@phdthesis{Subbarayal2015, author = {Subbarayal, Prema}, title = {The role of human Ephrin receptor tyrosine kinase A2 (EphA2) in Chlamydia trachomatis infection}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114778}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {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.}, subject = {Chlamydia trachomatis}, language = {en} } @phdthesis{Vollmuth2021, author = {Vollmuth, Nadine}, title = {Role of the proto-oncogene c-Myc in the development of Chlamydia trachomatis}, doi = {10.25972/OPUS-20365}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203655}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Chlamydia trachomatis, an obligate intracellular human pathogen, is the world's leading cause of infection related blindness and the most common, bacterial sexually transmitted disease. In order to establish an optimal replicative niche, the pathogen extensively interferes with the physiology of the host cell. Chlamydia switches in its complex developmental cycle between the infectious non-replicative elementary bodies (EBs) and the non-infectious replicative reticulate bodies (RBs). The transformation to RBs, shortly after entering a host cell, is a crucial process in infection to start chlamydial replication. Currently it is unknown how the transition from EBs to RBs is initiated. In this thesis, we could show that, in an axenic media approach, L glutamine uptake by the pathogen is crucial to initiate the EB to RB transition. L-glutamine is converted to amino acids which are used by the bacteria to synthesize peptidoglycan. Peptidoglycan inturn is believed to function in separating dividing Chlamydia. The glutamine metabolism is reprogrammed in infected cells in a c-Myc-dependent manner, in order to accomplish the increased requirement for L-glutamine. Upon a chlamydial infection, the proto-oncogene c-Myc gets upregulated to promote host cell glutaminolysis via glutaminase GLS1 and the L-glutamine transporter SLC1A5/ASCT2. Interference with this metabolic reprogramming leads to limited growth of C. trachomatis. Besides the active infection, Chlamydia can persist over a long period of time within the host cell whereby chronic and recurrent infections establish. C. trachomatis acquire a persistent state during an immune attack in response to elevated interferon-γ (IFN-γ) levels. It has been shown that IFN-γ activates the catabolic depletion of L-tryptophan via indoleamine 2,3-dioxygenase (IDO), resulting in the formation of non-infectious atypical chlamydial forms. In this thesis, we could show that IFN-γ depletes the key metabolic regulator c-Myc, which has been demonstrated to be a prerequisite for chlamydial development and growth, in a STAT1-dependent manner. Moreover, metabolic analyses revealed that the pathogen de routs the host cell TCA cycle to enrich pyrimidine biosynthesis. Supplementing pyrimidines or a-ketoglutarate helps the bacteria to partially overcome the persistent state. Together, the results indicate a central role of c-Myc induced host glutamine metabolism reprogramming and L-glutamine for the development of C. trachomatis, which may provide a basis for anti-infectious strategies. Furthermore, they challenge the longstanding hypothesis of L-tryptophan shortage as the sole reason for IFN-γ induced persistence and suggest a pivotal role of c-Myc in the control of the C. trachomatis dormancy.}, language = {en} } @article{PrustyChowdhuryGulveetal.2018, author = {Prusty, Bhupesh K. and Chowdhury, Suvagata R. and Gulve, Nitish and Rudel, Thomas}, title = {Peptidase Inhibitor 15 (PI15) Regulates Chlamydial CPAF Activity}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {8}, journal = {Frontiers in Cellular and Infection Microbiology}, number = {183}, issn = {2235-2988}, doi = {10.3389/fcimb.2018.00183}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-196918}, year = {2018}, abstract = {Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuole called inclusion. How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation. Silencing expression as well as over expression of PI15 affected normal development of Chlamydia. PI15 was transported into the chlamydial inclusion lumen where it co-localized with CPAF aggregates. We show that PI15 binds to the CPAF zymogen and potentially induces CPAF protease activity at low concentrations. However, at high concentrations PI15 inhibits CPAF activity possibly by blocking its protease domain. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia.}, language = {en} } @article{SubbarayalKarunakaranWinkleretal.2015, author = {Subbarayal, Prema and Karunakaran, Karthika and Winkler, Ann-Cathrin and Rother, Marion and Gonzalez, Erik and Meyer, Thomas F. and Rudel, Thomas}, title = {EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis}, series = {PLoS Pathogens}, volume = {11}, journal = {PLoS Pathogens}, number = {4}, doi = {10.1371/journal.ppat.1004846}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125566}, pages = {e1004846}, year = {2015}, abstract = {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.}, language = {en} } @article{KunzGoetzSaueretal.2019, author = {Kunz, Tobias C. and G{\"o}tz, Ralph and Sauer, Markus and Rudel, Thomas}, title = {Detection of chlamydia developmental forms and secreted effectors by expansion microscopy}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {9}, journal = {Frontiers in Cellular and Infection Microbiology}, number = {276}, issn = {2235-2988}, doi = {10.3389/fcimb.2019.00276}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195716}, year = {2019}, abstract = {Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.}, language = {en} } @article{RudelPrustySiegletal.2013, author = {Rudel, Thomas and Prusty, Bhupesh K. and Siegl, Christine and Hauck, Petra and Hain, Johannes and Korhonen, Suvi J. and Hiltunen-Back, Eija and Poulakkainen, Mirja}, title = {Chlamydia trachomatis Infection Induces Replication of Latent HHV-6}, series = {PLoS ONE}, journal = {PLoS ONE}, doi = {10.1371/journal.pone.0061400}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96731}, year = {2013}, abstract = {Human herpesvirus-6 (HHV-6) exists in latent form either as a nuclear episome or integrated into human chromosomes in more than 90\% of healthy individuals without causing clinical symptoms. Immunosuppression and stress conditions can reactivate HHV-6 replication, associated with clinical complications and even death. We have previously shown that co-infection of Chlamydia trachomatis and HHV-6 promotes chlamydial persistence and increases viral uptake in an in vitro cell culture model. Here we investigated C. trachomatis-induced HHV-6 activation in cell lines and fresh blood samples from patients having Chromosomally integrated HHV-6 (CiHHV-6). We observed activation of latent HHV-6 DNA replication in CiHHV-6 cell lines and fresh blood cells without formation of viral particles. Interestingly, we detected HHV-6 DNA in blood as well as cervical swabs from C. trachomatis-infected women. Low virus titers correlated with high C. trachomatis load and vice versa, demonstrating a potentially significant interaction of these pathogens in blood cells and in the cervix of infected patients. Our data suggest a thus far underestimated interference of HHV-6 and C. trachomatis with a likely impact on the disease outcome as consequence of co-infection.}, language = {en} }