EphA2 activated upon early infection co-localizes with <i>Ctr</i>.

<p>(A) Adherence assay: HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. The transfected cells were infected with <i>Ctr</i> (MOI-100) for 1 h at 4°C followed by immunostaining <i>Ctr</i>-EB (Hsp60, green) and pEphA2 (phospho EphA2 Ser897, red). (B) Co-localization of EB with pEphA2 was quantified. The graph was made similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004846#ppat.1004846.g001" target="_blank">Fig 1B</a>. The data are expressed as a mean percentage of pEphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C) Invasion assay: The transfected cells were infected with <i>Ctr</i> (MOI-20-25) for 4–5 h at 35°C followed by immunostaining as (A) including Actin filaments. (A, C) Arrows indicate the co-localization of <i>Ctr</i> with pEphA2 (yellow). Magnification is indicated in size bar.</p

<i>Ctr</i>-induces EphA2 activation and receptor internalization which associates with pPI3K during early infection.

<p>(A) HeLa cells uninfected (UN) or infected with <i>Ctr</i> (MOI-50-75) for 30 min or 3 h were analyzed via FACS for surface EphA2 under non permeabilised or total EphA2 under permeabilised condition. Controls were indicated on the left curve chart and corresponding samples on the right curve chart of the same experiment. (UN: uninfected, Iso: isotype and perm: permeabilised). (B) Result of experiment shown in A demonstrated as bar chart. Shown is the mean ± SD of three independent experiments normalized to UN perm. *P<0.05, ns: non-significant. Error bars show mean ± SD. (C) HeLa cells were UN or infected with <i>Ctr</i> (MOI-50) for the indicated time points. The cells were immunobloted against pEphA2 and Actin. The blot was stripped and reprobed for total EphA2. (D) HeLa cells were treated with control or rhEphrin-A1 for 3 h and were harvested for WB analysis. (E) HeLa cells were UN or infected with EB for the indicated time points and immunoprecipitated (IP) with α-EphA2 or α-p85-PI3K antibodies. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%) or (F) immunostained using α-EphA2 and α-pPI3K for 2 h at RT followed by secondary staining with anti-mouse Alexa fluor 488 and anti-rabbit Alexa fluor 647 for the microscopic analysis, respectively. Magnification is indicated in size bar.</p

<i>Ctr</i>-infected cells were sensitized to TNF-α induced apoptosis upon EphA2 knockdown.

<p>(A) The transfection efficiency of siRNA directed against EphA2 was monitored by WB analysis against total EphA2. In addition, levels of pAkt, IncA and Actin were verified. (B) siLuci or siEphA2-transfected cells were left uninfected or infected with <i>Ctr</i> for 16 h. Cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis. Rectangle boxes (black: before infection) or (red: after infection) denotes the difference in PARP cleavage after TNF-α induction in siLuci and siEphA2 transfected cells. (C) For quantification, TUNEL positive cells from each sample were counted from ten different fields. Shown is the mean ± SD of two independent experiments. **P<0.01, *P<0.05, ns: non-significant. Error bars show mean ± SD.</p

<i>Ctr</i>-serovar D utilize EphA2 signaling to invade the cells and to prevent apoptosis induced by TNF-α.

<p>(A) HeLa cells left UN or infected with <i>Ctr</i>-serovar D were centrifuged at 910 x g for 30 min and allowed to infect for the indicated time points. The cells were harvested and subjected to WB analysis. (B) HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. Adherence assay: The transfected cells were infected with <i>Ctr-</i>serovar D for 1 h at 4°C. Cells were immunostained against EB and Actin filaments. Number of extracellular EB was counted randomly from 30 different cells. Data are expressed as percentage of extracellular EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. Invasion assay: The transfected cells were infected with <i>Ctr-</i>serovar D for 4–5 h at 35°C. Cells were immunostained against EB and Actin filaments. Number of invaded EB was counted randomly from 30 different cells. Data are expressed as percentage of invaded EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.001. Error bars show mean ± SD. (C) FACS analysis was performed with <i>Ctr</i>-serovar D infected cells as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004846#ppat.1004846.g003" target="_blank">Fig 3A</a>. (UN: uninfected, Iso: isotype and perm: permeabilised). (D) Results of C were shown as bar charts. Shown is the mean ± SD of two independent experiments normalized to UN. ***P<0.001, *P<0.05, ns: non-significant. Error bars show mean ± SD. (E) HeLa cells were left UN or infected with <i>Ctr-</i>serovar D (MOI-1) for 14, 20 and 28 h and cells were analyzed via FACS as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004846#ppat.1004846.g004" target="_blank">Fig 4A</a>. (F) EphA2 knockdown followed by 16 h <i>Ctr</i>-serovar D infected cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis.</p

<i>Ctr</i>-induced EphA2 expression is prevented being re-translocated to the cell surface and is recruited to the inclusion membrane.

<p>(A) HeLa cells were infected with <i>Ctr-</i>EB (MOI-1) for 14, 20 and 26 h and cells were analyzed via FACS as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004846#ppat.1004846.g003" target="_blank">Fig 3A</a>. (B) HeLa cells were infected with <i>Ctr-</i>EB for 14, 24 and 36 h. Input (5%) was taken after homogenization step (before plasma membrane protein isolation) and subjected to WB analysis to determine pEphA2, Hsp60 and Actin. The blot was stripped and reprobed for total EphA2. Numbers under the blot represents fold activation for pEphA2 with respect to total EphA2. (C) Plasma membrane protein was isolated from UN or <i>Ctr</i>-infected cells at different time points and subjected to WB analysis. Pan Cadherin was used as a control for plasma membrane. IncA was used as a marker to test the quality of the isolated plasma membrane. * Unspecific bands detected by IncA polyclonal serum. (D) Culture medium of the UN as well as time course <i>Ctr-</i>infected cells (as indicated) was collected and TCA precipitated. The precipitated lysates were subjected to WB analysis against pEphA2, total EphA2 and GP96. (E, F) HeLa cells were transfected with EphA2-pcDNA3 at 37°C and the cells were infected with <i>Ctr</i>-pIncA-flag for 24 h at 35°C. (E) Cells were fixed and stained against Flag (red), EphA2 (green) and DNA (blue). (F) Cells were fixed and stained against Flag (red), pEphA2 (green) and DNA (blue). Magnification is indicated in size bar.</p

Small molecule EphA2 inhibitor dasatinib affects <i>Ctr</i> infection.

<p>(A) HUVEC cells were pretreated with DMSO(1) control or with 2.5 μM, 5 μM and 10 μM of DA, respectively, for 1 h at 37°C and were infected with <i>Ctr</i> (MOI-1) for 24 h. Or the cells were first infected with <i>Ctr</i> for 14 h and then treated with DMSO(2) control or with 5 μM, 10 μM and 20 μM DA, respectively, for 10 h. Cells were harvested for WB analysis. (B, C, D) Experiments were performed as (A) and immunostained for <i>Ctr</i> inclusion (Hsp60, green) and DNA (Draq5, blue). (B) The size of the inclusion was measured by ImageJ software. Shown is the mean ± SD of two independent experiments normalized to control. **P<0.01. Error bars show mean ± SD. Magnification is indicated in size bar.</p

Schematic model depicting the role of EphA2 as an invasion and intracellular signaling receptor for <i>C</i>. <i>trachomatis</i>.

<p>(1) <i>Ctr</i>-EB bind and activate EphA2 and enter the cell together with the activated receptor and PI3K recruited to EphA2. (2) <i>Ctr</i>-mediated ERK activation induces EphA2 upregulation. (3) Upregulated EphA2 does not appear at the cell surface. (4) Intracellular EphA2 is associated with the <i>Ctr</i> inclusion which interacts with pPI3K. (5, 6) EphA2-induced signaling cascades (pAkt) are required to inhibit apoptosis and enhance the infection.</p

EphA2 intracellular cytoplasmic domain is crucial for <i>Ctr</i> infection.

<p>(A) HeLa cells were left UN or infected with <i>Ctr</i> (MOI-2) for the indicated period of times and immunoprecipitated using α-EphA2 antibody. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%). (B) Cells were transfected with EphA2-pcDNA3 and p85-PI3K expression plasmid together for 15 h followed by <i>Ctr</i> infection for 24 h. Cells were stained against EphA2 (red), DNA (blue) and pPI3K (green). (C) Schematic representation of full length EphA2 plasmid, EphA2 kinase dead mutant plasmid (EphA2K645R) and EphA2 mutant plasmid missing the entire cytoplasmic domain (EphA2ΔIC). TM: transmembrane domain. (D) The plasmids having full length EphA2 or EphA2ΔIC were transfected and these cells were infected with <i>Ctr</i> for 20 h. Cells were fixed and stained against α-EphA2 antibody (red) and <i>Ctr</i> using α-Hsp60 (green). (E) Arrows were marked to illustrate the difference between the size of the inclusion of untransfected (white arrows) and transfected (red arrows) cells. (F) Size of the inclusion for (E) was determined by ImageJ software. Empty-plasmid transfected cells act as a control. Shown is the mean ± SD of two independent experiments normalized to empty-plasmid transfected cells. *P<0.05, **P<0.01. Error bars show mean ± SD. (G) Cells after transfection of the constructs (each 1.5 μg/ml) indicated above the lanes followed by infection (MOI-1) for 18 h were subjected to WB analysis to analyze the proteins as indicated. EphA2ΔIC was detected by N-terminal EphA2 antibody. Due to the high intensity of total EphA2 and phospho EphA2 from the EphA2-transfected infected cells, the upregulation of endogenous EphA2 cannot be visualized. (B, D, F) Magnification is indicated in size bar.</p

EphA2 overexpression and knockdown influences <i>Ctr</i> infection.

<p>(A) Infectivity assay performed for B) C) D) E) F): The transfected cells infected with <i>Ctr</i> (MOI-1) for 24 h at 35°C was referred as primary infection. The supernatant of the primary infected cells was taken to infect the fresh cells to determine the secondary infection (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004846#sec008" target="_blank">infectivity assay</a>-methods). (B) HeLa cells were left untransfected (control) or transfected with empty pcDNA3 or EphA2-pcDNA3 each 1 μg/ml and then left UN or infected with <i>Ctr</i> for 24 h. Cells were harvested for WB analysis. (C) HeLa cells were untransfected (control) or transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C and then left UN or infected with <i>Ctr</i> for another 24 h. Cells were harvested for WB analysis. (D, E) Primary infected cell lysates of (B) and (C) were taken to infect the fresh cells for secondary infection (Infectivity assay). (F) Number of the inclusion per cell (%) for the infectivity assay was determined by counting the inclusion on 10 independent fields. Shown is the mean ± SD of three independent experiments normalized to control. **P<0.01. Error bars show mean ± SD. (G) Cells were transfected with siRNA against luciferase (siLuci) or EphA2 (siEphA2) or PDGFRβ (siPDGFRβ) or both together for 40 h at 37°C and then infected with <i>Ctr</i>. UN: uninfected cells. Cells were harvested to determine the respective proteins by WB analysis. (H) Size of the inclusion per cell (%) for (F) was determined by ImageJ software by measuring cells out of four microscopic fields. Shown is the mean ± SD of two independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. In all of the above experiments, numbers under the blot represents fold activation for phospho specific proteins with respect to total proteins and fold change for the total proteins.</p