Der Einfluss des multifunktionalen extrazellulären Adhäsionsproteins (Eap) von Staphylococcus aureus auf die Morphologie und Funktion von eukaryotischen Zellen

Zusammenfassung Staphylococcus aureus ist eines der bedeutendsten Pathogene unserer Zeit und ein Hauptverursacher chronischer Wundinfektionen. Der Grund, warum dieses, normalerweise kommensalisch auf Schleimhäuten vorkommende, Bakterium einen so erfolgreichen opportunistischen Erreger darstellt, ist sein umfangreiches Arsenal aus zusammen wirkenden Virulenzfaktoren. Diese helfen dem Keim, durch Bindung an Wirtsstrukturen eine Infektion zu etablieren und diese durch Modulation des Immunsystems aufrechtzuerhalten. Einer dieser Faktoren ist das zu der Gruppe der SERAM (engl. "secretable expanded repertoire adhesive molecules") zählende, von S. aureus in das extrazelluläre Milieu sekretierte extrazelluläre Adhäsionsprotein (Eap). In früheren wissenschaftlichen Arbeiten konnte bereits gezeigt werden, dass Eap zum Erhalt tief eindringender, nicht-heilender Wunden beiträgt, indem es die Einwanderung und Proliferation von Immunzellen hemmt und durch diese biochemische Barriere die Bereinigung der sich in der Wunde vermehrenden Bakterien vermindert. Zudem inhibiert das in tiefen Wunden stark durch S. aureus exprimierte Protein die Neovaskularisierung des neu gebildeten Gewebes und unterbindet dadurch den Stofftransport dieses Wundgebiets, was in einem verzögerten Wundschluss resultiert. In meiner Arbeit wird mit Hilfe verschiedener Methoden gezeigt, dass Eap auf drei der wichtigsten, an der Wundheilung beteiligten Zelltypen - Keratinozyten, Endothelzellen und Fibroblasten - einen direkten proliferations- und migrationshemmenden Einfluss zeigt. Dies könnte in nativen Wunden dazu führen, dass es zu einer verminderten Ausbildung einer neuen extrazellulären Matrix durch Fibroblasten, einer eingeschränkten Bildung eines neuen Gefäßsystems durch Endothelzellen und zu einer deutlichen Verzögerung der abschließenden Überhäutung der Wunde durch Keratinozyten kommt. Ein Teil dieses Effekts besteht in der bereits in früheren Studien beschriebenen Unterbrechung der Signaltransduktion über die Mitogen-aktivierte Kinase-Kaskade (MAPKK), welche Zellfuktionen wie Zellvermehrung und Bewegung reguliert. Meine Arbeit zeigt jedoch auch, dass Eap die Morphologie und Adhäsion von Zellen am Substrat beeinflussen kann. Mit Eap behandelte Keratinozyten verändern ihre Form von elongiert-keilförmig zu rund, werden dabei flacher und zeigen eine verminderte, zelluläre Steifigkeit. Diese Änderungen gehen mit einer verminderten zellulären Mobilität und Teilungsfähigkeit einher und tragen so vermutlich zum Erhalt der chronischen Wunden bei. In einem zweiten Teil dieser Arbeit wird ein bis dato vollkommen neuer Aspekt der adhäsiven Fähigkeiten von Eap beschrieben. Es konnte gezeigt werden, dass das stark kationische Adhäsionsprotein in der Lage ist, DNA zu binden und diese zu aggregieren. Eap kann dabei jedoch nur an lineare DNA-Molekühle, nicht jedoch an zirkuläre Strukturen, wie Plasmide binden. Die Spezies aus der die DNA stammt, scheint dabei keine Rolle zu spielen. Ebenso wenig, wie die endständige Konfiguration mit "sticky", oder "blunt"-Enden der DNA-Moleküle. Diese Fähigkeit ist als weitere immunmodulatorische Fähigkeit dieses Proteins anzusehen, die dem Keim hilft, den aus dekondensiertem Chromatin bestehenden NETs (engl. "Neutrophil extrazellular traps") neutrophiler Granulozyten zu entkommen. Zusammen mit der bereits beschriebenen Inhibition der neutrophilen Serinproteasen, ist Eap damit in der Lage, diesen bedeutenden angeborenen Immunmechanismus auf verschiedenen Ebenen zu adressieren und S. aureus damit den Erhalt der Infektion zu ermöglichen. In einem dritten Forschungsschwerpunkt wird gezeigt, ob die zuvor beschriebenen anti-proliferativen und migrationshemmenden Eigenschaften von Eap das Protein zu einem potentiellen Kandidaten für ein Krebstherapeutikum machen. Mit der Blase als von außen zugängliches System, das keinen intravenösen Zugang erfordert, wurde hier im Hinblick auf eine zukünftige Anwendbarkeit das Blasenkarzinom gewählt. Urothelkarzinome stellen die fünfthäufigste Krebsart der westlichen Welt dar und betreffen in über 90% aller Fälle die Blase. Es entspricht der etablierten Vorgehensweise, nach der operativen Tumorresektion die Blase mit Chemotherapeutika zu spülen. Dabei wird davon ausgegangen, dass in der Blase zirkulierende Tumorzellen innerhalb der ersten drei post-operativen Tage dazu in der Lage sind, an das beschädigte Blasenepithel zu re-adhärieren und so neue Tumorherde zu entfachen. Da die etablierten Therapeutika stark variierende Effektivitäten aufweisen, ist die Entwicklung neuer, effektiverer Agenzien nach wie vor von größter Bedeutung. Die von mir gewonnen Erkenntnisse zu den Blasenkrebslinien RT-112 und 5637 zeigen, dass Eap auch bei diesen Zellen die Fähigkeit zur Migration und Proliferation beeinträchtigt. Mit Hilfe der Rasterkraftmikroskopie (AFM, "atomic force microscope") konnten für RT-112 Zellen zudem ähnliche, morphologische Effekte wie bei Keratinozyten beobachtet werden. Zusätzlich konnte mittels eines Antikörper-basierten Immunfluoreszenzassays (AlphaLisa) eine Eap induzierte Inhibition der MAPKK und einiger nachfolgender Zellfunktionen (Proliferation, Mobilität, Morphologie) in RT-112 Zellen nachgewiesen werden, wie sie bereits für Keratinozyten und Endothelzellen bekannt war. Die hier gewonnenen Erkenntnisse betonen erneut die Multifunktionalität des initial als Adhäsin beschriebenen Immunmodulators Eap. Seine Eigenschaften, die Teilung und Mobilität von Zellen verschiedener Abstammung zu beeinflussen, machen es zudem zu einem vielversprechenden Kandidaten zur Etablierung neuer antikanzerogener Therapien.Summary Staphylococcus aureus is one of the most important pathogens of our time and a major cause of chronic wound infections. One reason why this commensal bacterium, which usually persists on mucous membranes, represents such a successful opportunistic pathogen, is its extensive armamentarium of virulence factors. These factors contribute to the establishment of infections by binding to host structures and to its perpetuation by modulating the host immune system. One of these factors is the "extracellular adherence protein" (Eap), which belongs to the secreted expanded repertoire adhesive molecules (SERAM). Earlier work showed that Eap contributes to the maintenance of wounds by inhibiting the immigration and proliferation of immune cells, thereby reducing the clearance of bacteria in the wounded tissue. This protein, which is strongly expressed in deep-seated wounds, also inhibits the neovascularization of the newly formed tissue and thereby prevents the transport of nutrients into the wound area, resulting in a delayed wound closure. My thesis shows with the aid of different assays, that Eap has a direct proliferation- and migration-inhibiting influence on three of the most important cell types which are related to wound healing - keratinocytes, endothelial cells and fibroblasts. We hypothetize that in native wounds, these effects could have inhibiting impacts on the formation of a new extracellular matrix by fibroblasts, the formation of a new vascular system by endothelial cells and the final coverage of the wound by keratinocytes. At least part of this effect is mediated by the Eap-driven attenuation of the "mitogen-activated kinase cascade" (MAPKK) signal transduction pathway, which is known to regulate cell functions such as proliferation and movement. The present work also shows that Eap affects the morphology and adhesion properties of host cells to its substrate. Eap-treated keratinocytes change their shape from elongated-wedge-shaped to round cells, and this morphological change is accompanied by a flattening of the cell and a diminished cellular stiffness. These changes probably contribute to the marked reduction of cellular mobility and the ability to divide, thereby contributing to the preservation of chronic wounds. In a second part of my thesis, a completely new aspect of Eap's adhesive capabilities is described. It was shown that the cationic protein is able to bind to DNA and to aggregate it. Eap thereby only binds to linear DNA-molecules, but not to circular structures like plasmids. The species origin of the DNA, as well as the terminal configuration like "sticky", or "blunt" does not seem to matter in this background. This ability is considered to be a new immunomodulatory mechanism that contributes to the bacteriums ability to escape from entrappment by "neutrophil extracellular traps" (NETs), consisting of decondensed chromatin that is excreted by neutrophil granulocytes upon stimulation by a number of internal and external signals. This observation demonstrates that Eap, together with the previously described inhibition of neutrophil serine proteases, is able to modulate this important innate immune mechanism by various means, and thereby enables S. aureus to maintain the infection. A third focus of my thesis was to verify, if the afore mentioned anti-proliferative and anti-migration properties of Eap qualify this protein to become a potential candidate for new cancer therapeutic agents. With the bladder as an externally accessible system that does not require intravenous access, bladder cancer was chosen as a suitable target. Urothelial carcinomas are the fifth most common cancer forms of the western hemisphere, and affect the bladder in over 90% of all cases. The currently used operative procedure includes rinsing of the bladder with chemotherapeutic agents after surgical tumor resection. This procedure is based on the assumption that the resection process induces a certain release of tumor cells, which are subsequently circulating in the bladder and may re-adhere to the damaged bladder epithelium within the first three post-operative days leading in new tumor foci. Since all currently established therapeutics are afflicted by largely varying efficiencies, the development of new agents and/or adjuvant strategies is of high-ranking importance. My findings obtained for the bladder cancer lines RT-112 and 5637 demonstrate that Eap also impairs the migration and proliferation abilities of these cancer cell types. Atomic force microscope (AFM) studies confirmed that Eap also exerts a morphological effect on RT-112 cells similar to the one seen with keratinocytes. An antibody-based immunofluorescence assay (AlphaLisa) allowed to detect an Eap-dependent inhibition of MAPKK and all its subsequent functions, in line with previous findings made with keratinocytes and endothelial cells. The findings of my thesis emphasize the role of Eap as immunomodulator, which was initially described as an adhesin. Its ability to influence the proliferation and mobility of various cell types including tumor cells support the idea that Eap might constitute a promising candidate for the establishment of new anti-cancer therapies

Human infections caused by Staphylococcus argenteus in Germany: genetic characterisation and clinical implications of novel species designation

We report a series of Staphylococcus argenteus infections from Saarland, Germany. Travel histories were unremarkable for extra-European sojourns, indicating an autochthonous transmission mode. Multilocus sequence typing revealed that all isolates were members of the clonal complex CC2250. In only one case, guideline-adherent treatment with an isoxazolyl penicillin was prescribed. Our report illustrates the perils of novel species designations, which may lead to misconceptions and suboptimal treatment choices among clinicians

Bacterial MgrB peptide activates chemoreceptor Fpr3 in mouse accessory olfactory system and drives avoidance behaviour

Innate immune chemoreceptors of the formyl peptide receptor (Fpr) family are expressed by vomeronasal sensory neurons (VSNs) in the accessory olfactory system. Their biological function and coding mechanisms remain unknown. We show that mouse Fpr3 (Fpr-rs1) recognizes the core peptide motif f-MKKFRW that is predominantly present in the signal sequence of the bacterial protein MgrB, a highly conserved regulator of virulence and antibiotic resistance in Enterobacteriaceae. MgrB peptide can be produced and secreted by bacteria, and is selectively recognized by a subset of VSNs. Exposure to the peptide also stimulates VSNs in freely behaving mice and drives innate avoidance. Our data shows that Fpr3 is required for neuronal detection and avoidance of peptides derived from a conserved master virulence regulator of enteric bacteria

Predictive Role Of Body Composition Parameters In Operable Breast Cancer Patients Treated With Neoadjuvant Chemotherapy.

BACKGROUND: Fat tissue is strongly involved in BC tumorigenesis inducing insulin resistance, chronic inflammation and hormonal changes. Computed tomography (CT) imaging instead of body mass index (BMI) gives a reliable measure of skeletal muscle mass and body fat distribution. The impact of body composition parameters (BCPs) on chemosensitivity is still debated. We examined the associations between BCPs and tumor response to neoadjuvant chemotherapy (NC) in patients treated for operable breast cancer (BC). METHODS: A retrospective review of BC patients treated with NC in Modena Cancer Center between 2005 and 2017 was performed. BCPs, such as subcutaneous fat area (SFA), visceral fat area (VFA), lumbar skeletal muscle index (LSMI) and liver-to-spleen (L/S) ratio were calculated by Advance workstation (General Electric), software ADW server 3.2 or 4.7. BMI and BCPs were correlated with pathological complete response (pCR) and survival outcomes. RESULTS: 407 patients were included in the study: 55% with BMI < 25 and 45% with BMI 65 25. 137 of them had pre-treatment CT scan imagines. Overweight was significantly associated with postmenopausal status and older age. Hormonal receptor positive BC was more frequent in overweight patients (p<0.05). Postmenopausal women had higher VFA, fatty liver disease and obesity compared to premenopausal patients. No association between BMI classes and tumor response was detected. High VFA and liver steatosis were negative predictive factors for pCR (pCR rate: 36% normal VFA vs 20% high VFA, p= 0.048; no steatosis 32% vs steatosis 13%, p=0.056). Neither BMI classes nor BCPs significantly influenced overall survival and relapse-free survival. CONCLUSION: Visceral adiposity as well as steatosis were closely involved in chemosensitivity in BC patients treated with NC. Their measures from clinically acquired CT scans provide significant predictive information that outperform BMI value. More research is required to evaluate the relationship among adiposity site and survival outcomes

Simple Questionnaires to Improve Pooling Strategies for SARS-CoV-2 Laboratory Testing

Background: Liberal PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to contain the coronavirus disease 2019 (COVID-19) pandemic. Combined multi-sample testing in pools instead of single tests might enhance laboratory capacity and reduce costs, especially in low- and middle-income countries. Objective: The purpose of our study was to assess the value of a simple questionnaire to guide and further improve pooling strategies for SARS-CoV-2 laboratory testing. Methods: Pharyngeal swabs for SARS-CoV-2 testing were obtained from healthcare and police staff, hospital inpatients, and nursing home residents in the southwestern part of Germany. We designed a simple questionnaire, which included questions pertaining to a suggestive clinical symptomatology, recent travel history, and contact with confirmed cases to stratify an individual’s pre-test probability of having contracted COVID-19. The questionnaire was adapted repeatedly in face of the unfolding pandemic in response to the evolving epidemiology and observed clinical symptomatology. Based on the response patterns, samples were either tested individually or in multi-sample pools. We compared the pool positivity rate and the number of total PCR tests required to obtain individual results between this questionnaire-based pooling strategy and randomly assembled pools. Findings: Between March 11 and July 5, 2020, we processed 25,978 samples using random pooling (n = 6,012; 23.1%) or questionnaire-based pooling (n = 19,966; 76.9%). The overall prevalence of SARS-CoV-2 was 0.9% (n = 238). Pool positivity (14.6% vs. 1.2%) and individual SARS-CoV-2 prevalence (3.4% vs. 0.1%) were higher in the random pooling group than in the questionnaire group. The average number of PCR tests needed to obtain the individual result for one participant was 0.27 tests in the random pooling group, as compared to 0.09 in the questionnaire-based pooling group, leading to a laboratory capacity increase of 73% and 91%, respectively, as compared to single PCR testing. Conclusions: Strategies that combine pool testing with a questionnaire-based risk stratification can increase laboratory testing capacities for COVID-19 and might be important tools, particularly in resource-constrained settings

The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation

The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of “neutrophil extracellular traps” (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host

Una tonadilla escénica para la Navidad: "Los Payos y el Alcalde", del Señor Marcolini

Invasion and persistence of bacteria within host cells requires that they adapt to life in an intracellular environment. This adaptation induces bacterial stress through events such as phagocytosis and enhanced nutrient-restriction. During stress, bacteria synthesize a family of proteins known as heat shock proteins (HSPs) to facilitate adaptation and survival. Previously, we determined the Staphylococcus aureus HSP ClpC temporally alters bacterial metabolism and persistence. This led us to hypothesize that ClpC might alter intracellular survival. Inactivation of clpC in S. aureus strain DSM20231 significantly enhanced long-term intracellular survival in human epithelial (HaCaT) and endothelial (EA.hy926) cell lines, without markedly affecting adhesion or invasion. This phenotype was similar across a genetically diverse collection of S. aureus isolates, and was influenced by the toxin/ antitoxin encoding locus mazEF. Importantly, MazEF alters mRNA synthesis and/or stability of S. aureus virulence determinants, indicating ClpC may act through the mRNA modulatory activity of MazEF. Transcriptional analyses of total RNAs isolated from intracellular DSM20231 and isogenic clpC mutant cells identified alterations in transcription of α-toxin (hla), protein A (spa), and RNAIII, consistent with the hypothesis that ClpC negatively affects the intracellular survival of S. aureus in non-professional phagocytic cells, via modulation of MazEF and Agr

The Catabolite Control Protein E (CcpE) Affects Virulence Determinant Production and Pathogenesis of Staphylococcus aureus

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Image_1_The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation.PDF

<p>The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of “neutrophil extracellular traps” (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host.</p

Simple Questionnaires to Improve Pooling Strategies for SARS-CoV-2 Laboratory Testing

Background: Liberal PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to contain the coronavirus disease 2019 (COVID-19) pandemic. Combined multi-sample testing in pools instead of single tests might enhance laboratory capacity and reduce costs, especially in low- and middle-income countries. Objective: The purpose of our study was to assess the value of a simple questionnaire to guide and further improve pooling strategies for SARS-CoV-2 laboratory testing. Methods: Pharyngeal swabs for SARS-CoV-2 testing were obtained from healthcare and police staff, hospital inpatients, and nursing home residents in the southwestern part of Germany. We designed a simple questionnaire, which included questions pertaining to a suggestive clinical symptomatology, recent travel history, and contact with confirmed cases to stratify an individual's pre-test probability of having contracted COVID-19. The questionnaire was adapted repeatedly in face of the unfolding pandemic in response to the evolving epidemiology and observed clinical symptomatology. Based on the response patterns, samples were either tested individually or in multi-sample pools. We compared the pool positivity rate and the number of total PCR tests required to obtain individual results between this questionnaire-based pooling strategy and randomly assembled pools. Findings: Between March 11 and July 5, 2020, we processed 25,978 samples using random pooling (n = 6,012; 23.1%) or questionnaire-based pooling (n = 19,966; 76.9%). The overall prevalence of SARSCoV-2 was 0.9% (n = 238). Pool positivity (14.6% vs. 1.2%) and individual SARS-CoV-2 prevalence (3.4% vs. 0.1%) were higher in the random pooling group than in the questionnaire group. The average number of PCR tests needed to obtain the individual result for one participant was 0.27 tests in the random pooling group, as compared to 0.09 in the questionnaire-based pooling group, leading to a laboratory capacity increase of 73% and 91%, respectively, as compared to single PCR testing. Conclusions: Strategies that combine pool testing with a questionnaire-based risk stratification can increase laboratory testing capacities for COVID-19 and might be important tools, particularly in resource-constrained settings