TY - JOUR A1 - Steiner, Thomas A1 - Zachary, Marie A1 - Bauer, Susanne A1 - Müller, Martin J. A1 - Krischke, Markus A1 - Radziej, Sandra A1 - Klepsch, Maximilian A1 - Huettel, Bruno A1 - Eisenreich, Wolfgang A1 - Rudel, Thomas A1 - Beier, Dagmar T1 - Central Role of Sibling Small RNAs NgncR_162 and NgncR_163 in Main Metabolic Pathways of Neisseria gonorrhoeae JF - mBio N2 - Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C\(_1\)) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus. KW - sRNA KW - Neisseria gonorrhoeae KW - posttranscriptional regulation KW - amino acid transporter KW - bipartite metabolism Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313323 VL - 14 ER - TY - JOUR A1 - Herbert, Saskia-Laureen A1 - Fick, Andrea A1 - Heydarian, Motaharehsadat A1 - Metzger, Marco A1 - Wöckel, Achim A1 - Rudel, Thomas A1 - Kozjak-Pavlovic, Vera A1 - Wulff, Christine T1 - Establishment of the SIS scaffold-based 3D model of human peritoneum for studying the dissemination of ovarian cancer JF - Journal of Tissue Engineering N2 - Ovarian cancer is the second most common gynecological malignancy in women. More than 70% of the cases are diagnosed at the advanced stage, presenting as primary peritoneal metastasis, which results in a poor 5-year survival rate of around 40%. Mechanisms of peritoneal metastasis, including adhesion, migration, and invasion, are still not completely understood and therapeutic options are extremely limited. Therefore, there is a strong requirement for a 3D model mimicking the in vivo situation. In this study, we describe the establishment of a 3D tissue model of the human peritoneum based on decellularized porcine small intestinal submucosa (SIS) scaffold. The SIS scaffold was populated with human dermal fibroblasts, with LP-9 cells on the apical side representing the peritoneal mesothelium, while HUVEC cells on the basal side of the scaffold served to mimic the endothelial cell layer. Functional analyses of the transepithelial electrical resistance (TEER) and the FITC-dextran assay indicated the high barrier integrity of our model. The histological, immunohistochemical, and ultrastructural analyses showed the main characteristics of the site of adhesion. Initial experiments using the SKOV-3 cell line as representative for ovarian carcinoma demonstrated the usefulness of our models for studying tumor cell adhesion, as well as the effect of tumor cells on endothelial cell-to-cell contacts. Taken together, our data show that the novel peritoneal 3D tissue model is a promising tool for studying the peritoneal dissemination of ovarian cancer. KW - ovarian cancer KW - 3D tissue model KW - co-culture KW - peritoneal metastasis KW - cancer dissemination Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301311 SN - 2041-7314 VL - 13 ER - TY - JOUR A1 - Vollmuth, Nadine A1 - Schlicker, Lisa A1 - Guo, Yongxia A1 - Hovhannisyan, Pargev A1 - Janaki-Raman, Sudha A1 - Kurmasheva, Naziia A1 - Schmitz, Werner A1 - Schulze, Almut A1 - Stelzner, Kathrin A1 - Rajeeve, Karthika A1 - Rudel, Thomas T1 - c-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis JF - eLife N2 - Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3β axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism. KW - Chlamydia trachomatis Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301385 VL - 11 ER - TY - JOUR A1 - Peters, Simon A1 - Fohmann, Ingo A1 - Rudel, Thomas A1 - Schubert-Unkmeir, Alexandra T1 - A Comprehensive Review on the Interplay between Neisseria spp. and Host Sphingolipid Metabolites JF - Cells N2 - Sphingolipids represent a class of structural related lipids involved in membrane biology and various cellular processes including cell growth, apoptosis, inflammation and migration. Over the past decade, sphingolipids have become the focus of intensive studies regarding their involvement in infectious diseases. Pathogens can manipulate the sphingolipid metabolism resulting in cell membrane reorganization and receptor recruitment to facilitate their entry. They may recruit specific host sphingolipid metabolites to establish a favorable niche for intracellular survival and proliferation. In contrast, some sphingolipid metabolites can also act as a first line defense against bacteria based on their antimicrobial activity. In this review, we will focus on the strategies employed by pathogenic Neisseria spp. to modulate the sphingolipid metabolism and hijack the sphingolipid balance in the host to promote cellular colonization, invasion and intracellular survival. Novel techniques and innovative approaches will be highlighted that allow imaging of sphingolipid derivatives in the host cell as well as in the pathogen. KW - sphingolipids KW - host–pathogen interaction KW - Neisseria meningitidis KW - Neisseria gonorrhoeae Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250203 SN - 2073-4409 VL - 10 IS - 11 ER - TY - JOUR A1 - Kunz, Tobias C. A1 - Rühling, Marcel A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kozjak-Pavlovic, Vera A1 - Rudel, Thomas A1 - Fraunholz, Martin T1 - The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy JF - Frontiers in Cellular and Infection Microbiology N2 - Expansion Microscopy (ExM) is a novel tool improving the resolution of fluorescence microscopy by linking the sample into a hydrogel that gets physically expanded in water. Previously, we have used ExM to visualize the intracellular Gram-negative pathogens Chlamydia trachomatis, Simkania negevensis, and Neisseria gonorrhoeae. Gram-positive bacteria have a rigid and thick cell wall that impedes classic expansion strategies. Here we developed an approach, which included a series of enzymatic treatments resulting in isotropic 4× expansion of the Gram-positive pathogen Staphylococcus aureus. We further demonstrate the suitability of the technique for imaging of planktonic bacteria as well as endocytosed, intracellular bacteria at a spatial resolution of approximately 60 nm with conventional confocal laser scanning microscopy. KW - high-resolution imaging KW - endosomes KW - autophagosomes KW - host-pathogen interaction KW - expansion microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232292 SN - 2235-2988 VL - 11 ER - TY - JOUR A1 - Stelzner, Kathrin A1 - Boyny, Aziza A1 - Hertlein, Tobias A1 - Sroka, Aneta A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kessie, David A1 - Mehling, Helene A1 - Potempa, Jan A1 - Ohlsen, Knut A1 - Fraunholz, Martin J. A1 - Rudel, Thomas T1 - Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A. KW - Staphylococcus aureus KW - Staphylococcal infection KW - host cells KW - HeLa cells KW - cytotoxicity KW - intracellular pathogens KW - apoptosis KW - epithelial cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263908 VL - 17 IS - 9 ER - TY - JOUR A1 - Eisenreich, Wolfgang A1 - Rudel, Thomas A1 - Heesemann, Jürgen A1 - Goebel, Werner T1 - Persistence of Intracellular Bacterial Pathogens—With a Focus on the Metabolic Perspective JF - Frontiers in Cellular and Infection Microbiology N2 - Persistence has evolved as a potent survival strategy to overcome adverse environmental conditions. This capability is common to almost all bacteria, including all human bacterial pathogens and likely connected to chronic infections caused by some of these pathogens. Although the majority of a bacterial cell population will be killed by the particular stressors, like antibiotics, oxygen and nitrogen radicals, nutrient starvation and others, a varying subpopulation (termed persisters) will withstand the stress situation and will be able to revive once the stress is removed. Several factors and pathways have been identified in the past that apparently favor the formation of persistence, such as various toxin/antitoxin modules or stringent response together with the alarmone (p)ppGpp. However, persistence can occur stochastically in few cells even of stress-free bacterial populations. Growth of these cells could then be induced by the stress conditions. In this review, we focus on the persister formation of human intracellular bacterial pathogens, some of which belong to the most successful persister producers but lack some or even all of the assumed persistence-triggering factors and pathways. We propose a mechanism for the persister formation of these bacterial pathogens which is based on their specific intracellular bipartite metabolism. We postulate that this mode of metabolism ultimately leads, under certain starvation conditions, to the stalling of DNA replication initiation which may be causative for the persister state. KW - persistence KW - mechanisms of persister formation KW - intracellular bacterial pathogens KW - stress conditions KW - ATP-DnaA complex KW - DNA replication initiation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222348 SN - 2235-2988 VL - 10 ER - TY - JOUR A1 - Heydarian, Motaharehsadat A1 - Schweinlin, Matthias A1 - Schwarz, Thomas A1 - Rawal, Ravisha A1 - Walles, Heike A1 - Metzger, Marco A1 - Rudel, Thomas A1 - Kozjak-Pavlovic, Vera T1 - Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity JF - Journal of Tissue Engineering N2 - Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment. KW - Triple co-culture KW - biomimetic 3D tissue model KW - Neisseria gonorrhoeae KW - perfusion-based bioreactor system KW - neutrophil transmigration Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259032 VL - 12 ER - TY - JOUR A1 - Götz, Ralph A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Solger, Franziska A1 - Schlegel, Jan A1 - Seibel, Jürgen A1 - Kozjak-Pavlovic, Vera A1 - Rudel, Thomas A1 - Sauer, Markus T1 - Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy JF - Nature Communications N2 - Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 +/- 7.7nm. Imaging of lipid bilayers using light microscopy is challenging. Here the authors label cells using a short chain click-compatible ceramide to visualize mammalian and bacterial membranes with expansion microscopy. KW - nanoscale imaging KW - bacterial infection KW - sphingolipid expansion microscopy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231248 VL - 11 ER - TY - JOUR A1 - Solger, Franziska A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Paprotka, Kerstin A1 - Pfister, Pauline A1 - Hagen, Franziska A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Seibel, Jürgen A1 - Rudel, Thomas T1 - A Role of Sphingosine in the Intracellular Survival of Neisseria gonorrhoeae JF - Frontiers in Cellular and Infection Microbiology N2 - Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells. KW - sphingosine KW - sphingolipids KW - sphingosine kinases KW - invasion KW - survival KW - click chemistry Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204111 SN - 2235-2988 VL - 10 ER -