TY - JOUR A1 - Groma, Michaela A1 - Horst, Sarah A. A1 - Das, Sudip A1 - Huettel, Bruno A1 - Klepsch, Maximilian A1 - Rudel, Thomas A1 - Medina, Eva A1 - Fraunholz, Martin T1 - Identification of a Novel LysR-Type Transcriptional Regulator in Staphylococcus aureus That Is Crucial for Secondary Tissue Colonization during Metastatic Bloodstream Infection JF - mbio N2 - Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection in secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used an S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used an S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched-chain amino acid biosynthesis, a methionine sulfoxide reductase, and a copper transporter as well as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, is induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enables a rapid adaptation of S. aureus to the changing host microenvironment. IMPORTANCE Staphylococcus aureus is an important pathogen that can disseminate via the bloodstream and establish metastatic infections in distant organs. To achieve a better understanding of the bacterial factors facilitating the development of these metastatic infections, we used in this study a Staphylococcus aureus transposon mutant library in a murine model of intravenous infection, where bacteria first colonize the liver as the primary infection site and subsequently progress to secondary sites such as the kidney and bones. We identified a novel LysR-type transcriptional regulator (LTTR), which was specifically required by S. aureus for efficient colonization of secondary organs. We also determined the transcriptional activation as well as the regulon of LTTR, which suggests that this regulator is involved in the metabolic adaptation of S. aureus to the host microenvironment found in secondary infection sites. KW - Staphylococcus aureus KW - metabolic adaptation KW - secondary site infection KW - transcriptional regulation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230473 VL - 11 IS - 4 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 - TY - JOUR A1 - Blättner, Sebastian A1 - Das, Sudip A1 - Paprotka, Kerstin A1 - Eilers, Ursula A1 - Krischke, Markus A1 - Kretschmer, Dorothee A1 - Remmele, Christian W. A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Schuelein-Voelk, Christina A1 - Hertlein, Tobias A1 - Mueller, Martin J. A1 - Huettel, Bruno A1 - Reinhardt, Richard A1 - Ohlsen, Knut A1 - Rudel, Thomas A1 - Fraunholz, Martin J. T1 - Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes JF - PLoS Pathogens N2 - Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. KW - cell death KW - cytotoxicity KW - Staphylococcus aureus KW - host cells KW - neutrophils KW - macrophages KW - transposable elements KW - epithelial cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180380 VL - 12 IS - 9 ER - TY - JOUR A1 - Eisenreich, Wolfgang A1 - Rudel, Thomas A1 - Heesemann, Jürgen A1 - Goebel, Werner T1 - How viral and intracellular bacterial pathogens reprogram the metabolism of host cells to allow their intracellular replication JF - Frontiers in Cellular and Infection Microbiology N2 - Viruses and intracellular bacterial pathogens (IBPs) have in common the need of suitable host cells for efficient replication and proliferation during infection. In human infections, the cell types which both groups of pathogens are using as hosts are indeed quite similar and include phagocytic immune cells, especially monocytes/macrophages (MOs/MPs) and dendritic cells (DCs), as well as nonprofessional phagocytes, like epithelial cells, fibroblasts and endothelial cells. These terminally differentiated cells are normally in a metabolically quiescent state when they are encountered by these pathogens during infection. This metabolic state of the host cells does not meet the extensive need for nutrients required for efficient intracellular replication of viruses and especially IBPs which, in contrast to the viral pathogens, have to perform their own specific intracellular metabolism to survive and efficiently replicate in their host cell niches. For this goal, viruses and IBPs have to reprogram the host cell metabolism in a pathogen-specific manner to increase the supply of nutrients, energy, and metabolites which have to be provided to the pathogen to allow its replication. In viral infections, this appears to be often achieved by the interaction of specific viral factors with central metabolic regulators, including oncogenes and tumor suppressors, or by the introduction of virus-specific oncogenes. Less is so far known on the mechanisms leading to metabolic reprogramming of the host cell by IBPs. However, the still scant data suggest that similar mechanisms may also determine the reprogramming of the host cell metabolism in IBP infections. In this review, we summarize and compare the present knowledge on this important, yet still poorly understood aspect of pathogenesis of human viral and especially IBP infections. KW - metabolic adaptation KW - viruses KW - intracellular bacterial pathogens KW - metabolism of infected and uninfected host cells KW - reprogamming of host cell metabolism Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197188 SN - 2235-2988 VL - 9 ER - TY - JOUR A1 - Auer, Daniela A1 - Hügelschäffer, Sophie D. A1 - Fischer, Annette B. A1 - Rudel, Thomas T1 - The chlamydial deubiquitinase Cdu1 supports recruitment of Golgi vesicles to the inclusion JF - Cellular Microbiology N2 - Chlamydia trachomatis is the main cause of sexually transmitted diseases worldwide. As obligate intracellular bacteria Chlamydia replicate in a membrane bound vacuole called inclusion and acquire nutrients for growth and replication from their host cells. However, like all intracellular bacteria, Chlamydia have to prevent eradication by the host's cell autonomous system. The chlamydial deubiquitinase Cdu1 is secreted into the inclusion membrane, facing the host cell cytosol where it deubiquitinates cellular proteins. Here we show that inactivation of Cdu1 causes a growth defect of C. trachomatis in primary cells. Moreover, ubiquitin and several autophagy receptors are recruited to the inclusion membrane of Cdu1‐deficient Chlamydia . Interestingly, the growth defect of cdu1 mutants is not rescued when autophagy is prevented. We find reduced recruitment of Golgi vesicles to the inclusion of Cdu1 mutants indicating that vesicular trafficking is altered in bacteria without active deubiquitinase (DUB). Our work elucidates an important role of Cdu1 in the functional preservation of the chlamydial inclusion surface. KW - autophagy KW - Cdu1 KW - ChlaDUB1 KW - Chlamydia trachomatis KW - DUB KW - Golgi KW - xenophagy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-208675 VL - 22 IS - 5 ER - TY - JOUR A1 - Prusty, Bhupesh K. A1 - Chowdhury, Suvagata R. A1 - Gulve, Nitish A1 - Rudel, Thomas T1 - Peptidase Inhibitor 15 (PI15) Regulates Chlamydial CPAF Activity JF - Frontiers in Cellular and Infection Microbiology N2 - 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. KW - chlamydia KW - CPAF activation KW - peptidase inhibitor PI15 KW - chlamydial inclusion KW - chlamydia serine proteases Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-196918 SN - 2235-2988 VL - 8 IS - 183 ER - TY - JOUR A1 - Nadella, Vinod A1 - Mohanty, Aparna A1 - Sharma, Lalita A1 - Yellaboina, Sailu A1 - Mollenkopf, Hans-Joachim A1 - Mazumdar, Varadendra Balaji A1 - Palaparthi, Ramesh A1 - Mylavarapu, Madhavi B. A1 - Maurya, Radheshyam A1 - Kurukuti, Sreenivasulu A1 - Rudel, Thomas A1 - Prakash, Hridayesh T1 - Inhibitors of Apoptosis Protein Antagonists (Smac Mimetic Compounds) Control Polarization of Macrophages during Microbial Challenge and Sterile Inflammatory Responses JF - Frontiers in Immunology N2 - Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity. KW - apoptosis KW - macrophages immunobiology KW - inflammation mediators KW - polarization KW - infection KW - hypothalamus Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197484 SN - 1664-3224 VL - 8 IS - 1792 ER - TY - JOUR A1 - Kunz, Tobias C. A1 - Götz, Ralph A1 - Sauer, Markus A1 - Rudel, Thomas T1 - Detection of chlamydia developmental forms and secreted effectors by expansion microscopy JF - Frontiers in Cellular and Infection Microbiology N2 - 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. KW - expansion microscopy KW - chlamydia KW - secreted effectors KW - developmental forms KW - superresolution KW - imaging Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195716 SN - 2235-2988 VL - 9 IS - 276 ER - TY - JOUR A1 - Heydarian, Motaharehsadat A1 - Yang, Tao A1 - Schweinlin, Matthias A1 - Steinke, Maria A1 - Walles, Heike A1 - Rudel, Thomas A1 - Kozjak-Pavlovic, Vera T1 - Biomimetic human tissue model for long-term study of Neisseria gonorrhoeae infection JF - Frontiers in Microbiology N2 - Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions. KW - 3D tissue model KW - small intestinal submucosa scaffold KW - co-culture KW - infection KW - Neisseria gonorrhoeae Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197912 SN - 1664-302X VL - 10 IS - 1740 ER - TY - JOUR A1 - Yang, Manli A1 - Rajeeve, Karthika A1 - Rudel, Thomas A1 - Dandekar, Thomas T1 - Comprehensive Flux Modeling of Chlamydia trachomatis Proteome and qRT-PCR Data Indicate Biphasic Metabolic Differences Between Elementary Bodies and Reticulate Bodies During Infection JF - Frontiers in Microbiology N2 - Metabolic adaptation to the host cell is important for obligate intracellular pathogens such as Chlamydia trachomatis (Ct). Here we infer the flux differences for Ct from proteome and qRT-PCR data by comprehensive pathway modeling. We compare the comparatively inert infectious elementary body (EB) and the active replicative reticulate body (RB) systematically using a genome-scale metabolic model with 321 metabolites and 277 reactions. This did yield 84 extreme pathways based on a published proteomics dataset at three different time points of infection. Validation of predictions was done by quantitative RT-PCR of enzyme mRNA expression at three time points. Ct’s major active pathways are glycolysis, gluconeogenesis, glycerol-phospholipid (GPL) biosynthesis (support from host acetyl-CoA) and pentose phosphate pathway (PPP), while its incomplete TCA and fatty acid biosynthesis are less active. The modeled metabolic pathways are much more active in RB than in EB. Our in silico model suggests that EB and RB utilize folate to generate NAD(P)H using independent pathways. The only low metabolic flux inferred for EB involves mainly carbohydrate metabolism. RB utilizes energy -rich compounds to generate ATP in nucleic acid metabolism. Validation data for the modeling include proteomics experiments (model basis) as well as qRT-PCR confirmation of selected metabolic enzyme mRNA expression differences. The metabolic modeling is made fully available here. Its detailed insights and models on Ct metabolic adaptations during infection are a useful modeling basis for future studies. KW - metabolic modeling KW - metabolic flux KW - infection biology KW - elementary body KW - reticulate body KW - Chlamydia trachomatis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189434 SN - 1664-302X VL - 10 IS - 2350 ER -