00128] Herein, data is presented to support a mechanism whereby E. chaffeensis T1S effector protein TRP120 can manipulate Notch signaling to regulate immune recognition through inhibition of TLR expression to promote survival. TRP120 is found on the surface of the infectious DC form, but is also secreted into the host cell where it interacts with a variety of host cell targets and DNA (Zhu et al., 2011; Luo et al., 2011; Luo and McBride 2012). TRPs can also be released from the E. chaffeensis infected cell during infection (Luo et al., 2008). Data presented in this study demonstrated that TRP120 bound to a substrate or in soluble form can activate Notch signaling pathway and modulate PU.1 and TLR2/4 expression. These findings support the idea that the effects of E. chaffeensis TRP120 on Notch pathway activation not only occur through direct bacterium-host interactions, but may have systemic effects through the release of soluble TRP120 that could interact with uninfected cells to downregulate innate immunity and promote infection. [00129] The limited understanding of the molecular pathogen-host interactions and cellular pathways usurped by Ehrlichia as well as those of other obligate intracellular microbes is a major impediment to defining the mechanisms that enable ehrlichial intracellular survival and development of next-generation therapeutics aimed at mechanistically defined targets. Without wishing to be bound by any theory, these experiments reveal a novel effector- dependent mechanism, which involves interaction with the ADAM17 and Notch1, and activation of canonical Notch signaling pathway on monocytes, a primary target of E. chaffeensis, to modulate ERK1/2, p38 MAPK pathway and regulate TLR2/4 expression (FIG. 17). Hence, these experiments demonstrate the importance of Notch pathway in ehrlichial survival, and therapeutic modulation of this pathway bay be used to treat an ehrlichial infection and/or other intracellular pathogens in which exploitation of such conserved cellular pathways is important or required for pathogen survival. * * * [00130] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. U.S. Patent No.5,399,363

U.S. Patent No.5,466,468

U.S. Patent No.5,543,158

U.S. Patent No.5,580,579

U.S. Patent No.5,629,001

U.S. Patent No.5,641,515

U.S. Patent No.5,725,871

U.S. Patent No.5,756,353

U.S. Patent No.5,780,045

U.S. Patent No.5,792,451

U.S. Patent No.5,804,212

U.S. Patent No.6,613,308 Artavanis-Tsakonas et al., Notch signaling: cell fate control and signal integration in

development. Science 284:770-776, 1999.

Barrick and Kopan, The Notch transcription activation complex makes its move. Cell

124:883-885, 2006.

Brou et al., 2000. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5:207-216., 2000.

Chen et al., Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat Chem Biol 5: 100-107, 2009.

Croce et al., Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development. Development 133: 547-557, 2006.

Doyle et al., Differentially expressed and secreted major immunoreactive protein orthologs of Ehrlichia canis and E. chaffeensis elicit early antibody responses to epitopes on glycosylated tandem repeats. Infect Immun 74: 711-720, 2006.

Dunphy et al., Ehrlichia moonlighting effectors and interkingdom interactions with the mononuclear phagocyte. Microbes Infect 15: 1005-1016, 2013. Dunphy et al., Ehrlichia chaffeensis exploits host SUMOylation pathways to mediate effector- host interactions and promote intracellular survival. Infect Immun 82:4154-4168, 2014. Espinoza and Miele, Notch Inhibitors for Cancer Treatment. Pharmacol Ther., 139(2): 95–110, 2013.

Fischer and Gessler, Delta-Notch--and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids Res 35:4583-4596, 2007. Fortini, ME, Introduction--Notch in development and disease. Semin Cell Dev Biol023:419- 420, 2012.

Gooz M., ADAM-17: the enzyme that does it all. Crit Rev Biochem Mol Biol 45:146-169, 2010.

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

Habas and Dawid, Dishevelled and Wnt signaling: is the nucleus the final frontier? J Biol 4: 2, 2005.

Hoyne GF, Notch signaling in the immune system. J Leukoc Biol 74:971-981, 2003.

Hwang et al., Crit. Rev. Ther. Drug Carrier Syst., 15(3):243-284, 1998.

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

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

Kovall RA, Structures of CSL, Notch and Mastermind proteins: piecing together an active transcription complex. Curr Opin Struct Biol 17:117-127, 2007.

Kumagai et al., Cyclic di-GMP signaling regulates invasion by Ehrlichia chaffeensis of human monocytes. J Bacteriol 192: 4122-4133, 2010.

Kuriakose et al., Molecular basis of antibody mediated immunity against Ehrlichia chaffeensis involves species-specific linear epitopes in tandem repeat proteins. Microbes Infect 14: 1054-1063, 2012.

Lafont et al., Bacterial invasion via lipid rafts. Cell Microbiol 7: 613-620, 2005.

Lin and Rikihisa, Obligatory intracellular parasitism by Ehrlichia chaffeensis and Anaplasma phagocytophilum involves caveolae and glycosylphosphatidylinositol-anchored proteins. Cell Microbiol 5: 809-820, 2003.

Lin et al., Rapid activation of protein tyrosine kinase and phospholipase C-gamma2 and increase in cytosolic free calcium are required by Ehrlichia chaffeensis for internalization and growth in THP-1 cells. Infect Immun 70: 889-898, 2002. Lin and Rikihisa Y, Ehrlichia chaffeensis downregulates surface Toll-like receptors 2/4, CD14 and transcription factors PU.1 and inhibits lipopolysaccharide activation of NF- kappa B, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol 6:175-186, 2004.

Lippincott et al., 2005.

Logan and Nusse, The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20: 781-810, 2004.

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

Luo et al., Ehrlichia chaffeensis TRP120 interacts with a diverse array of eukaryotic proteins involved in transcription, signaling, and cytoskeleton organization. Infect Immun 79: 4382-4391, 2011.

Luo et al., Major species-specific antibody epitopes of the Ehrlichia chaffeensis p120 and E. canis p140 orthologs in surface-exposed tandem repeat regions. Clin Vaccine Immunol 16: 982-990, 2009.

Luo et al., A variable-length PCR target protein of Ehrlichia chaffeensis contains major

species-specific antibody epitopes in acidic serine-rich tandem repeats. Infect Immun 76:1572-1580, 2008.

MacDonald et al., Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17: 9-26, 2009.

Maiti et al., The Wingless homolog Wnt5a stimulates phagocytosis but not bacterial killing.

Proc Natl Acad Sci USA 109: 16600-16605, 2012.

Mathiowitz et al., Nature, 386(6623):410-414, 1997.

McBride and Walker, Molecular and cellular pathobiology of Ehrlichia infection: targets for new therapeutics and immunomodulation strategies. Expert Rev Mol Med 13: e3, 2011. Moellering et al., Direct inhibition of the NOTCH transcription factor complex. Nature

462:182-188, 2009.

Mohan et al., Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells. PLoS Pathog 9: e1003666, 2013.

Morohashi et al., C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]- S-phenylglycine t-butyl ester). J Biol Chem 281:14670-14676, 2006. Nusse and Varmus, Three decades of Wnts: a personal perspective on how a scientific field developed. EMBO J 31: 2670-2684, 2012.

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

Nusse et al., Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307: 131-136, 1984.

Olivier et al., The Notch ligand delta-1 is a hematopoietic development cofactor for plasmacytoid dendritic cells. Blood 107:2694-2701, 2006.

Paddock and Childs, Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin Microbiol Rev 16: 37-64, 2003.

Popov et al., The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry. Microb Pathog 28: 71-80, 2000.

Radtke et al., Regulation of innate and adaptive immunity by Notch. Nat Rev Immunol

13:427-437, 2013.

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

Remington: The Science and Practice of Pharmacy, 21^st Ed.

Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580 Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329.

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

Sasamura et al., Neurotic, a novel maternal neurogenic gene, encodes an O- fucosyltransferase that is essential for Notch-Delta interactions. Development 130:4785-4795, 2013.

Schmidt et al., Wnt6 controls amniote neural crest induction through the non-canonical signaling pathway. Dev Dyn 236: 2502-2511, 2007.

Schulte and Bryja, The Frizzled family of unconventional G-protein-coupled receptors. Trends Pharmacol Sci 28: 518-525, 2007.

Seveau et al., A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway. Cell Microbiol 9: 790-803, 2007.

Shafer et al., Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1- mediated feedback inhibition in growth cone guidance. Dev Cell 20: 177-191, 2011. Shi and Stanley, Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc Natl Acad Sci U S A 100:5234-5239, 2003.

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

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

Sumi et al., The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Commun 181: 968-975, 1991.

Takenaga et al., 1998

Thorne et al., Small-molecule inhibition of Wnt signaling through activation of casein kinase 1alpha. Nat Chem Biol 6: 829-836, 2010.

Venerando et al., Pyrvinium pamoate does not activate protein kinase CK1, but promotes Akt/PKB down-regulation and GSK3 activation. Biochem J 452: 131-137, 2013. Wakeel et al., An Ehrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptional regulation, and vesicle trafficking. Infect Immun 77: 1734-1745, 2009.

Wakeel et al., Ehrlichia chaffeensis tandem repeat proteins and Ank200 are type 1 secretion system substrates related to the repeats-in-toxin exoprotein family. Front Cell Infect Microbiol 1: 22, 2011

Wakeel et al., New insights into molecular Ehrlichia chaffeensis-host interactions. Microbes Infect 12: 337-345, 2010

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

Wolfe and Kopan, Intramembrane proteolysis: theme and variations. Science 305:1119-1123, 2004..

Yin and Janmey, Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol 65:

761-789, 2003.

Zhang et al., The developmental cycle of Ehrlichia chaffeensis in vertebrate cells. Cell Microbiol 9: 610-618, 2007.

Zhang et al., Notch signal suppresses Toll-like receptor-triggered inflammatory responses in macrophages by inhibiting extracellular signal-regulated kinase 1/2-mediated nuclear factor kappaB activation. J Biol Chem 287:6208-6217, 2012.

Zheng et al., Role of WNT7B-induced noncanonical pathway in advanced prostate cancer. Mol Cancer Res 11: 482-493, 2013. Zhu et al., Ehrlichia chaffeensis TRP120 binds a G+C-rich motif in host cell DNA and exhibits eukaryotic transcriptional activator function. Infect Immun 79:4370-4381, 2011.

Claims

WHAT IS CLAIMED IS: 1. A method of treating a bacterial infection in a mammalian subject, comprising administering a therapeutically effective amount of a Wnt inhibitor to said subject, wherein said bacterial infection comprises an obligately intracellular bacteria.

2. The method of claim 1, wherein the obligately intracellular bacteria is Ehrlichia, Chlamydia, Rickettsia, Coxiella, Orientia, or Mycobacteria.

3. The method of claim 1, wherein the obligately intracellular bacteria is Ehrlichia chaffeensis.

4. The method of claim 1, wherein the obligately intracellular bacteria is Ehrlichia canis.

5. The method of claim 1, wherein the subject is a human.

6. The method of claim 1, wherein the subject is a dog.

7. The method of any of claims 1-6, wherein the Wnt inhibitor is a small interfering RNA (RNAi), an antibody, or a small molecule Wnt inhibitor.

8. The method of claim 7, wherein the Wnt inhibitor is administered orally, intravenously, or parenterally.

9. The method of any one of claims 1-7, wherein the Wnt inhibitor is an inhibitor of a canonical Wnt pathway.

10. The method of claim 9, wherein the Wnt inhibitor selectively inhibits PI3K, CKII, CK1ε, or β-catenin/TCF/LEF, or the Wnt inhibitor selectively activates casein kinase Iα/GSK3β.

11. The method of claim 10, wherein the Wnt inhibitor selectively activates casein kinase Iα/GSK3β.

12. The method of claim 9, wherein the Wnt inhibitor is pyrvinium pamoate, TBCA, SB202190, LY294002, or FH535.

13. The method of claim 12, wherein the Wnt inhibitor is pyrvinium pamoate.

14. The method of any one of claims 1-7, wherein the Wnt inhibitor is an inhibitor of a non- canonical Wnt pathway.

15. The method of any one of claims 1-7, wherein the Wnt inhibitor is an inhibitor of a Wnt/Ca²⁺ pathway.

16. The method of claim 15, wherein the Wnt inhibitor selectively inhibits calmodulin kinase II (CaMKII) or IKK.

17. The method of claim 16, wherein the Wnt inhibitor selectively inhibits calmodulin kinase II.

18. The method of claim 15, wherein the Wnt inhibitor is KN93.

19. The method of claim 14, wherein the Wnt inhibitor is an inhibitor of a Wnt/PCP pathway.

20. The method of claim 19, wherein the Wnt inhibitor selectively inhibits PI3K, Akt, or IKK.

21. The method of claim 20, wherein the Wnt inhibitor selectively inhibits Akt.

22. The method of claim 19, wherein the Wnt inhibitor is pyrvinium pamoate, LY294002, or BAY 11-7082.

23. The method of claim 22, wherein the Wnt inhibitor is pyrvinium pamoate.

24. The method of claim 1, wherein the Wnt inhibitor selectively inhibits Wnt secretion.

25. The method of claim 24, wherein the Wnt inhibitor selectively inhibits PORCN.

26. The method of claim 24, wherein the Wnt inhibitor is IWP-2.

27. The method of any one of claims 1-26, wherein the Wnt inhibitor is comprised in a pharmaceutical preparation.

28. The method of claim 27, wherein the pharmaceutical preparation is formulated for oral, intravenous, topical, or parenteral administration.

29. A method of treating a bacterial infection in a mammalian subject, comprising administering a therapeutically effective amount of a Notch inhibitor to said subject, wherein said bacterial infection comprises an obligately intracellular bacteria.

30. The method of claim 29, wherein the obligately intracellular bacteria is Ehrlichia, Chlamydia, Rickettsia, Coxiella, Orientia, or Mycobacteria.

31. The method of claim 29, wherein the obligately intracellular bacteria is Ehrlichia chaffeensis.

32. The method of claim 29, wherein the obligately intracellular bacteria is Ehrlichia canis.

33. The method of claim 29, wherein the subject is a human.

34. The method of claim 29, wherein the subject is a dog.

35. The method of any of claims 29-34, wherein the Notch inhibitor is a small interfering RNA (RNAi), an antibody, or a small molecule Notch inhibitor.

36. The method of claim 35, wherein the Notch inhibitor is administered orally, intravenously, or parenterally.

37. The method of any of claims claim 29-36, wherein the Notch inhibitor selectively inhibits γ-secretase, Notch transcription factor complex, Notch1, ADAM17, or ADAM10.

38. The method of claim 37, wherein the Notch inhibitor selectively inhibits γ-secretase.

39. The method of claim 38, wherein the Notch inhibitor is DAPT, BMS-906024, MK0752, PF-03084014, MRK0003, or RO4929097.

40. The method of claim 37, wherein the Notch inhibitor is an antibody, an antibody fragment, or a blocking peptide.

41. The method of any one of claims 29-40, wherein the Notch inhibitor is comprised in a pharmaceutical preparation.

42. The method of claim 41, wherein the pharmaceutical preparation is formulated for oral, intravenous, topical, or parenteral administration.