The Role of Human Ephrin Receptor Tyrosine Kinase A2 (EphA2) in Chlamydia trachomatis Infection

Chlamydia trachomatis (Ctr), an obligate intracellular gram negative human pathogen, causes sexually transmitted diseases and acquired blindness in developing countries. The infectious elementary bodies (EB) of Ctr involved in adherence and invasion processes are critical for chlamydial infectivity and subsequent pathogenesis which requires cooperative interaction of several host cell factors. Few receptors have been known for this early event, yet the molecular mechanism of these receptors involvement throughout Ctr infection is not known. Chlamydial inclusion membrane serves as a signaling platform that coordinates Chlamydia-host cell interaction which encouraged me to look for host cell factors that associates with the inclusion membrane, using proteome analysis. The role of these factors in chlamydial replication was analyzed by RNA interference (RNAi) (in collaboration with AG Thomas Meyer). Interestingly, EphrinA2 receptor (EphA2), a cell surface tyrosine kinase receptor, implicated in many cancers, was identified as one of the potential candidates. Due to the presence of EphA2 in the Ctr inclusion proteome data, I investigated the role of EphA2 in Ctr infection. EphA2 was identified as a direct interacting receptor for adherence and entry of C. trachomatis. Pre-incubation of Ctr-EB with recombinant human EphA2, knockdown of EphA2 by siRNA, pretreatment of cells with anti-EphA2 antibodies or the tyrosine kinase inhibitor dasatinib significantly reduced Ctr infection. This marked reduction of Ctr infection was seen with both epithelial and endothelial cells used in this study. Ctr activates EphA2 upon infection and invades the cell together with the activated EphA2 receptor that interacts and activates PI3K survival signal, promoting chlamydial replication. EphA2 upregulation during infection is associated with Ctr inclusion membrane inside the cell and are prevented being translocated to the cell surface. Ephrins are natural ligands for Ephrin receptors that repress the activation of the PI3K/Akt pathway in a process called reverse signaling. Purified Ephrin-A1, a ligand of EphA2, strongly interferes with chlamydial infection and normal development, supporting the central role of these receptors in Chlamydia infection. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Ctr infection induces EphA2 upregulation and is mediated by activation of ERK signaling pathway. Interfering with EphA2 upregulation sensitizes Ctr-infected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-α) suggesting the importance of intracellular EphA2 signaling. Collectively, these results revealed the first Ephrin receptor “EphA2” that functions in promoting chlamydial infection. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism how Chlamydia subverts the host cell and induces apoptosis resistance. By applying the natural ligand Ephrin-A1 and targeting EphA2 offers a promising new approach to interfere with Chlamydia infection. Thus, the work provides the evidence for a host cell surface tyrosine kinase receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate the chlamydial replication

Die Rolle der humanen Rezeptor-Tyrosinkinase EphrinA2 (EphA2) in der Chlamydia trachomatis Infektion

Chlamydia trachomatis (Ctr), an obligate intracellular gram negative human pathogen, causes sexually transmitted diseases and acquired blindness in developing countries. The infectious elementary bodies (EB) of Ctr involved in adherence and invasion processes are critical for chlamydial infectivity and subsequent pathogenesis which requires cooperative interaction of several host cell factors. Few receptors have been known for this early event, yet the molecular mechanism of these receptors involvement throughout Ctr infection is not known. Chlamydial inclusion membrane serves as a signaling platform that coordinates Chlamydia-host cell interaction which encouraged me to look for host cell factors that associates with the inclusion membrane, using proteome analysis. The role of these factors in chlamydial replication was analyzed by RNA interference (RNAi) (in collaboration with AG Thomas Meyer). Interestingly, EphrinA2 receptor (EphA2), a cell surface tyrosine kinase receptor, implicated in many cancers, was identified as one of the potential candidates. Due to the presence of EphA2 in the Ctr inclusion proteome data, I investigated the role of EphA2 in Ctr infection. EphA2 was identified as a direct interacting receptor for adherence and entry of C. trachomatis. Pre-incubation of Ctr-EB with recombinant human EphA2, knockdown of EphA2 by siRNA, pretreatment of cells with anti-EphA2 antibodies or the tyrosine kinase inhibitor dasatinib significantly reduced Ctr infection. This marked reduction of Ctr infection was seen with both epithelial and endothelial cells used in this study. Ctr activates EphA2 upon infection and invades the cell together with the activated EphA2 receptor that interacts and activates PI3K survival signal, promoting chlamydial replication. EphA2 upregulation during infection is associated with Ctr inclusion membrane inside the cell and are prevented being translocated to the cell surface. Ephrins are natural ligands for Ephrin receptors that repress the activation of the PI3K/Akt pathway in a process called reverse signaling. Purified Ephrin-A1, a ligand of EphA2, strongly interferes with chlamydial infection and normal development, supporting the central role of these receptors in Chlamydia infection. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Ctr infection induces EphA2 upregulation and is mediated by activation of ERK signaling pathway. Interfering with EphA2 upregulation sensitizes Ctr-infected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-α) suggesting the importance of intracellular EphA2 signaling. Collectively, these results revealed the first Ephrin receptor “EphA2” that functions in promoting chlamydial infection. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism how Chlamydia subverts the host cell and induces apoptosis resistance. By applying the natural ligand Ephrin-A1 and targeting EphA2 offers a promising new approach to interfere with Chlamydia infection. Thus, the work provides the evidence for a host cell surface tyrosine kinase receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate the chlamydial replication.Chlamydia trachomatis (Ctr) ist ein obligat intrazellulär lebendes Gram negatives Bakterium, das Geschlechtskrankheiten verursachen kann. In Entwicklungsländern führt es zudem häufig zu erworbener Blindheit. Die infektiösen Elementarkörper (EB) sind für die Anheftung an die Wirtszelle sowie die Aufnahme von Ctr in die Wirtzelle verantwortlich. Dies ist ein wichtiger Schritt, da nur so die sich anschließende Krankheitsentwicklung stattfinden kann. Diese ist auch abhängig vom engen Zusammenspiel der Ctr Proteine mit den Wirtszellfaktoren. Obgleich dieser Schritt so wichtig ist, wurden erst wenige Wirtszellrezeptoren gefunden und welche Rolle diese Rezeptoren im weiteren Verlauf der Infektion spielen, ist noch nicht richtig verstanden. Die chlamydiale Inklusionsmembran fungiert als Signalplattform, die das Zusammenspiel von Chlamydien und Wirtszelle koordiniert. In dieser Arbeit wurden die Wirtszellproteine, die an der Inklusionsmembran lokalisiert sind, mit Hilfe einer Proteomanalyse identifiziert. Anschließend wurde die Rolle dieser Proteine bei der Chlamydienvermehrung in einem RNAi screen untersucht (in Zusammenarbeit mit der AG Thomas Meyer). Hier wurde überraschenderweise der EphrinA2 Rezeptor, eine sich auf der Oberfläche der Zellen befindliche Rezeptor Tyrosin Kinase, die vor allem mit Krebs in Verbindung gebracht wird, als ein potentieller Kandidat identifiziert. Da die Proteomdaten gezeigt haben, dass EphrinA2 an der Inklusionsmembran lokalisiert ist, wurde die Rolle von EphrinA2 während der Ctr Infektion hier näher untersucht. Es konnte gezeigt werden, dass EphrinA2 ein direkter Rezeptor für Ctr ist, der sowohl die Adhärenz als auch die Aufnahme von Ctr in die Wirtszelle bewerkstelligt. Vorinkubation von Ctr- EB mit rekombinantem menschlichen EphrinA2, das herunterregulieren von EphrinA2 mit Hilfe einer siRNA oder das Vorinkubieren der menschlichen Zelle mit Antikörpern gegen EphrinA2 oder dem Tyrosinkinase Inhibitor Dasatinib, reduzierten die Ctr Infektion signifikant. Diese drastische Reduktion der Ctr Infektion wurde sowohl in Epithelzellen als auch in Endothelzellen beobachtet. Ctr aktiviert EphrinA2 während der Infektion und invadiert die Wirtszelle zusammen mit dem aktivierten Rezeptor, dieser interagiert mit dem aktivierten PI3K Überlebenssignal, was die Replikation der Chlamydien ermöglicht. An der Inklusionsmembran akkumuliert EphrinA2, da der Transport von neuem Rezeptor zur Zellmembran unterbunden ist. Ephrine sind die natürlichen Liganden der Ephrinrezeptoren, sie unterdrücken die Aktivierung des PI3K/Akt Signalweges in einem Prozess, der reverse Signalübertragung genannt wird. Aufgereinigtes Ephrin-A1, ein Ligand des EphrinA2 Rezeptors, verhindert eine normale Chlamydieninfektion, was eine zentrale Rolle dieses Rezeptors weiterhin bestätigt. Die Überexpression von EphrinA2, erhöhte die PI3K Aktivierung und Ctr Infektion. Dies war nicht der Fall, wenn eine Mutante, der die intrazelluläre Domäne fehlt, überexprimiert wurde. Eine Ctr Infektion induziert die Hochregulierung von EphrinA2, welche durch die Aktivierung des ERK Signalwegs bewerkstelligt wird. Wenn die Hochregulierung von EphrinA2 verhindert wird, werden Ctr infizierte Zellen sensitiver für Apoptose induziert durch tumor necrosis factor-alpha (TNF-α), was ein weiter Hinweis für die Bedeutung der intrazellulären EphrinA2 Signalübermittlung ist. Insgesamt haben diese Ergebnisse den ersten Ephrin Rezeptor "EphA2" offenbart, der in der Förderung chlamydialer Infektionen fungiert. Hinzu kommt, dass die Bindung eines Oberflächenrezeptors an die Inklusionsmembran ein neuer Mechanismus ist, die Wirtszelle zu verändern und eine Apoptoseresistenz in der Zelle zu induzieren. Die Zugabe des natürlichen Liganden Ephrin-A1 eröffnet eine neue vielversprechende Möglichkeit Chlamydieninfektionen zu bekämpfen. Daher liefert diese Arbeit erste Hinweise, das eine Wirtszelltyrosinkinase, die sich an der Zelloberfläche befindet, notwendig ist für die Invasion und die intrazelluläre Signalübermittlung, welche für die chlamydiale Replikation notwendig ist, essentiell ist

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance

Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. IMPORTANCE Chlamydia trachomatis is a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections. C. trachomatis can develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal "coats" or "cages," whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell

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>-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>-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

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

Purification and proteomics of pathogen-modified vacuoles and membranes

Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation