Effect of β-catenin knock-down on <i>C</i>. <i>pneumoniae</i> anti-apoptotic activity.

<p>HeLa cells infected with <i>C</i>. <i>pneumoniae</i> AR39 at a m.o.i. of 0.5 for 24h were transfected with control siRNA or β-catenin-targeted siRNA. (A) 24h after transfection, lysates were collected from a portion of the culture samples for detecting β-catenin (panel a), Bcl-2 (b) and β-actin (c) using the corresponding antibodies as listed in the materials and methods section. Note that cells transfected with β-catenin-targeted siRNA (right lane), but not control siRNA (left lane), displayed reduced levels of both β-catenin and Bcl-2. (B) The parallel cultures including the uninfected normal HeLa cells were also fixed for immunofluorescence assay for labeling <i>C</i>. <i>pneumoniae</i> AR39 (green), β-catenin (red) and DNA (blue) using the corresponding antibodies and dyes as listed in the materials and methods section. Note that the β-catenin SiRNA treatment significantly reduced the β-catenin protein level in both the AR-39-infected and normal uninfected cells. (C) The parallel cultures were further induced to undergo apoptosis using staurosporine (STS) stimulation for 4h. The apoptotic cells were identified using DNA staining. 10 random views were counted from each coverslip. Note that the <i>C</i>. <i>pneumoniae</i> AR39-infected cells were significantly resistant to apoptosis induction (column 1 v.s. 2) and the treatment with β-catenin-targeted siRNA but not control siRNA significantly neutralized the <i>C</i>. <i>pneumoniae</i> anti-apoptotic activity (column 2 v.s. column 4). Asterisks indicates significant differences of <i>P</i><0.05; paired Student’s <i>t</i>-test. The data came from 3 independent experiments each with duplicates.</p

<i>C</i>. <i>pneumoniae</i> interaction with host GSK3β.

<p>(A) HeLa cells infected with <i>C</i>. <i>pneumoniae</i> AR39 organisms were processed for co-staining with a mouse anti-GSK3β plus a Cy3-conjugated goat anti-mouse IgG (red) and a rabbit anti-IncA antiserum plus a Cy2-conjugated goat anti-rabbit IgG (green). The DNA was labeled with DAPI (blue). Note that the endogenous GSK3β was detected around the chlamydial inclusions. The region marked with a white square in panel a was magnified and the magnified area was shown in panels b (3 color-overlay), c (GSK3β alone, red), d (IncA alone, green) and e (DNA alone, blue) respectively. (B) Lysates from HEK 293T cells transfected with pDsRed vector alone (expressing RFP alone, left lanes) or pDsRed-Cpn1027 plasmid [expressing RFP-Cpn1027, right lanes] were immunoprecipitated without (panel a) or with an anti-RFP antibody (b & c). The lysates or precipitates were analyzed by Western blot using an anti-RFP (panels a & b) or an anti-GSK3α/β (c) antibodies. Note that RFP-Cpn1027but not RFP alone co-precipitated the endogenous GSK3β. (C) The parallel lysates were precipitated without (panel d) or with an anti-GSK3α/β antibody (e & f). The lysate or precipitates were analyzed by using αGSK3α/β (panels d & e) or αRFP antibodies. Note that the endogenous GSK3β co-precipitated RFP-Cpn1027, but not RFP alone (f).</p

<i>C</i>. <i>pneumoniae</i> interaction with host protein Caprin2.

<p>(A) Yeasts co-transformed with pEXP-AD502-Caprin2(245–1128) as prey plus pDBLeu-Cpn1027(210–527) as bait (row 1) or the positive control prey pGal4AD-TRAF3 plus bait pGal4DB-LMP1 (row 2) or the negative control prey pGal4AD plus bait pGal4DB-LMP1 (row 3) were grown on plates with selective medium without Leu and Trp (-LW, left column) or without Leu, Trp and His but supplemented with 50mM <i>3-</i>Amino<i>-1</i>,<i>2</i>,<i>4-</i>triazole or 3AT (-LWH 50mM 3AT, middle column) or without Leu, Trp and Uracil (-LWU, right column). Note that bait-prey interactions occurred in rows 1 & 2 but not 3. (B) HEK 293T cells co-transfected with pFLAG-Cpn1027(210–527) and pDsRed vector (RFP alone, left lane), pDsRed-APC4 (RFP-APC4, middle lane) or pDsRed-Caprin2(245–1128) [RFP-Caprin2(245–1128), right lane] were harvested as whole cell lysates for immunoprecipitation using an anti-RFP antibody and the precipitates were probed with an anti-Cpn1027 antibody (panel a). The whole cell lysates were also directly probed with anti-Cpn1027 antibody (b) and anti-RFP antibody (c) respectively. Note that the anti-RFP antibody co-precipitated FLAG-Cpn1027(210–527) from cells co-transfected with pDsRed-Caprin2 but not pDsRed-APC4 or pDsRed vector alone. (C) Glutathione-S-transferase (GST) and GST-Caprin2(245–1128) fusion proteins were purified using glutathione-agarose beads as described in Materials and Methods. The GST (lanes 3 & 4) or GST-Caprin2(245–1128; lanes 5 & 6) beads were incubated overnight with lysates of HEK 293T cells transfected with pDsRed vector clone (expressing RFP tag alone, lanes 3 & 5) or pDsRed-Cpn1027 [expressing RFP-Cpn1027, lanes 4 & 6]. The bead-associated materials were resolved in SDS-PAGE for Western blot detection of Cpn1027 (a) and the tags RFP and GST (b). Note that the GST-Caprin2(245–1128) fusion protein but not GST alone pulled down RFP-Cpn1027. Samples loaded in lanes 1 & 2 were lysates without the GST pull down, thus marked as input on top of the figure. All GST or RFP tag-containing proteins were detected in panel b. (D) <i>C</i>. <i>pneumoniae</i> AR39-infected HeLa cells were transfected with pDsRed (expressing RFP alone, panels a-c) or pDsRed-Caprin2(245–1128) [expressing RFP-Caprin2(245–1128), d-f) as described in Materials and Methods. The chlamydial organisms were visualized with a rabbit anti-AR39 antiserum (αAR39 in green, stock# R12AR39) plus a Cy2-conjugated goat anti-rabbit IgG (green) and DNA with DAPI (blue). The areas marked with white squares in panels a & d were magnified and the magnified areas were shown in panels b (3 color-merged) & c (RFP alone in black and white) and e (3 color-merged) & f (RFP-Caprin2 in black and white) respectively. Note that the RFP-Caprin2 fusion protein but not RFP alone was detected around the inclusion membrane in the <i>C</i>. <i>pneumoniae</i> AR39<i>-</i>infected cells.</p

Identification of the Cpn1027-Caprin2 interaction domains.

<p>(A) Bead-immobilized GST (lane 2) or various GST-Cpn1027 fragments (from fragments 1 or F1, lane 3 to F2, lane 4, F3 lane 5 and F4, lane 6) as listed on the top of the panel were incubated overnight at 4°C with lysates made from HEK 293T cells transfected with pDsRed-Caprin2(245–1128) [expressing RFP-Caprin2(245–1128); The Caprin2(245–1128) fragment is the length of the cDNA clone identified during yeast two-hybrid screening). The precipitates pulled down by the beads were subjected to Western blot detection with anti-RFP (panel a) or anti-GST (b) antibodies. Note that GST-Cpn1027(366–527) or F4 was the shortest fragment that still pulled down a significant amount of RFP-Caprin2(245–1128). (B) Schematic representation of GST-Cpn1027 fragments (F1 to F4) and summary of their Caprin2 binding activities on the right side. Numbers indicate amino acid residue positions. The conserved domains include: TM, transmembrane domain; Coil, coil-coil domains; NGR, non-globular domain. The dashed line indicate the regions deleted from the corresponding constructs. (C) GST and GST-Caprin2 fragments as listed along the X-axis in the bottom of the figure were immobilized onto glutathione-coated microplates. 1μg of purified Cpn1027(210–527) protein (no GST tag, cleaved off from a GST fusion protein) was added to each well. The Cpn1027(210–527) bound to the plate-immobilized Caprin2 fragments was probed using an anti-Cpn1027 antibody followed by detection with a secondary goat anti-mouse IgG antibody conjugated with HRP plus a soluble substrate. The results were expressed as OD readings obtained at a wavelength of 405nm. Data from three independent experiments are shown. Note that GST-Caprin2 (927–1128) still maintained the ability to bind to Cpn1027. (D) Schematic representation of GST-Caprin2 fragments and summary of their Cpn1027 binding activities on the right side. WT Caprin2 depicts full-length Caprin2. Numbers indicate amino acid residue positions. The conserved domains include HR1, homologous region 1, HR2, homologous region 2 and CRD, C1q related domain.</p

A working model for <i>C</i>. <i>pneumoniae</i>-mediated activation of the Wnt signaling transduction pathway.

<p>A. In the absence of Wnt signals, cytosolic β-catenin is degraded by a β-catenin destruction complex composed of Axin, adenomatosis polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α). Both GSK3β and CK1α can phosphorylate β-catenin, which triggers ubiquitination and subsequent degradation of β-catenin by proteasomes. B. The Wnt signaling pathway is activated by binding of Wnt ligands, to Frizzled and LRP5/6 at the cell surface. LRP5/6 is activated by phosphorylation, which recruits the β-catenin destruction complex to the plasma membrane. The cytoplasmic activation/proliferation-associated protein 2 (Caprin2) facilitates LRP5/6 phosphorylation by GSK3β and enhances the interaction between Axin and the cytoplasmic tail of LRP5/6, which promotes the sequestration of the β-catenin destruction complex. The cytoplasmic pool of β-catenin rises and translocates into the nucleus, where it binds to the TCF/LEF family of transcription factors, displacing co-repressors Groucho and HDAC and acts as a co-activator to stimulate the transcription of anti-apoptotic, Bcl-2. C. In the <i>C</i>. <i>pneumoniae</i> AR39-infected cells, signalosomes are formed around the inclusion possibly through the chlamydial Inc protein Cpn1027 interacting with the β-catenin destruction complex, composed of Caprin2, Axin, GSK3β and CKIα resulting in the activation of the Wnt signaling transduction pathway in the absence of extracellular stimulation of Wnt. This leads to an increase in the cytoplasmic pool of β-catenin, nuclear β-catenin and membrane-associated β-catenin. The β-catenin-transactivated anti-apoptosis genes may promote the survival of the <i>C</i>. <i>pneumoniae</i> AR39-infected cells, hence the survival of AR39.</p

<i>Chlamydia trachomatis</i> acquisition of host sphingomyelin is independent of PRAK.

<p>(A) HeLa cells with (panels e–h) or without (a–d) <i>C. trachomatis</i> infection (MOI = 0.5) were treated without (a & e) or with EGCG (1 µM, b & f; 10 µM, c & g) or rottlerin (1 µM, d & h) 16 h post infection. Eight hours later, the cultures were subjected to BODIPY-FL-C5-ceremide labeling and visualized under a fluorescence microscope. Note that EGCG failed to block the accumulation of BODIPY-FL-sphingomyelin in the chlamydial inclusions (panel f & g) while rottlerin did (h). (B) MEF without (panels a & c) or with (b & d) PRAK deficiency (PRAK−/−) were infected with <i>C. trachomatis</i> (MOI = 0.5) and 24 h post infection, the cultures were labeled with BODIPY-FL-C5-ceremide and observed as described above. Note that <i>C. trachomatis</i> organisms can take up BODIPY-FL-sphingomyelin from MEF cells with or without PRAK. The thick arrows point to chlamydial inclusions with while thin arrows point to the inclusions without the fluorescent sphingomyelin.</p

EGCG fails to inhibit chlamydial growth.

<p>HeLa cells infected with <i>C. trachomatis</i> (MOI = 0.5) were treated with EGCG (1 µM, panel b & 10 µM, c) or rottlerin (1 µM, d) 16 h post infection. The cultures were processed 44 h post infection for immuno-labeling with a rabbit antibody for labeling the <i>C. trachomatis</i> organisms (green) and a mouse monoclonal antibody (clone BB2) for inclusion membrane protein IncA (red). Note that EGCG at 1 µM failed to inhibit inclusion expansion and even at 10 µM only slightly reduced inclusion size while rottlerin at 1 µM completely blocked inclusion expansion.</p

Rottlerin inhibits chlamydial growth in the absence of PRAK.

<p>MEF without (panels a–c) or with (d–f) PRAK deficiency (PRAK−/−) were infected with <i>C. trachomatis</i> (MOI = 0.5) and at oh (b & e) or 16 h (c & f) post infection, parallel cultures were treated with (b, c, e & f) or without (a & d) rottlerin at 1 µM. The cultures were processed 44 h post infection for immunofluorescence assay as described in Fig. 3 legend. Note that rottlerin inhibited chlamydial growth in both wild type and PRAK-deficient MEF cells.</p

Replication of <i>C. trachomatis</i> organisms in PRAK-deficient mouse embryo fibroblast cells.

<p>Mouse embryo fibroblast cells (MEF) without (panels a–d) or with (e–h) PRAK deficiency (PRAK−/−) were infected with (b–d & f–h) or without (a & e) <i>C. trachomatis</i> (MOI = 0.5) for various periods of time as indicated on top of the figure. The cultures were processed for immunofluorescence assay with a rabbit antibody for visualizing chlamydial organisms (green), Alexa-Fluor 568 Phalloidin for host cell F-actin (red) and Hoechst dye for DNA (blue). Note that the inclusion sizes were similar in MEF with or without PRAK deficiency.</p

Reduced spreading of <i>Chlamydia muridarum</i> from the oviduct/ovary into uterine horn after intrabursal injection.

<p>The wild type <i>C</i>. <i>muridarum</i> organisms were intrabursally inoculated into the right oviduct/ovary of female C57BL/6J mice with 2 x 10⁵ inclusion forming units (IFUs) per mouse. At various time points post inoculation as indicated along the X-axis, groups of mice (n = 4 to 5) were sacrificed for harvesting the right oviduct/ovary (RO, solid bar) and right uterine horn (RU, open) respectively. The tissue samples were homogenized for titrating <i>C</i>. <i>muridarum</i> live organisms, and the titers were expressed as Log10 IFUs displayed along the Y-axis. Please note that titers of live organisms recovered from RO that received direct injection were always significantly higher than those from RU that is on the opposite side of the uterotubal junction (**p<0.01, *p<0.05, Wilcoxon), suggesting a functional barrier between the oviduct and uterine horn. The lower recovery during the first 24h is consistent with the concept that the inoculum needs time to replicate and to differentiate the replicating but non-infectious reticulate bodies into the infectious elementary bodies.</p