TY - THES A1 - Aumann, Ralf T1 - Vorkommen und Expression des opcA Gens in Meningokokkenstämmen von Erkrankten und asymptomatischen Trägern T1 - Prevalence and expression of the opcA gene in meningococci from invasive and carrier strains N2 - Das Opc-Protein ist ein Außenmembranprotein von Meningokokken, das über extrazelluläre Matrixproteine mit Integrinen der Wirtszelle interagiert. Opc ist in Menschen immunogen und induziert bakterizide Antikörper. Das Opc-Protein wurde daher als aussichtsreicher Impfstoff-Kandidat angesehen, da es außerdem relativ gut konserviert ist. Allerdings wird das Opc-Protein nicht von allen Meningokokkenstämmen exprimiert. Einerseits fehlt das opc-Gen in einigen klonalen Komplexen (z.B. ST-8, ST-11, ST-53), andererseits ist die Opc-Expression nicht konstitutiv wegen einer phasenvariablen Transkription, die auf einem Poly-Cytidin-Bereich im Promotor des opc-Gens beruht. In dieser Arbeit wurde die Präsenz des opc-Gens und die Opc-Expression in zwei großen Sammlungen deutscher Meningokokkenisolate von invasiven Erkrankungen (n=1141) und gesunden Trägern (n=792) untersucht. Das opc-Gen war bei 71% der invasiven und 77% der Trägerstämme nachweisbar. Der größte Teil der opc-Gen negativen Stämme gehörte zu den klonalen Komplexen ST-8, ST-11, ST-213, ST-231, ST-334 und ST-53. Der Anteil opc-positiver Stämme, die Opc in vitro exprimieren, war bei den invasiven Stämmen kleiner als bei den Trägerstämmen (13% vs. 29%, p<0,001, Chi-square-Test). Der größere Anteil Opc-exprimierender Trägerstämme ist u.a. am ehesten mit der Überrepräsentation von wenig pathogenen klonalen Komplexen (ST-23, ST-35, ST-198) mit einer hohen Opc-Expressionsrate zu erklären. 24 von den 176 invasiven Stämmen mit einer Anzahl von 11 - 14 Cs in der Promotor-Region, die die Opc-Expression begünstigt, zeigten weder im ELISA noch im Westernblot eine Opc-Expression. Bei 14 dieser 24 Stämme wurde als Ursache ein phasenvariabler, intragenischer Poly-Adenin-Bereich identifiziert, der zu einer Leserasterverschiebung führte. Die Vermutung mehrerer Autoren, dass die Opc-Expression mit dem klinischen Bild der Meningitis verknüpft ist, konnte mit der hier genutzten großen Stammsammlung nicht bestätigt werden. Invasive Stämme, die das Opc-Protein exprimierten, wurden genauso häufig von Patienten mit dem klinischen Bild der Meningitis isoliert wie Stämme, die das Opc-Protein nicht exprimierten (46% vs. 47%, Chi-square-Test: p<0,9). Allerdings gibt es eine starke Assoziation der Gegenwart des opc-Gens mit dem klinischen Merkmal Meningitis. Dieser Befund gibt Anlass zu der Hypothese, dass in vitro und in vivo Expression von Opc sich unterscheiden. Zusammenfassend lässt sich festhalten, dass das Opc-Protein nur in 19,8% aller Isolate (invasive und Trägerstämme zusammengenommen) exprimiert wurde. Es zeigte sich eine Tendenz zu häufigerer Opc-Expression in apathogenen Trägerisolaten. Das Vorhandensein des opc-Gens, nicht aber die in vitro Expression konnten mit dem klinischen Merkmal Meningitis assoziiert werden. Zusätzlich wurde ein weiterer Mechanismus der intragenischen Phasenvariation beschrieben. N2 - Presence of opc was associated with meningitis, mostly because ST-11/ST-8 cc meningococci with low meningitis rates were consistently opc negative. On the other hand, lack of opc did not exclude meningitis. Opc was expressed in only 13% of all invasive isolates. In vitro Opc expression was not associated with meningitis. Limitation: Definite conclusion about expression in vivo is not possible with cultured isolates. Evidence for intragenic opc phase-variation was provided. KW - Neisseria meningitidis KW - opc KW - Medizin KW - Mikrobiologie KW - Meningitis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157278 ER - TY - JOUR A1 - Herrmann, Johannes A1 - Muenstermann, Marcel A1 - Strobel, Lea A1 - Schubert-Unkmeir, Alexandra A1 - Woodruff, Trent M. A1 - Gray-Owen, Scott D. A1 - Klos, Andreas A1 - Johswich, Kay O. T1 - Complement C5a receptor 1 exacerbates the pathophysiology of N. meningitidis sepsis and is a potential target for disease treatment JF - mBio N2 - Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1\(^{-/-}\) mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1\(^{-/-}\) mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy. Importance: The devastating consequences of N. meningitidis sepsis arise due to the rapidly arising and self-propagating inflammatory response that mobilizes antibacterial defenses but also drives the immunopathology associated with meningococcemia. The complement cascade provides innate broad-spectrum protection against infection by directly damaging the envelope of pathogenic microbes through the membrane attack complex and triggers an inflammatory response via the C5a peptide and its receptor C5aR1 aimed at mobilizing cellular effectors of immunity. Here, we consider the potential of separating the bactericidal activities of the complement cascade from its immune activating function to improve outcome of N. meningitidis sepsis. Our findings demonstrate that the specific genetic or pharmacological disruption of C5aR1 rapidly ameliorates disease by suppressing the pathogenic inflammatory response and, surprisingly, allows faster clearance of the bacterial infection. This outcome provides a clear demonstration of the therapeutic benefit of the use of C5aR1-specific inhibitors to improve the outcome of invasive meningococcal disease. KW - C5aR1 KW - whole-blood model KW - Neisseria meningitidis KW - anaphylatoxins KW - complement system KW - inflammation KW - invasive disease KW - mouse model KW - neutrophils KW - sepsis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175792 VL - 9 IS - 1 ER - TY - JOUR A1 - Silwedel, Christine A1 - Speer, Christian P. A1 - Haarmann, Axel A1 - Fehrholz, Markus A1 - Claus, Heike A1 - Buttmann, Mathias A1 - Glaser, Kirsten T1 - Novel insights into neuroinflammation: bacterial lipopolysaccharide, tumor necrosis factor α, and Ureaplasma species differentially modulate atypical chemokine receptor 3 responses in human brain microvascular endothelial cells JF - Journal of Neuroinflammation N2 - Background: Atypical chemokine receptor 3 (ACKR3, synonym CXCR7) is increasingly considered relevant in neuroinflammatory conditions, in which its upregulation contributes to compromised endothelial barrier function and may ultimately allow inflammatory brain injury. While an impact of ACKR3 has been recognized in several neurological autoimmune diseases, neuroinflammation may also result from infectious agents, including Ureaplasma species (spp.). Although commonly regarded as commensals of the adult urogenital tract, Ureaplasma spp. may cause invasive infections in immunocompromised adults as well as in neonates and appear to be relevant pathogens in neonatal meningitis. Nonetheless, clinical and in vitro data on Ureaplasma-induced inflammation are scarce. Methods: We established a cell culture model of Ureaplasma meningitis, aiming to analyze ACKR3 variances as a possible pathomechanism in Ureaplasma-associated neuroinflammation. Non-immortalized human brain microvascular endothelial cells (HBMEC) were exposed to bacterial lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), and native as well as LPS-primed HBMEC were cultured with Ureaplasma urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). ACKR3 responses were assessed via qRT-PCR, RNA sequencing, flow cytometry, and immunocytochemistry. Results: LPS, TNF-α, and Ureaplasma spp. influenced ACKR3 expression in HBMEC. LPS and TNF-α significantly induced ACKR3 mRNA expression (p < 0.001, vs. control), whereas Ureaplasma spp. enhanced ACKR3 protein expression in HBMEC (p < 0.01, vs. broth control). Co-stimulation with LPS and either Ureaplasma isolate intensified ACKR3 responses (p < 0.05, vs. LPS). Furthermore, stimulation wielded a differential influence on the receptor’s ligands. Conclusions: We introduce an in vitro model of Ureaplasma meningitis. We are able to demonstrate a pro-inflammatory capacity of Ureaplasma spp. in native and, even more so, in LPS-primed HBMEC, underlining their clinical relevance particularly in a setting of co-infection. Furthermore, our data may indicate a novel role for ACKR3, with an impact not limited to auto-inflammatory diseases, but extending to infection-related neuroinflammation as well. AKCR3-induced blood-brain barrier breakdown might constitute a potential common pathomechanism. KW - atypical chemokine receptor 3 KW - human brain microvascular endothelial cells KW - meningitis KW - neuroinflammation KW - Ureaplasma species Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175952 VL - 15 IS - 156 ER - TY - JOUR A1 - Strobel, Lea A1 - Johswich, Kay O. T1 - Anticoagulants impact on innate immune responses and bacterial survival in whole blood models of Neisseria meningitidis infection JF - Scientific Reports N2 - Neisseria meningitidis (meningococcus) causes invasive diseases such as meningitis or septicaemia. Ex vivo infection of human whole blood is a valuable tool to study meningococcal virulence factors and the host innate immune responses. In order to consider effects of cellular mediators, the coagulation cascade must be inhibited to avoid clotting. There is considerable variation in the anticoagulants used among studies of N. meningitidis whole blood infections, featuring citrate, heparin or derivatives of hirudin, a polypeptide from leech saliva. Here, we compare the influence of these three different anticoagulants, and additionally Mg/EGTA, on host innate immune responses as well as on viability of N. meningitidis strains isolated from healthy carriers and disease cases, reflecting different sequence types and capsule phenotypes. We found that the anticoagulants significantly impact on cellular responses and, strain-dependently, also on bacterial survival. Hirudin does not inhibit complement and is therefore superior over the other anticoagulants; indeed hirudin-plasma most closely reflects the characteristics of serum during N. meningitidis infection. We further demonstrate the impact of heparin on complement activation on N. meningitidis and its consequences on meningococcal survival in immune sera, which appears to be independent of the heparin binding antigens Opc and NHBA. KW - infection KW - pathogens KW - Neisseria meningitidis KW - anticoagulants Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176226 VL - 8 IS - 10225 ER - TY - JOUR A1 - Ruf, Dominik A1 - Brantl, Victor A1 - Wagener, Johannes T1 - Mitochondrial Fragmentation in \(Aspergillus\) \(fumigatus\) as Early Marker of Granulocyte Killing Activity JF - Frontiers in Cellular and Infection Microbiology N2 - The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae. KW - Aspergillus fumigatus KW - killing KW - assay KW - PMNs KW - granulocytes KW - mitochondria KW - mitochondrial morphology KW - fungicidal activity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227133 VL - 8 IS - 128 ER - TY - JOUR A1 - Prauße, Maria T. E. A1 - Lehnert, Teresa A1 - Timme, Sandra A1 - Hünniger, Kerstin A1 - Leonhardt, Ines A1 - Kurzai, Oliver A1 - Figge, Marc Thilo T1 - Predictive Virtual Infection Modeling of Fungal Immune Evasion in Human Whole Blood JF - Frontiers in Immunology N2 - Bloodstream infections by the human-pathogenic fungi Candida albicans and Candida glabrata increasingly occur in hospitalized patients and are associated with high mortality rates. The early immune response against these fungi in human blood comprises a concerted action of humoral and cellular components of the innate immune system. Upon entering the blood, the majority of fungal cells will be eliminated by innate immune cells, i.e., neutrophils and monocytes. However, recent studies identified a population of fungal cells that can evade the immune response and thereby may disseminate and cause organ dissemination, which is frequently observed during candidemia. In this study, we investigate the so far unresolved mechanism of fungal immune evasion in human whole blood by testing hypotheses with the help of mathematical modeling. We use a previously established state-based virtual infection model for whole-blood infection with C. albicans to quantify the immune response and identified the fungal immune-evasion mechanism. While this process was assumed to be spontaneous in the previous model, we now hypothesize that the immune-evasion process is mediated by host factors and incorporate such a mechanism in the model. In particular, we propose, based on previous studies that the fungal immune-evasion mechanism could possibly arise through modification of the fungal surface by as of yet unknown proteins that are assumed to be secreted by activated neutrophils. To validate or reject any of the immune-evasion mechanisms, we compared the simulation of both immune-evasion models for different infection scenarios, i.e., infection of whole blood with either C. albicans or C. glabrata under non-neutropenic and neutropenic conditions. We found that under non-neutropenic conditions, both immune-evasion models fit the experimental data from whole-blood infection with C. albicans and C. glabrata. However, differences between the immune-evasion models could be observed for the infection outcome under neutropenic conditions with respect to the distribution of fungal cells across the immune cells. Based on these predictions, we suggested specific experimental studies that might allow for the validation or rejection of the proposed immune-evasion mechanism. KW - immune evasion KW - state-based model KW - innate immune response KW - polymorphonuclear neutrophils KW - whole-blood infection assay Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197493 SN - 1664-3224 VL - 9 IS - 560 ER -