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Bats (Chiroptera) form the second largest order of mammals and with over 1,250 species, they represent about 20% of all mammalian species worldwide. They are the only mammals with true and sustained flight and distributed all over the world except the arctic regions. Moreover, bats entered specific ecological niches and with their food spectra, they reduce different arthropod populations as well as disperse seeds and pollen of plant species in various regions and habitats.
Bats also have a crucial role in spreading high-pathogenic and zoonotic viruses, harbor in general more viruses (zoonotic and non-zoonotic), and, related to the species, number even more than rodents. However, clinical symptoms of viral diseases are rarely reported in bat communities. Also seroconversions after infection were not reported for a variety of viruses found in bats. Since the incidence of virus-positive bats estimated in passive surveillance studies is usually very low, it is a question how such viruses can use bats as reservoir hosts. There is obviously a special evolutionary relationship between the pathogens and bats as hosts, which are based on possibly physiologic adaptations also in resistance and immunity.
In this thesis, the two lyssaviruses, European Bat Lyssavirus 1 and 2 (EBLV-1 and -2) were chosen as a model to investigate the immune response of European bats against viral infection in vitro. Lyssaviruses are the causative agents of rabies, a fatal zoonotic disease with neurotropic characteristics.
One main question to investigate was in which way bats act as reservoir host and developed a high disease resistance. The present thesis is based on three hypotheses about innate immune response against lyssavirus infection:
A) In bats specific peripheral resistance mechanisms evolved which reduce the risk of systemic viral infection after a hypothesized airborne transmission and infection via nasal epithelium supported by the social structure of and communication within bat communities.
B) The co-evolution of EBLV and the innate resistance of bats resulted in a very effective type I interferon response to inhibit a systemic lyssavirus infection.
C) The specific physiology of body temperature of bats with daily torpor depresses the viral replication but favours the type I interferon response.
To analyze the interferon-based resistance mechanisms, the type I interferon (IFN) genes of two European bats species (Eptesicus serotinus and Myotis myotis) were cloned and sequenced. Using established cell lines from the respiratory nasal epithelium (MmNep), olfactory nasal epithelium (MmNol), and Bulbus olfactorius brain (MmBr), the type I IFN response along a possible airborne infection route was investigated. The anti-viral effects and induction of IFNs/interferon stimulated genes (ISGs) in each cell line were also investigated in detail after infection in vitro. Finally, the influence of different temperatures on lyssavirus replication was analyzed in cell culture experiments.
The results indicated that (a) along the hypothesized airborne infection route the susceptibility for lyssavirus infections is decreased, (b) the type I IFN activity in contrast is increased contributing to a limitation of lyssavirus replication and (c) an obvious influences of varying cultivation temperatures on the resistance against lyssavirus infections, which favor the IFN response and repressing lyssavirus replication.
The result from these in vitro studies supports the hypothesis of a special co-evolution between lyssaviruses and bats. However, in vivo studies on the relevance in infected animals are missing so far. This model could also explain the generally limited pathogenicity of bat-associated viruses.
Cardiovascular diseases are the most common cause of death in industrial nations. The basis of these diseases is a dysfunction in the interaction between the cells the heart is composed of. The main types of cells making up the human heart are cardiomyocytes that build the myocardium and provide the contraction properties, endothelial cells that delimit the blood flowing through the inner chambers and coronary arteries from the myocardial tissue, and fibroblasts, which build the connective tissue. A common process in the development of cardiovascular diseases is the formation of fibrosis due to injury of the endothelium and subsequent infiltration of the cardiac tissue by immune cells, and inflammatory agents like cytokines. Cytokines exert different functions in cardiac cells. Tumor necrosis factor α (TNFα) is an inducer of apoptosis. Transforming growth factor ß (TGFß) is known for activation of proliferation. Other cytokines like C-X-C motif chemokine 11 (CXCL11), interleukin-6 (IL-6), or brain-derived neurotrophic factor (BDNF) have not yet been investigated or their impact on such cells is unknown. Eventually, however, fibrotic scar tissue arises from the transition from fibroblasts to myofibroblasts leading to a stiffening of the cardiac muscle and impaired pump function. In order to prevent the occurrence of these events the balance of proliferation, migration, and differentiation of cardiac cells needs to be controlled very delicately.
The mechanisms controlling these interactions are still not well understood, which is why this work aimed at the elucidation of molecular mechanisms within the three main cell types that might play a role in the regulation of cardiac function. A proteomic approach using mass spectrometry was used to identify alterations in protein levels that could provide hints about the involved pathways and find new players as candidates for more detailed investigation. Initially, the proteomic composition of HL-1 cardiomyocytes, L929 fibroblasts, and human umbilical vein endothelial cells (HUVECs) that were cultivated in standard growth conditions without stress was investigated. Half of the total protein intensity was made up by only 42 to 53 proteins, depending on the cell type. More than a third of all proteins were identified in all three cell types, which may be proteins performing common cell functions. Indeed, the proteins displaying the highest abundance seem to be predominantly involved in such common cellular functions as the regulation of glucose metabolism or the cytoskeleton. More specific functions like heart development and muscle contraction were found enriched in cardiomyocytes as were mitochondrial proteins. The proportion of proteins with extracellular localization and function was higher in fibroblasts and endothelial cells.
Secondly, the impact of cytokines on the proliferative behavior and the proteomic composition of cardiomyocytes and fibroblasts was analyzed. HL-1 cardiomyocytes and L929 fibroblasts were treated with different concentrations of cytokines with a cytotoxic, proliferative, or yet unknown effect on these cells. While HL-1 cells exhibited no macroscopic reaction to any of the cytokines used, cytotoxic/growth inhibitory (TNFα, CXCL11) and proliferative (TGFß, IL6, BDNF) effects were observed for L929 cells. The latter also showed CXCL11-induced upregulated EIF2 signaling, pointing to a higher need of protein synthesis.
The third aim was the examination of proteome adaptations in endothelial cells due to different kinds of stress, as these cells are the first line of defense against inflammatory agents or injury and therefore prone to wounding. The role of the growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in wounding and starvation was another object of this study as they are known for their angiogenic and cell survival supporting properties. Additionally, the impact of the cellular sex on the response to stress and growth factors was examined, because a person’s sex plays an important role in susceptibility, risk factors, and outcome of cardiovascular diseases. This has mainly been attributed to the different hormone levels, especially the higher levels of estrogen in premenopausal women, which exerts cardioprotective properties, but also genetic background was reported to play an important role. Only few studies that examined the molecular properties of HUVECs considered the cellular sex and if so, the genetic bias of unrelated samples was not taken into account. This is why Lorenz and colleagues at the Charité in Berlin collected HUVECs from newborn twins of opposite sex, cultivated them without stress in standard growth medium, exposed them to wounding and serum starvation, and investigated the impact of the growth factors and the sex on migrational behavior and metabolic issues. The current work focused on the alterations of not only the intra- but also the extracellular proteome, because paracrine signaling is crucial for intercellular communication in order to cope with stress. General differences between male and female cells were observed for proteins encoded on the X chromosome with higher levels in females (DDX3X, UBA1, EIF1AX, RPS4X, HDHD1), except for one protein with higher levels in male cells (G6PD). A Y-chromosomal protein was, for the first time, identified in endothelial cells (DDX3Y). Wounding, starvation, and growth factor treatment led to alterations and sex-specific different levels in an unexpectedly high number of proteins, with VEGF showing a stronger impact than bFGF. Many proteins with alterations observed without taking the sex into account, were actually only changed in male or female cells. Some proteins were regulated in opposite directions, or growth factors inhibited their secretion in a sex-specific way by unknown mechanisms. Tissue factor pathway inhibitor 2 (TFPI2) should be emphasized as a protein with sex-specific differences, especially in the extracellular space and with increased levels after starvation and VEGF treatment. These observations suggest a temporal lack in TFPI2 synthesis and secretion in male cells, which might explain the enhanced adaptation of females to wounding.
The results of this work lay the basis for future investigation by providing a database of intra- and extracellular proteome changes due to different environmental circumstances. It strongly suggests the investigation of male and female HUVECs, and other cells, separately to avoid the impact of the sex observed in this work. Essentially, the observations suggest a number of candidate proteins for more detailed investigations of endothelial and cardiovascular diseases.
Untersuchungen zur Immunantwort gegen potenzielle Vakzinkandidaten von Streptococcus pneumoniae
(2022)
Das Gram-positive Bakterium Streptococcus pneumoniae (Pneumokokken) stellt vor allem bei der sehr jungen und älteren sowie immungeschwächten Bevölkerung einen immer mehr an Bedeutung gewinnenden Krankheitserreger dar. Krankheitsbilder wie die Pneumokokken-Meningitis, -Pneumonie und -Sepsis, um nur einige zu nennen, können z.T. schwere Verläufe nehmen. Neue Serotyp-unabhängige Impfstoffe sollen flächendeckenden Schutz vor Infektionen mit S. pneumoniae bieten. Das Hauptaugenmerk der Impfstoffforschung liegt unter anderem auf der Untersuchung von Pneumokokken Protein-basierten Impfstoffen als Bestandteil eines Konjugat- bzw. Multikomponenten Impfstoffes. In diesem Zusammenhang wurde in dieser Arbeit die Immunantwort humaner Immunzellen gegenüber den vier Pneumokokken Oberflächen-Lipoproteinen MetQ, DacB, PnrA und PsaA untersucht. Diese Lipoproteine wurden sowohl in lipidierter als auch in nicht-lipidierter Form als heterologe Proteine generiert, um den Einfluss der Lipidierung auf die Immunreaktion beurteilen zu können. Zur Untersuchung der Reaktion des menschlichen Immunsystems wurden humane Blut-Monozyten (hPBMCs) isoliert und zu pro-inflammatorischen Makrophagen (M1) einerseits und anti-inflammatorischen Makrophagen (M2a) andererseits differenziert. Die Zellen wurden mit den lipidierten bzw. nicht-lipidierten Proteinen stimuliert. In den Überständen wurden zur Beurteilung der abgelaufenen Immunreaktion die Konzentrationen von IL-1β, IL-2, IL-6, IL-8 und TNF bestimmt. Eine Produktion von IL-1β und IL-2 durch die Makrophagen konnte hierbei nicht nachgewiesen werden. M1 Makrophagen zeichneten sich durch eine kaum messbare Cytokin-Produktion auf beide Proteinstimuli aus. Anti-inflammatorische Makrophagen zeigten eine signifikant verstärkte IL-6, IL-8 und TNF Produktion (p< 0,05) nach Stimulation mit allen vier lipidierten Proteinen im Gegensatz zur Stimulation mit nicht-lipidierten Proteinen. Das stärkere Stimulationspotenzial der lipidierten Proteine konnte auf deren Agonismus am TLR2 zurückgeführt werden. Weiterhin konnte die starke Reaktion der M2a Makrophagen mit deren Plastizität sowie mit dem Potenzial von Lipoproteinen zur Umpolarisation von Makrophagen aufgrund des TLR2 Agonismus erklärt werden. Die Ergebnisse zeigen, dass die eingesetzten lipidierten Proteine einen suffizienten und signifikant potenteren Immunstimulus im menschlichen Organismus darstellen, als die gleichen Proteine in nicht-lipidierter Form. Somit könnten diese lipidierten Proteine geeignete Vakzinkandidaten gegen S. pneumoniae darstellen.
In den letzten Jahren gewannen ω-Transaminasen zunehmend an Bedeutung. Ihr breites Substratspektrum, das sowohl Aminosäuren als auch Amine umfasst, macht sie interessant für biotechnologische Anwendungen. Im Gegensatz zu α-Aminotransferasen sind ω-Aminotransferasen nicht auf α-Aminosäuren als Aminodonor bzw. α-Ketosäuren als Aminoakzeptoren beschränkt. Auch sind einige ω-Transaminasen in der Lage, Aldehyde oder Ketone zu aminieren. Dadurch sind sie vielseitig einsetzbar. Seit ihrer Entdeckung wurden ω-Transaminasen in einer Vielzahl von Organismen nachgewiesen. Viele dieser Enzyme stammen aus Pilzen und Bakterien. Da es ständig Bedarf an neuen Transaminasen gibt, wurden verschiedene Organismen auf das Vorhandensein solcher Enzyme untersucht. Die Hefe Blastobotrys raffinosifermentans LS3 ist einer dieser Organismen. Für diese Hefe existiert bereits eine Vielzahl biotechnologischer Anwendungen, was unter anderem an ihren vielseitigen physiologischen Möglichkeiten liegt. Um das Spektrum dieses Stammes noch zu erweitern, wurde sein Genom auf ORFs gescannt. Die ermittelten ORFs wurden translatiert und die so erhaltenen, theoretischen Proteine in einer Proteindatenbank gespeichert. Die Einträge dieser Datenbank wurden einem „hmmerscan“ (hmm ist kurz für „hidden Markov model“) unterzogen. Dabei werden die Proteine in sogenannte Pfams, kurz für Proteinfamilien, eingeteilt. Drei Proteine wurden der Familie PF00202.21 zugeordnet. Das ist die sogenannte Aminotran_3 Familie. In dieser Familie befinden sich eukaryotische ω-Transaminasen. Die Gene brota1, brota2 und brota3 codieren für diese Enzyme. Jeweils eins der Gene wurde in den Vektor XPLOR®3 kloniert, damit die potentiellen ω-Transaminasen in B. raffinosifermentans G1212 [aleu2 atrp1:ALEU2] [1] überexprimiert werden können. Eine Besonderheit von BroTA1 ist, dass es neben der Aminotran_3 Domäne noch eine AAA Domäne aufweist. Deshalb ist es mit etwa 85 kDa auch deutlich größer als die meisten ω-Transaminasen, die meist zwischen 45 und 50 kDa liegen. BroTA2 und BroTA3 beinhalten nur die Aminotran_3 Domäne. Alle drei Enzyme zeigen niedrige Aktivität bei der kinetischen Auflösung racemischer β-Aminosäuren.
Neben den eukaryotischen ω-Transaminasen wurden auch einige bakterielle Enzyme untersucht. Literatursuche und das Screenen der Stammsammlung der Arbeitsgruppe Hefegenetik des Leibniz-Instituts für Pflanzengenetik und Kulturpflanzenforschung führten zu mehreren potentiellen bakteriellen ω-Transaminasen. Das zu Beginn dieser Arbeit noch als hypothetisches Protein bezeichnete Enzym von Variovorax boronicumulans hat sich als ω-Transaminase herausgestellt. Das Enzym wurde detailliert hinsichtlich des Substratspektrums und seiner biochemischen Eigenschaften charakterisiert. Es handelt sich hierbei um eine ω-Transaminase mit β-Aktivität. Diese Transaminase akzeptiert sowohl aromatische als auch aliphatische β-Aminosäuren als Substrat. Sequenzvergleiche dieses Enzyms mit anderen ω-Transaminasen, die nur aliphatische Aminosäuren akzeptieren, führten zu tieferen Einblicken in konservierte Bereiche dieser beiden Gruppen von ω-Transaminasen.
Der dritte Ansatz war das Anpassen einer bekannten ω-Transaminase des thermophilen Bakteriums Sphaerobacter thermophilus an ein potentielles Motiv für aromatische ω-Transaminasen. Dadurch sollte die Aktivität des Enzyms erhöht werden. Dieser Ansatz führte zu 7 Varianten des Enzyms mit höherer Aktivität als der Wildtyp. Durch diese Versuche wurden einige für die Transferaseaktivität wichtige Aminosäurereste offenbart. So hat sich zum Beispiel herausgestellt, dass N70 offenbar wichtig für den Umsatz von γ-Aminosäuren ist, da ein Austausch gegen Glutamat zu einer verminderten Aktivität mit γ-Aminopentansäure führte.
Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation
(2018)
Streptococcus pneumoniae is endowed with a variety of surface-exposed proteins representing putative vaccine candidates. Lipoproteins are covalently anchored to the cell membrane and highly conserved among pneumococcal serotypes. Here, we evaluated these lipoproteins for their immunogenicity and protective potential against pneumococcal colonisation. A multiplex-based immunoproteomics approach revealed the immunogenicity of selected lipoproteins. High antibody titres were measured in sera from mice immunised with the lipoproteins MetQ, PnrA, PsaA, and DacB. An analysis of convalescent patient sera confirmed the immunogenicity of these lipoproteins. Examining the surface localisation and accessibility of the lipoproteins using flow cytometry indicated that PnrA and DacB were highly abundant on the surface of the bacteria. Mice were immunised intranasally with PnrA, DacB, and MetQ using cholera toxin subunit B (CTB) as an adjuvant, followed by an intranasal challenge with S. pneumoniae D39. PnrA protected the mice from pneumococcal colonisation. For the immunisation with DacB and MetQ, a trend in reducing the bacterial load could be observed, although this effect was not statistically significant. The reduction in bacterial colonisation was correlated with the increased production of antigen-specific IL-17A in the nasal cavity. Immunisation induced high systemic IgG levels with a predominance for the IgG1 isotype, except for DacB, where IgG levels were substantially lower compared to MetQ and PnrA. Our results indicate that lipoproteins are interesting targets for future vaccine strategies as they are highly conserved, abundant, and immunogenic.
Immunogenicity and protectivity of surface-localized lipoproteins of Streptococcus pneumoniae
(2019)
Steptococcus pneumoniae (pneumococcus) represents a common colonizer of the human upper respiratory tract (URT). However, under certain conditions, for example following viral infections, or in indiciduals with a weakened immune system, including young children, elderly and immunocompromised persons, it can cause a wide range of life-threatening diseases, such as pneumonia, meningitis or sepsis. Based on the polysaccharide capsule that surrounds the bacterium, pneumococci are classified into so far 98 different serotypes. Prevention of S. pneumoniae infections was achieved by the development of pneumococcal polysaccharide-based (PPSV) vaccines. However, these vaccines have important limitations, including high manufacturing costs and restricted serotype coverage facilitating replacement by non-vaccine serotypes. Aiming for the development of a serotype-independent vaccine, the potential of surface-exposed and highly conserved pneumococcal lipoproteins was evaluated for being targeted as a future protein-based vaccine. Therefore, selected lipoproteins were examined i) for their surface abundance and accessibility, ii) for their presence in clinically relevant S. pneumoniae strains, and iii) for their immunogenicity. Finally, based on these initial screenings, the most promising candidates were selected to analyze their protective efficacy in a moude model of colonization. DacB and PnrA were identified as highly abundant lipoproteins on the pneumococcal surface. They showed to be immunogenic both during natural infection using convalescent patient sera and when given to mice as a subunit vaccine formulation. Following intranasal immunization and challenge of mice with two heterologous S. pneumoniae strains, both proteins reduced the pneumococcal load in the nasopharynx. The protection correlated with increased production of IL-17A indicative for a Th17-mediated immunity, which is strongly suggested to play a critical role in preventing pneumococcal colonization and infection. Lipoproteins are triggering innate receptors on antigen-presenting cells, thereby linking innate with adaptive immune responses. Therefore, lipidated proteins were evaluated for their potential to be used as an adjuvant for vaccination. Lipidation clearly enhanced humoral immune responses to DacB and PnrA without the need of an additional adjuvant. However, an additional adjuvant was required to confer protection against pneumococcal colonization. In conclusion, Lipoproteins are interesting candidates for future protein-based vaccine strategies because they are highly conserved, abundant and immunogenic. PnrA and DacB were identified as potential candidates, since they induced protection against pneumococcal colonization, which in turn may lead to a decline in infections and transmission.
Geistige Behinderung ist eine der häufigsten Formen von erblich bedingten kognitiven Beeinträchtigungen. Definiert wird sie durch einen Intelligenzquotienten unter 70 und obwohl ihre genetischen Ursachen sehr heterogen sein können, gibt es unter ihnen eine beachtliche Menge Gene, die am Aufbau der Glycocalyx beteiligt sind [1, 2]. Die Glycocalyx besteht aus Zuckerbausteinen, die Teil von Lipiden und Proteinen der Zelloberfläche oder der extrazellulären Matrix sind. Vor kurzem konnten wir belegen, dass Mutationen im ST3GAL3-Gen, welches für die Golgi-lokalisierte β-Galactosid-α2,3-sialyltransferase-III codiert, zu verschiedenen klinischen Befunden führt. Zwei unabhängige Mutationen (p.Ala13Asp and p.Asp370Tyr), gefunden in iranischen Familien, konnten mit relativ milden Formen nicht-syndromaler geistiger Behinderung (NSARID) in Verbindung gebracht werden [5]. Eine dritte Punktmutation (p.Ala320Pro), gefunden in einer palästinensischen Familie, verursachte hingegen eine schwere, altersabhängige epileptische Enzephalopathie, das West-Syndrom. Dieses Syndrom ist mit einem Arrest der geistigen Entwicklung oder sogar, wie in unserem Fall, einer Regression assoziiert [3, 4]. ST3GAL3 bildet im Menschen unter anderem das Sialyl Lewis-a (sLea)-Epitop auf Proteinen. Exogene Expression der Volllängen-c-MYC-Fusionsproteine, der Mutationsvarianten in LMTK—Zellen, zeigte, dass alle Varianten eine gestörte subzellulare Lokalisierung zeigen und zwei von ihnen (p.Ala13ASp und p.ALA320Pro) kaum mehr messbare Aktivität besitzen [5]. Um die molekularen und zellulären Mechanismen näher zu beleuchten, die dem ST3GAL3-bedingten West-Syndrom zugrunde liegen, haben wir erfolgreich ein patientenspezifisches, induzertes pluripotentes Stammzellmodell etabliert. Hierfür wurden Fibroblasten der Patientin, die eine Mutation im Exon 12 (c.958G>C, p.Ala320Pro) des ST3GAL3-Gens trägt, und einer gesunden Schwester mittels eines lentiviralen Vektorsystems reprogrammiert. Da ST3GAL3 die höchsten Expressionswerte im frontalen Kortex zeigte, und dies auch in Übereinstimmung mit dem vorgeschlagenen Ursprung epileptischer Anfälle steht, wurde ein Differenzierungsprotokoll für kortikale Neuronen etabliert und erfolgreich für beide Zelllinien durch geführt. Einer der größten Vorteile dieses Protokolls ist, dass hier die Neurogenese in vitro nach demselben temporalen Muster abläuft wie die Neurogenese in vivo. Die iPSC und die daraus differenzierten Neuronen wurden anschließend mittels Lectinblot, mRNA-Sequenzierung, Adhärenzassays und FACS untersucht. Während keine Unterschiede zwischen den iPSC und den Fibroblasten festgestellt wurden, konnten für die kortikalen Neuronen der Patientin eine zusätzliche Bande im Lektinblot (70 kDa), ein verändertes Adhärenzverhalten auf poly-L-Orinithin/Laminin-beschichteter Oberfläche und eine deutlich reduzierte Menge T-box-transcription factor-brain-1-exprimierende Neuronen festgestellt werden. Unsere Ergebnisse zeigen, dass die ST3GAL3-Aktivität wichtig für die normale Entwicklung und Funktion des Gehirns ist.
During infections, innate immune cells are crucial for initiating a pro-inflammatory immune response and clearing the invading pathogen. Delay in pathogen clearance or initiation of an immune response due to impaired functionality of immune cells can result in devastating consequences. The cellular compartment of the innate immune system comprises an array of specialized cell types: Macrophages are tissue-resident professional phagocytes that clear cellular debris, pathogens, and foreign objects. Dendritic cells (DCs) are immune sentinels specialized in antigen uptake and subsequent T cell priming. They are primary sources of cytokines in response to infection. Neutrophils are efficient effector cells that respond rapidly to infection and clear bacteria by different mechanisms. If effector mechanisms of these cells are affected by either bacterial or other factors, infections might not be resolved and can spread throughout the host. Cobalt-chromium-molybdenum biomaterial is widely used in arthroplasty. Implant-derived wear particles and ions lead to macrophage-driven adverse local tissue reactions: Such reactions have been linked to an increased risk of periprosthetic joint infection after revision arthroplasty. While metal-induced cytotoxicity is well characterized in human macrophages, direct effects on their functionality remain elusive. In Paper I, we show that local peri-implant tissue is exposed to Co and Cr in situ. Influx of macrophages is also evident. Exposure of isolated human monocytes/macrophages to Cr3+ in vitro had only minor effects. However, exposure of monocytes/macrophages to pathologic concentrations of Co2+ significantly impaired both phenotype and functionality. High concentrations of Co2+ induced loss of surface markers, including CD14 and CD16. Both Co2+ and Cr3+ impaired macrophage responses to Staphylococcus aureus infection. Co2+ -exposed macrophages, in particular, showed decreased phagocytic activity. These findings demonstrate the immunosuppressive effects of locally elevated metal ions on the innate immune response. Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild to severe necrotizing soft tissue infections (NSTIs). In the host environment hypervirulent GAS variants carrying mutations within the genes encoding for control of virulence (Cov)R/S two component system are enriched. This adaptation is associated with loss of SpeB secretion. In Paper II, we show that in vitro infections with hyper-virulent GAS variants harboring dysfunctional CovR/S suppress secretion of IL-8 and IL-18 by human monocytic cells. This phenotype was mediated by a caspase-8 dependent mechanism. Knockout of streptococcal SLO in a GAS strain carrying functional CovR/S even increased secretion of IL1β and IL-18 by moDCs. Of 67 fully sequenced GAS NSTI isolates, 28 contained covS or covR mutations that rendered the TCS dysfunctional. However, no differences in systemic IL-8 and IL-18 were detected in these patients. GAS isolates recovered from patients often display a mixed phenotype, consisting of SpeB positive (SpeB+ ) and SpeB negative (SpeB- ) clones. Irreversible loss of SpeB expression is often caused by loss of function mutations in regulatory components (CovR/S, RopB). Loss of SpeB is often associated with hyper-virulence. In Paper III, we show that the host environment induces transiently abrogated secretion of SpeB by GAS. Tissue inflammation, neutrophil influx, and degranulation correlated with increased frequencies of SpeB- GAS clones. Isolates recovered from tissue expressed but did not secrete SpeB, which was reversible. Neutrophilderived ROS were identified as the main factor responsible for abrogated SpeB secretion. Hyper-virulent SpeB- clones also exhibit better survival within and induce excessive degranulation of neutrophils.
Die Pankreatitis ist gekennzeichnet durch den Selbstverdau des Organs. Dabei werden pankreatische Proteasen aktiviert und es entstehen lokale Entzündungsherde. Diese aktivieren die Immunantwort durch Ausschüttung pro- inflammatorischer Mediatoren und der Rekrutierung von Immunzellen in das geschädigte Gewebe. Als erste Schutzinstanz reagiert das angeborene Immunsystem inklusive der Neutrophilen, Granulozyten und Makrophagen. Es ist bekannt, dass diese Immunzellen Mustererkennungsrezeptoren nutzen, um die Infektion zu erkennen. Zu diesen gehören die Toll-like Rezeptoren, welche u.a. über den MyD88/IRAK Signalweg den Transkriptionsfaktor NF-κB aktivieren können. In dieser Signalkaskade existiert ein negativer Feedback Regulator IRAK-M, auch bekannt als IRAK-3. Dieser ist in der Lage die Signalweiterleitung zu inhibieren. In dieser Arbeit wurde untersucht ob und inwieweit IRAK-M Einfluss auf den Verlauf einer experimentell induzierten Pankreatitis in Mäusen hat. Bisherige Studien zeigten die Expression von IRAK-M in verschiedenen Zelltypen und Geweben, jedoch nicht im Pankreas sowie den Azinuszellen. In dieser Arbeit konnte nachgewiesen werden, dass IRAK-M in Pankreasgewebe sowie isolierten Azini von C57BL/6 Mäusen exprimiert wird. Die Stimulation von isolierten C57BL/6-Azinuszellen mit CCK hatte eine Expressionserhöhung von IRAK-M zur Folge. Es konnte zudem gezeigt werden, dass Toll-like Rezeptoren (TLR), insbesondere 2, 3, 4 und 9, in bzw. auf Azini exprimiert werden. Die TLR1, 2, 3, 7 und 9 zeigten ein höheres Expressionslevel in den Azinuszellen der defizienten Tiere. Die Caerulein induzierte akute Pankreatitis zeigte einen milderen Verlauf in Bezug auf die Schweregradmarker Amylase und Lipase im Serum der IRAK-M -/- Tiere sowie einen geringen lokalen pankreatischen Schaden. Die Entzündungsreaktion erhöhte die MPO-Aktivität in der Lunge der defizienten Tiere. Zudem zeigten die Tiere eine erhöhte T-Zellaktivierung und Sekretion pro-inflammatorischer Zytokine wie TNFα und IL12 sowie des anti-inflammatorischen Zytokins IL10. Tendenziell wanderten mehr Neutrophile, M1- sowie M2- Makrophagen in das Pankreasgewebe während der Entzündung. Der Transkriptionsfaktor NF-κB konnte nach 8h akuter Pankreatitis, transloziert im Zellerkern, vermehrt in den IRAK-M defizienten Tieren nachgewiesen werden. Die Untersuchung von IRAK-M -/- BMDM zeigte, dass das Zytokin Milieu zur Differenzierung des M1 -Phänotyps dominierend war. Zudem lag eine verstärkte Phagozytose vor und die Makrophagen wiesen eine verstärkte Sekretion und Expression von TNFα, IL6, IL10 und IL12 auf. Nach 3d schwerer akuter Pankreatitis (SAP) wurde eine höhere Konzentration an Serumlipase sowie ein stärkerer pankreatischer Schaden beobachtet. Die MPO-Aktivität in der Lunge der defizienten Tiere war vermindert. Dennoch konnten vermehrt pro-inflammatorische Zytokine wie TNFα, IL6, IL12 und MCP1 im Serum gemessen werden. Die T-Zellen der defizienten Tiere zeigten zudem eine erhöhte Aktivierung. Die Visualisierung von infiltrierten Zellen im Pankreas zeigte keine Unterschiede. Der Vergleich der beiden experimentellen Pankreatitis-Modelle zeigte, dass die Caerulein induzierte Pankreatitis bei den IRAK-M -/- Tieren zu einer lokal begrenzten Entzündung im Pankreas führte. Wohingegen die Pankreatitis nach Gangligatur einen deutlich stärkeren Schaden aufwies und in einer signifikant erhöhten Zytokinsekretion resultierte. Der Vergleich von IL6 zeigte, dass die defizienten Tieren das 14-fache ins Serum sekretieren nach SAP, während die Kontrolltiere nur einen Anstieg um das 4,5- fache zeigten. Somit lässt sich zusammenfassen, dass eine kontrollierte und in Maßen ablaufende Immunantwort protektiv bzw. förderlich für den Krankheitsverlauf ist. Andererseits kann eine überschießende Immunantwort zu systemischen Komplikationen sowie Multiorganversagen führen. IRAK-M nimmt dabei eine regulierende Rolle ein und verhindert u.a., dass die Immunreaktion überschießt.
Overall, the present thesis provides tools for virus characterization. Importantly, the application of the developed tools contributed to the fundamental knowledge of selected, veterinary relevant viruses in terms of their underlying biology and virus-host interaction.
By using in vitro models and full-genome sequencing, important new findings were gained that contributed to the deeper understanding of the selected viruses. Results show that in vitro models can be successfully modified to enable study of specific host factors that are important for viral entry. By genetically modifying a bovine cell line using CRISPR/CAS9 technology , a stable cell culture model was established that is now available to the research community, to study the virus-host interaction of pestiviruses. The model was further used to elucidate the adaptability of bovine viral diarrhea the virus and impact on infectivity and growth. By using deep sequencing, genetic changes that occurred during the adaption process of bovine viral diarrhea virus were identified and linked to the phenotype, allowing the characterization of genetic regions important for virus binding to the host cell.
Whole-genome analysis using deep-sequencing was further used to characterize circulating rabbit haemorrhagic disease virus (RHDV) strains from Germany. The study provides more than 50 full genomes of RHDV strains sampled between 2013 and 2020. Since the virus family is drastically under sampled, in particular in central Europe, these sequences represent a very valuable addition to the field. The investigation led further to the discovery of a novel recombinant virus strain in hares, that is likely still circulating today. This finding is of special interest, since it is the first detection of a recombination event between the genogroups RHDV and European brown hare syndrome virus (EBHSV) of Lagoviruses. It highlights the importance of full genome virus surveillance and the potential risk of virus variants that might evade diagnostic detection.
Serological assay were used to study the persistency of antibodies developed during a natural infection with Schmallenberg virus. It could be shown that these antibodies are long lasting and therefore, re-emergence of this virus in Europe is likely favoured by introduction of naïve animals into a herd and not by decreasing antibody-titers over time.
Overall, the discoveries described in this thesis underline the importance of adequate tools for virus characterization and they give valuable answers to fundamental questions regarding the biology of the different viruses.
Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.
The human innate response plays a pivotal role in detection of pathogen- or damage-associated molecular patterns (PAMPs and DAMPs) and contributes to a crucial inflammatory response. PAMPs or DAMPs are recognized by the host immune system via pattern recognition receptors (PRRs). NLR family pyrin domain-containing 3 (NLRP3) inflammasome is one of these PRRs. NLRP3 is a cytoplasmic immune sensor that upon activation produce pro-inflammatory cytokines such as IL-1β and IL-18. These cytokines induce a diverse range of protective host pathways aiming to eradicate the pathogen. However, excessive or chronic inflammasome activation are implicated in the pathogenesis of several autoimmune and auto-inflammatory disorders. Pharmacologic inhibitors of IL-1 are commonly used to combat these disorders. In paper I, we explore the currently available IL-1β inhibiting therapies and how patients undergoing these treatments are at a disproportionate risk to experience invasive bacterial infections. We also summarize the limited knowledge on the role of NLRP3 inflammasome in pneumococcal pathogenesis.
Hydrogen peroxide (H2O2) is a physiological metabolite and an important virulence determinant produced by pneumococci. It is highly cytotoxic to host cells. However, not much is known about its impact on host cell death pathways such as NLRP3 inflammasome mediated pyroptosis. In Paper II, we examined the effect of pneumococci-derived H2O2 on epithelial cells by analyzing the interplay between two key cell death pathways, namely apoptosis and pyroptosis. We show that H2O2 can prime as well as activate the NLRP3 inflammasome. Furthermore, we demonstrate that pneumococcal H2O2 initiates cell death via the activation of both apoptotic as well as pyroptotic pathways, mediated by the activation of caspase-3/7 and caspase-1, respectively. H2O2 mediated inflammasome activation results in caspase-1 dependent IL 1β production. However, we show that the final IL-1β release is independent of gasdermin-D (GSDMD) and mainly dependent on the apoptotic cell lysis.
In paper III, we focused on understanding the host metabolic responses to infections with pathogens which cause respiratory diseases. We performed metabolome profiling of in vitro single bacterial and viral as well as co-infections of bronchial epithelial cells with Influenza A virus (IAV), Streptococcus pneumoniae, and Staphylococcus aureus. We show that IAV and S. aureus use the host resources for survival and multiplication and have minimal effects on the host metabolome. In contrast, pneumococci significantly alter various host metabolome pathways, including glycolysis, tricarboxylic acid (TCA) cycle and amino-acid metabolism. A hallmark of pneumococcal infections was the intracellular citrate accumulation, which was directly attributed to the action of pneumococci-derived H2O2.
Host cell death during an infection results in the release of pro-inflammatory cytokines and danger signals such as ATP. Released ATP can induce neutrophil chemotaxis mediated via purinergic signaling. Neutrophils are typically the first leukocytes to be recruited to the site of infection and are key players in bacterial clearance. However, excessive neutrophil activation is associated with further tissue injury. In paper IV, we investigated the role of ATP in neutrophil response to pneumococcal infections. We show that pneumolysin (Ply), a highly effective pore-forming toxin produced by pneumococci, is a potent activator of neutrophils. Microscale Thermophoresis analysis revealed that Ply and ATP bind to each other. Subsequently, ATP binding neutralizes Ply-mediated neutrophil degranulation, suggesting that Ply-ATP interactions are potentially beneficial during the course of the infection as this could limit the lung injury resulting from excessive Ply-mediated neutrophil activation.
Abstract
Background
The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard‐to‐transfect cells remains challenging.
Methods
In a proof‐of‐principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high‐fidelity Cas9 variant was then transfected into patient‐derived BOECs using a ribonucleoprotein complex and a single‐strand DNA oligonucleotide. In addition, patient‐specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation.
Results
Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9‐induced mutations in predicted off‐target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1‐deficient BOECs displayed increased resistance to stress‐induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1‐deficient BOECs largely replaced corrected as well as heterozygous BOECs.
Conclusion
We here demonstrate that a non‐viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard‐to‐transfect endothelial cells (ECs). Comparing patient‐derived isogenic CCM1+/+, CCM1+/−, and CCM1−/− ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.
Bei der familiären Form zerebraler kavernöser Malformationen (CCMs) handelt es sich um eine autosomal-dominant erbliche Erkrankung, die durch pathogene Keimbahnvarianten in den Genen CCM1/KRIT1, CCM2 bzw. CCM3/PDCD10 ausgelöst wird und mit einer Prävalenz von 1:3300 bis 1:3800 in der Allgemeinbevölkerung vorkommt. Neben Punktmutationen und kleinen Indels werden auch Strukturvarianten (SVs) in den Krankheitsgenen beobachtet. Diese sind jedoch mithilfe von Short-Read-basierten Genpanel- oder Exomsequenzierungen in der Routinediagnostik zum Teil nur schwer zu identifizieren.
Im Rahmen der vorliegenden Arbeit wurde daher eine neue CRISPR/Cas9-basierte Nanopore-Sequenzierung der Gene CCM1, CCM2 und CCM3 sowie der flankierenden genomischen Regionen etabliert. Die Abdeckung der Zielregionen mit Reads länger als 20 kb erlaubte die zuverlässige Detektion von Kopienzahlveränderungen. Darüber hinaus konnte anhand einer interchromosomalen Insertion und einer 24 kb großen Inversion gezeigt werden, dass die Methode auch komplexere SVs nachweisen kann. Das Protokoll einer Cas9-basierten Sequenzierung ausgewählter genomischer Zielregionen auf der MinION-Plattform kann gut auf andere Fragestellungen übertragen werden und lässt sich mit vergleichsweise geringem Aufwand in vielen Laboren etablieren.
Ein zweiter Schwerpunkt der Promotionsarbeit lag auf der Entwicklung neuer, vielseitig einsetzbarer Zellkulturmodelle der CCM-Erkrankung. Mithilfe CRISPR/Cas9-vermittelter Genomeditierungen in fluoreszenzmarkierten humanen induzierten pluripotenten Stammzellen (hiPSCs) konnten komplexe Kokulturmodelle sowie dreidimensionale vaskuläre Organoidkulturen etabliert werden. Anhand von aus CCM3-defizienten hiPSCs differenzierten humanen Endothelzellen (ECs) und vaskulären Mosaikorganoiden ließ sich ein klonaler Überlebensvorteil von CCM3-/--Zellen in Kokultur mit Wildtyp-Zellen nachweisen, welcher kürzlich auch in CCM-Mausmodellen als Schlüsselelement der CCM-Pathogenese beschrieben wurde. In den neuen Zellkulturmodellen ließ sich auch der positive Effekt des in der Greifswalder Arbeitsgruppe identifizierten Kandidatenwirkstoffs NSC59984 auf die tumorähnliche Proliferation von CCM3-/--Zellen in Kokultur validieren. Durch systematische RNA-Sequenzierungen konnte zudem nachgewiesen werden, dass CCM1 für die Differenzierung von ECs aus hiPSCs nicht erforderlich ist, jedoch die physiologische Funktion von ECs aufrechterhält.
Die Ergebnisse der vorliegenden Arbeiten belegen nicht nur die Praktikabilität und Sicherheit der Identifizierung komplexer genomischer Strukturvarianten mittels CRISPR/Cas9-vermittelter Nanopore-Sequenzierungen, sondern sie ebnen zudem den Weg zur effektiven Testung neuer CCM-Therapieansätze im Hochdurchsatzverfahren und erlauben einen neuen Einblick in die noch immer unvollständig verstandene CCM-Pathogenese.
Group A streptococcus (GAS) and Streptococcus pneumoniae are both Gram-positive bacteria that asymptomatically colonise various human body parts. Both microbes cause diseases ranging from mild to severe invasive infections. The later are associated with high mortality. GAS is the major microbial aetiology of type II necrotising skin and soft tissue infections (NSTIs). Type II NSTIs typically affect the lower and upper limbs of healthy young adults and often require debridement as a surgical intervention to prevent the spread of infection. S. pneumoniae is the major cause of respiratory tract infections including community-acquired pneumonia in young children and the elderly. Although most respiratory tract infections are successfully treated with antibiotics, emerging antibiotic resistance is a major cause of concern. Secreted virulence factors of Gram-positive bacteria play a major role in the successful invasion of host tissues causing different diseases. Additionally, they facilitate the spread of infection, contribute to tissue pathology, and potentially act as immune evasion mechanisms. This thesis summarises the consequences of streptococcal pyrogenic exotoxin B (SpeB), a potent cysteine protease secreted by GAS and pneumococci-derived hydrogen peroxide (H2O2) on host responses.
GAS have developed genetic or phenotypic ways of adapting to the immune response to escape immune clearance. Analysis of GAS clones recovered from NSTI patient biopsies exhibit a mixed SpeB phenotype, with most clones being SpeB negative. SpeB negative clones have been associated with hyper-virulence. In Paper II, we showed that SpeB negative GAS clones recovered from tissue exhibit reversible impaired SpeB secretion due to environmental factors. In addition, mutations in covS and ropB, the major transcriptional regulators of SpeB expression, were responsible for the irreversible loss of SpeB expression. Immunohistochemistry analysis demonstrated that neutrophil degranulation, necrosis and excessive inflammation observed in NSTIs patient biopsies correlated with bacterial load and SpeB negativity of clones. Proteomic data analysis showed that SpeB negative GAS recovered from neutrophil infection harboured the protease intracellularly suggesting that the bacteria expressed but did not secrete SpeB. We have also shown that neutrophil-derived reactive oxygen species, H2O2 and hypochlorous acid, drive the SpeB negative phenotype. The SpeB negative clones survived neutrophil-mediated antimicrobial killing and induced excessive degranulation when compared with SpeB positive clones. These results provide new insights into GAS fitness induced by host factors in tissue and may be useful for the development of new treatment strategies in NSTIs.
Pneumococci produce H2O2 as a by-product of carbohydrate metabolism in a reaction catalysed by pyruvate oxidase SpxB. However, very little is known about the effects of pneumococcal H2O2 as a virulence factor. Our study aimed to investigate the role of H2O2 in initiating epithelial cell death, focusing on apoptosis and pyroptosis. In Paper III, we showed that pneumococci-derived H2O2 caused epithelial cell cytotoxicity by priming and activating the NLRP3 inflammasome resulting in subsequent IL-1β production and release. Additionally, H2O2 caused apoptotic and pyroptotic cell death as evidenced by activation of caspase-3/7 and caspase-1, respectively. However, the release of IL-1β was dependent on apoptosis and not pyroptosis since inactive gasdermin D was detected post-infection. These observations were not detected in the absence of H2O2. Overall, we showed the damaging effects of pneumococci-derived H2O2 on human bronchial epithelial cells.
Mechanically ventilated patients are at risk of ventilator-associated pneumonia, a serious infection of the lungs. Not every ventilated patient develops pneumonia due to a combination of the protective layer of mucus in the airways, the immune system and prophylactic antibiotic therapy. To date, only little was known about the antimicrobial factors produced by humans that protect the lungs against infection. Research described in this thesis was therefore aimed at investigating to what extent the lungs of ventilated patients can inhibit the growth of bacteria, the major causative agent of pneumonia Streptococcus pneumoniae in particular. To this end, the accumulated mucus in the patients’ lungs, sputum, was investigated. The most important conclusion was that sputum can indeed possess antimicrobial activity, explained either by a combination of antibiotics and S. pneumoniae-specific antibodies, or by the innate immune defenses. Thus, sputum may serve as a valuable source of information to unravel the complex interactions between the human host, antimicrobial factors and the microbiome of the lower respiratory tract. A possible consequence of pneumonia is the dissemination of bacteria from the lungs to the bloodstream and the brain, which may lead to meningitis. This thesis describes how this process takes place, and how the so-called choline-binding protein CbpL contributes to invasive pneumococcal infections. In addition, possible future approaches to prevent meningitis caused by this bacterium are proposed.
Zerebrale kavernöse Malformationen (CCMs) sind vaskuläre Fehlbildungen des zentralen Nervensystems, welche sich klinisch durch epileptische Anfälle, fokale neurologische Ausfälle und Kopfschmerzen äußern. Sie treten sowohl sporadisch als auch im Rahmen einer autosomal-dominant erblichen Form auf. Krankheitsassoziierte Varianten wurden in drei Genen beschrieben: CCM1, CCM2 und CCM3. Patienten mit pathogenen Varianten im CCM3-Gen fallen häufig durch einen schwereren Phänotyp und ein früheres Manifestationsalter auf. Der genaue Verlauf der molekularen CCM-Pathogenese ist jedoch bisher nicht ausreichend verstanden. In dieser Arbeit wurde deshalb die Entwicklung eines humanen in vitro Modells in den Fokus gestellt. Im Gegensatz zu bisher publizierten Studien, die auf einer transienten Herunterregulation von CCM3 beruhen, wurden hier die Folgen eines Langzeit-CCM3-Verlustes untersucht. Unter Verwendung der Komponenten des CRISPR/Cas9-Systems wurde ein Modell etabliert, welches die komplette Inaktivierung von CCM3 in kavernösen Endothelzellen von Trägern heterozygoter pathogener Keimbahnvarianten nachahmt.
In humanen Endothelzellen führte die CRISPR/Cas9-vermittelte CCM3-Inaktivierung zu veränderten endothelialen Eigenschaften, welche die Morphologie, die Apoptoseinduktion, die Stabilität kapillarähnlicher Strukturen sowie die Sphäroidorganisation umfassen. Zudem kam es zu einer vermehrten Aktin-Stressfaserbildung. Auf molekularer Ebene war eine veränderte Expression von Genen zu beobachten, deren Produkte eine Rolle in der Regulation von Zellverbindungen, der Zellmembran oder der extrazellulären Matrix spielen. Insbesondere das unter Basalbedingungen stark in Endothelzellen exprimierte Fibronektin war signifikant herunterreguliert. Die Zugabe exogenen Fibronektins konnte die Zellmorphologie, die Sphäroidorganisation und die kortikale Anordnung des Aktin-Zytoskelettes normalisieren. Da auch nach CRISPR/Cas9-induzierter Inaktivierung von CCM1 und CCM2 diese Effekte beobachtet werden konnten, deuten diese Ergebnisse darauf hin, dass der Aufbau einer intakten fibronektinhaltigen endothelialen Matrix von der Funktionalität der CCM-Proteine abhängt. Künftige Studien werden die Modulation der gestörten endothelialen Apoptoseinduktion als neuen Ansatz zur Identifizierung medikamentöser Therapieoptionen adressieren.
The human brain is distinguished by its remarkable size, high energy consumption, and cognitive abilities compared to all other mammals and non-human primates. However, little is known about what has accelerated brain evolution in the human lineage. One possible explanation is that the appearance of advanced communication skills and language has been a driving force of human brain development. The phenotypic adaptations in brain structure and function which occurred on the way to modern humans may be associated with specific molecular signatures in today’s human genome and/or transcriptome. Genes that have been linked to language, reading, and/or autism spectrum disorders are prime candidates when searching for genes for human-specific communication abilities. The database and genome-wide expression analyses we present here revealed a clustering of such communication-associated genes (COAG) on human chromosomes X and 7, in particular chromosome 7q31-q36. Compared to the rest of the genome, we found a high number of COAG to be differentially expressed in the cortices of humans and non-human primates (chimpanzee, baboon, and/or marmoset). The role of X-linked genes for the development of human-specific cognitive abilities is well known. We now propose that chromosome 7q31-q36 also represents a hot spot for the evolution of human-specific communication abilities. Selective pressure on the T cell receptor beta locus on chromosome 7q34, which plays a pivotal role in the immune system, could have led to rapid dissemination of positive gene variants in hitchhiking COAG.
Lung dendritic cells facilitate extrapulmonary bacterial dissemination during pneumococcal pneumonia
(2013)
Streptococcus pneumoniae is a leading cause of bacterial pneumonia worldwide. Given the critical role of dendritic cells (DCs) in regulating and modulating the immune response to pathogens, we investigated here the role of DCs in S. pneumoniae lung infections. Using a well-established transgenic mouse line which allows the conditional transient depletion of DCs, we showed that ablation of DCs resulted in enhanced resistance to intranasal challenge with S. pneumoniae. DCs-depleted mice exhibited delayed bacterial systemic dissemination, significantly reduced bacterial loads in the infected organs and lower levels of serum inflammatory mediators than non-depleted animals. The increased resistance of DCs-depleted mice to S. pneumoniae was associated with a better capacity to restrict pneumococci extrapulmonary dissemination. Furthermore, we demonstrated that S. pneumoniae disseminated from the lungs into the regional lymph nodes in a cell-independent manner and that this direct way of dissemination was much more efficient in the presence of DCs. We also provide evidence that S. pneumoniae induces expression and activation of matrix metalloproteinase-9 (MMP-9) in cultured bone marrow-derived DCs. MMP-9 is a protease involved in the breakdown of extracellular matrix proteins and is critical for DC trafficking across extracellular matrix and basement membranes during the migration from the periphery to the lymph nodes. MMP-9 was also significantly up-regulated in the lungs of mice after intranasal infection with S. pneumoniae. Notably, the expression levels of MMP-9 in the infected lungs were significantly decreased after depletion of DCs suggesting the involvement of DCs in MMP-9 production during pneumococcal pneumonia. Thus, we propose that S. pneumoniae can exploit the DC-derived proteolysis to open tissue barriers thereby facilitating its own dissemination from the local site of infection.
Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import. Bacillus subtilis imports a large set of osmostress protectants via five osmotically controlled transport systems (OpuA to OpuE). Biosynthesis of the particularly effective osmoprotectant glycine betaine requires the exogenous supply of choline. While OpuB is rather specific for choline, OpuC imports a broad spectrum of compatible solutes, including choline and glycine betaine. One previously mapped antisense RNA of B. subtilis, S1290, exhibits strong and transient expression in response to a suddenly imposed salt stress. It covers the coding region of the opuB operon and is expressed from a strictly SigB-dependent promoter. By inactivation of this promoter and analysis of opuB and opuC transcript levels, we discovered a time-delayed osmotic induction of opuB that crucially depends on the S1290 antisense RNA and on the degree of the imposed osmotic stress. Time-delayed osmotic induction of opuB is apparently caused by transcriptional interference of RNA-polymerase complexes driving synthesis of the converging opuB and S1290 mRNAs. When our data are viewed in an ecophysiological framework, it appears that during the early adjustment phase of B. subtilis to acute osmotic stress, the cell prefers to initially rely on the transport activity of the promiscuous OpuC system and only subsequently fully induces opuB. Our data also reveal an integration of osmostress-specific adjustment systems with the SigB-controlled general stress response at a deeper level than previously appreciated.
Bei dem weit verbreiteten, Gram-positiven Bakterium Bacillus subtilis handelt es sich um einen der am besten charakterisierten Organismen. Drei Faktoren haben insbesondere zur Etablierung von B. subtilis als Modellorganismus beigetragen: I.) die einfache genetische Manipulierbarkeit und Handhabung im Labor, II.) das Interesse an zellulären Differenzierungsprozessen wie der Bildung von Endosporen und III.) die biotechnologische Bedeutung von Bacillus-Arten. Deshalb sind die zellulären Komponenten, wie Proteine, Metabolite und regulatorische RNAs, und physiologischen Prozesse von B. subtilis, einschließlich seiner Anpassungsmechanismen an verschiedene Nährstoff- und Stressbedingungen, bereits sehr gut charakterisiert. Im Mittelpunkt der vorliegenden Arbeit stand die Anpassung der Genexpression von B. subtilis an hyperosmotische Bedingungen, mit denen das Bakterium in seinen natürlichen Habitaten häufig konfrontiert ist.
Die Konstruktion genomreduzierter Stämme ist ein Ansatz, die Interaktionen zwischen den zellulären Komponenten sowie die Funktion aller Proteine und funktionellen RNAs eines Organismus besser zu verstehen. Das MiniBacillus-Projekt verfolgt das Ziel, durch schrittweise Deletion aller nicht-essentiellen genomischen Regionen einen B. subtilis-Stamm zu entwickeln, der mit einem minimalen Set an Genen in komplexen Medien wachsen kann. In einer von der Arbeitsgruppe um Prof. J. Stülke (Georg-August-Universität Göttingen) koordinierten Studie erfolgte die bisher umfassendste Multi-Omics-basierte Charakterisierung von genomreduzierten Stämmen. Die zwei analysierten Stämme wiesen eine Genomreduzierung von ca. 35 % auf. Im Rahmen der vorliegenden Arbeit wurden das Transkriptom der Stämme im Vergleich zum Ausgangsstamm unter Verwendung von strangspezifischen Tiling-Arrays untersucht sowie phänotypische Merkmale der Zellen mittels Fluoreszenzmikroskopie analysiert. Die Studie lieferte wichtige Befunde zur Verteilung der zellulären Translationskapazität und zeigte beispielsweise, dass der Anteil der essentiellen Proteine am gesamten Proteom in den drei B. subtilis-Stämmen größer als 50 % ist, während Proteine mit unbekannter Funktion weniger als 3 % des Proteoms ausmachen. Weiterhin wurden die Auswirkungen der Deletionen auf die Genexpression und den Metabolismus untersucht, wobei sich die Unterschiede zwischen den genomreduzierten Stämmen und dem Ausgangsstamm zum größten Teil mit Veränderungen der Menge oder der Aktivität von Transkriptionsfaktoren erklären lassen. Die Multi-Omics-Studie trug nicht nur zum besseren Verständnis der zellulären Netzwerke bei, sondern identifizierte auch Ansatzpunkte für die gezielte Deletion weiterer genomischer Bereiche.
Eine weitere Transkriptomanalyse wurde im Rahmen eines Projektes mit der Arbeitsgruppe um Prof. J. Stülke zur Charakterisierung der physiologischen Rolle des sekundären Botenstoffs c-di-AMP durchgeführt. c-di-AMP ist ein essentielles Signalnukleotid vieler Bakterien, das bei Gram-positiven Organismen eine wichtige Rolle bei der zellulären Kalium-Homöostase spielt, aber auch weitere Funktionen wie den Zellwandmetabolismus beeinflusst. Im Rahmen dieser Arbeit wurden die Genexpressionsmuster des B. subtilis-Stammes 168 und einer Mutante, die c-di-AMP nicht abbauen kann, miteinander verglichen, um die Auswirkungen einer Akkumulation von c-di-AMP auf globaler Ebene zu untersuchen. Zu den stärksten Effekten gehörte die Repression der beiden Operons, die für die Biofilmbildung entscheidend sind. In weiteren Experimenten in Göttingen wurde mit einem nicht domestizierten Stamm gezeigt, dass B. subtilis bei hohen intrazellulären c-di-AMP-Konzentrationen keinen Biofilm ausbilden kann.
Ebenfalls im Rahmen eines Kooperationsprojektes mit der Arbeitsgruppe um Prof. J. Stülke wurde eine Transkriptomanalyse durchgeführt, um den Zusammenhang zwischen Veränderungen der DNA-Topologie und dem Glutamatstoffwechsel von B. subtilis aufzuklären. Der Ausgangspunkt dieser Analyse waren Studien der Göttinger Arbeitsgruppe zum zentralen Regulator der Kohlenstoff-Katabolitenrepression, CcpA, dessen Inaktivierung dazu führt, dass B. subtilis Glutamat nicht selbst synthetisieren kann. Die Ursache dafür ist die konstitutive Expression des rocG-Gens, das einen negativen Effektor des Transkriptionsaktivators GltC kodiert. Bestimmte Supressormutanten, die ohne Zugabe von Glutamat wachsen können, besitzen aufgrund einer Punktmutation im topA-Gen eine hyperaktive Form der Topoisomerase I, die mit einer Reduktion des negativen DNA-Supercoilings verbunden ist. In der durchgeführten Transkriptomanalyse wurden mehr als 1100 Gene identifiziert, die in der ccpA topAhyper-Mutante eine im Vergleich zur ccpA-Mutante veränderte Expression aufwiesen. Die damit verbundenen Auswirkungen auf das metabolische Netzwerk von B. subtilis führen dazu, dass RocG seine regulatorische Funktion nicht mehr ausüben kann und GltC dadurch in der Lage ist, die Transkription des Glutamat-Biosynthese-Operons gltAB zu aktivieren. Insgesamt hat die Studie Verbindungen zwischen der DNA-Topologie und dem metabolischen Netzwerk gezeigt, die unter physiologischen und biotechnologischen Aspekten von Interesse sind.
Um ein Absinken des Turgors unter hyperosmotischen Bedingungen zu vermeiden, akkumulieren Bakterien kompatible Solute im Zytoplasma. B. subtilis kann mit Hilfe von fünf osmotisch induzierbaren Aufnahmesystemen (OpuA bis OpuE, engl. osmostress protectant uptake) mindestens 15 verschiedene osmoprotektive Substanzen aus der Umgebung aufnehmen. Die aus einem Genduplikationsereignis hervorgegangenen Transporter OpuB und OpuC weisen eine Sequenzhomologie von über 70 % auf, unterscheiden sich aber dennoch deutlich hinsichtlich ihres Substratspektrums. Während OpuB weitgehend spezifisch für das Glycin-Betain-Vorläufermolekül Cholin ist, kann der OpuC-Transporter eine Vielzahl an verschiedenen Substanzen, einschließlich Glycin-Betain und Cholin, aufnehmen. Für das opuB-Operon wurde in einer vorangegangenen Transkriptomstudie ein Antisense-Transkript mit der Bezeichnung S1290 identifiziert, welches eine starke transiente Induktion nach Einwirkung eines Salzschocks zeigte. Im Rahmen der vorliegenden Arbeit wurde die bioinformatisch vorhergesagte Abhängigkeit der S1290-Expression vom alternativen RNA-Polymerase-Sigmafaktor SigB, dem zentralen Regulator der generellen Stressantwort von B. subtilis, experimentell bestätigt und die potentielle regulatorische Rolle der asRNA (engl. antisense RNA) untersucht. Es wurde gezeigt, dass die S1290-asRNA für die zeitlich verzögerte Induktion von opuB nach einem Salzschock verantwortlich ist. Diese Ergebnisse lassen darauf schließen, dass B. subtilis die effektive Nutzung der vorhandenen osmoprotektiven Substanzen und der energetischen Ressourcen erreicht, indem zunächst vorrangig die Gene induziert werden, die für den relativ unspezifischen OpuC-Transporter kodieren, und erst danach die Synthese des Cholin-spezifischen Transporters OpuB aktiviert wird.
Neben der Akkumulation von osmoprotektiven Substanzen sind andauernde hyperosmotische Bedingungen mit vielen weiteren Veränderungen der Genexpression und Physiologie von B. subtilis verbunden. Dazu zählen unter anderem Veränderungen im Zellwandmetabolismus, die Induktion einer Eisenlimitationsantwort, eine reduzierte Motilität und die Unterdrückung der Sporulation. Um zusätzliche Facetten der Anpassung von B. subtilis an ein kontinuierliches Wachstum unter hyperosmotischen Bedingungen aufzudecken, erfolgte im Rahmen dieser Arbeit eine globale Analyse des Transkriptoms unter Verwendung strangspezifischer Tiling-Arrays. Der Vergleich der Transkriptmengen unter Hochsalzbedingungen (1,2 M NaCl) und unter Standardbedingungen zeigte Veränderungen der Expression von mehr als einem Viertel der proteinkodierenden Gene sowie zahlreicher nicht-kodierender RNAs. Es wurden bisher nicht bekannte Anpassungsreaktionen von B. subtilis an hyperosmotische Bedingungen identifiziert, wie beispielsweise eine anhaltende nicht-maximale Induktion des SigB-Regulons und eine erhöhte Expression CymR-abhängiger Gene. Ein weiteres Ergebnis der durchgeführten Studie war, dass eine hohe Osmolarität der Umgebung nicht nur die Motilität der B. subtilis-Zellen negativ beeinflusst, sondern auch zu einer stark verringerten Biofilmbildung führt. Die Akkumulation von osmoprotektiven Substanzen wie Glycin-Betain wirkt der Dehydrierung des Zytoplasmas entgegen, kann aber zelluläre Prozesse auch durch eine stabilisierende Wirkung auf Proteine und Nukleinsäuren beeinflussen. Die genomweiten Effekte von Glycin-Betain auf die Genexpression von B. subtilis wurden in dieser Arbeit zum ersten Mal untersucht. In Gegenwart von 1,2 M NaCl und 1 mM Glycin-Betain wiesen 40 % der Gene, die unter Hochsalzbedingungen im Vergleich zur Kontrolle induziert waren, eine signifikant verringerte Expression auf. Darunter befanden sich Gene, die für Opu-Aufnahmesysteme kodieren, sowie viele Gene des SigB-Regulons. Demgegenüber wird die bei hohen Salzkonzentrationen unterdrückte Expression der Sporulationsgene durch die Supplementation des Mediums mit Glycin-Betain nicht aufgehoben. Die Transkriptmengen von 150 Genen waren durch Glycin-Betain sowohl unter Hochsalz- als auch unter Kontrollbedingungen beeinflusst. Mit dieser Studie wurden umfassende Daten zur Genexpression von B. subtilis unter hyperosmotischen Bedingungen generiert, wodurch neue Facetten der zellulären Anpassung sowie globale Effekte der osmoprotektiven Substanz Glycin-Betain aufgedeckt werden konnten.
Hintergrundinformationen: Bakterien gehören zu den ältesten Lebensformen und sind ein elementarer Bestandteil aller ökologischen Lebensräume auf der Erde. Der Mensch als Holobiont ist ein eigenständiges Ökosystem mit einer Vielzahl von ökologischen Nischen und einer großen bakteriellen Vielfalt. Durch innere oder äußere Einflüsse kann es zu Veränderungen der Umweltbedingungen kommen, die eine veränderte Zusammensetzung des Mikrobioms zur Folge haben. Eine solche Dysbiose wirkt sich auf den Gesundheitszustand des Menschen aus und kann zu schweren Krankheiten führen. Das orale Mikrobiom gehört mit zu den komplexesten Mikrobiomen des Menschen. Es bildet eine natürliche Barriere gegen Krankheitserreger und beugt somit u.a. lokalen Krankheiten wie Karies oder Parodontitis vor. Die Metaproteomik ermöglicht es, die exprimierten Proteine des Mikrobioms und deren Interaktion mit dem Wirt zu untersuchen. Diese Technologie überwindet somit die Beschränkung auf Laborkulturen und ermöglicht die Untersuchung des Mikrobioms direkt in seinem natürlichen Lebensraum. Die Metaproteomik bietet eine Reihe von Instrumenten zur Vertiefung des Verständnisses des oralen Mikrobioms hinsichtlich des Gesundheitszustandes des Menschen.
Ziele: Ein Ziel dieser Dissertation war es einen Arbeitsablauf für die Durchführung von Metaproteomstudien des oralen Mikrobioms zu erarbeiten, beginnend bei der Probensammlung über die Präparation der Proben für die Massenspektrometrie bis hin zur bioinformatischen Auswertung. Diesen Arbeitsablauf galt es für das Mikrobiom des Speichels sowie für die Biofilme auf der Zunge und des supragingivalen Plaques zu etablieren bzw. zu adaptieren. Darauf aufbauend wurden Metaproteomstudien durchgeführt, um die drei Mikrobiome bei gesunden Probanden hinsichtlich ihrer exprimierten Proteine, deren metabolischer Bedeutung und Interaktionen mit dem Wirt sowie deren taxonomische Zuordnung zu studieren.
Studiendesign: Die Dissertation umfasst drei Studien mit drei unterschiedlichen Kohorten. Allen Studien ist gemein, dass die Kohorten sich aus oral gesunden Probanden im Alter von 20-30 Jahren zusammensetzten.
In der ersten Studie verglichen wir die Salivette® sowie den Paraffinkaugummi anhand von fünf Probanden, um die effektivste Methode zur Sammlung von Speichel für Metaproteomstudien zu identifizieren.
In der zweiten Studie wurden die Mikrobiome von Speichel und Zunge anhand von 24 Probanden miteinander verglichen und dafür eine Auswertestrategie entwickelt, um der Komplexität dieser Metaproteomstudie gerecht zu werden.
Im Rahmen unserer dritten randomisierten Einzelblindstudie, die auf einem Cross-over-Design basierte, erhielten 16 Probanden vier unterschiedliche lokale Behandlungsschemata, um deren Auswirkung auf das Plaque-Mikrobiom zu untersuchen. Die Behandlungen bestanden aus zwei Lutschtabletten, die Bestandteile des Lactoperoxidase-Systems in unterschiedlichen Konzentrationen enthielten, einer Lutschtablette mit einem Placebo-Wirkstoff sowie Listerine® Total Care™ Mundspülung als Positivkontrolle.
Alle Proben wurden, basierend auf einem Bottom-Up-Ansatz, unter Verwendung von nano LC-MS/MS Massenspektrometern in einer datenabhängigen Messstrategie (DDA, data- dependant acquisition mode) vermessen. Die bioinformatische Auswertung erfolgte für die erste Studie mit Hilfe der Proteome Discoverer Software. Für die Studien zwei und drei wurde die Trans-Proteomic Pipeline eingesetzt. Die taxonomische sowie funktionelle Zuordnung der identifizierten Proteine erfolgte für alle Studien anhand der Prophane Software.
Ergebnisse:
Für den Paraffinkaugummi konnten wir mit 1.005 bakteriellen Metaproteinen dreimal so viele Metaproteine identifizieren im Vergleich zur Salivette® mit 313 Metaproteinen. 76,5 % der Metaproteine der Salivette® wurden ebenfalls mit dem Paraffinkaugummi gefunden. Insgesamt wurden 38 Genera und 90 Spezies identifiziert, wovon 13 Genera und 44 Spezies nur mit dem Paraffinkaugummi identifiziert werden konnten. Die größte funktionelle Diversität wurde ebenfalls mit dem Paraffinkaugummi detektiert.
Das Metaproteom des Speichel- und Zungen-Mikrobioms basiert auf 3.969 bakteriellen Metaproteinen sowie 1.857 humanen Proteinen. Die Anzahl der nur für das Zungen-Mikrobiom identifizierten Metaproteine, war doppelt so hoch, im Vergleich zum Speichel.
Die Metaproteine konnten 107 Genera sowie 7 Phyla zugeordnet werden. Funktionell wurden für das Speichel-Mikrobiom signifikant höhere Metaproteinabundanzen für die Zellmotilität gefunden. Beim Zungen-Mikrobiom hingegen wiesen die Metaproteine der Biosynthese von sekundären Metaboliten, Signaltransduktion oder der Replikation höhere Abundanzen auf.
Im Rahmen der Plaque-Studie identifizierten wir durchschnittlich 1.916 (± 465) bakterielle Metaproteine je Probe, die wir taxonomisch und funktionell 116 Genera sowie 1.316 Proteinfunktionen zuordnen konnten. Die Plaque inhibierende Wirkung von Listerine® zeigte sich durch eine Reduktion der Metaproteinidentifikation von durchschnittlich 23,5 % nach der Behandlung. Darüber hinaus zeigte die Mehrheit der bakteriellen Metaproteine reduzierte relative Abundanzen während für die Metaproteine humanen Ursprungs eine Erhöhung der Proteinabundanzen gegenüber der Kontrolle vor Behandlung zu verzeichnen war. Aus funktioneller Sicht waren insbesondere metabolische Prozesse, welche für das Zellwachstum und die Zellteilung wichtig sind, betroffen. Im Gegensatz dazu erhöhten sich durch die LPO Lutschtabletten sowohl die Identifikation der Metaproteine als auch die relative Abundanz für die Mehrheit der Proteine. Nach den durch die Metaproteomdaten erhaltenen funktionellen Informationen liegen Hinweise für einen wachsenden Biofilm vor. Die Metaproteine, die eine erhöhte Abundanz nach Behandlung mit den LPO-Dragees zeigten, wurden taxonomisch hauptsächlich Erst- (S. gordonii) und Zweitbesiedlern (F. nucleatum) sowie Bakterien zugeordnet, die einem gesunden Biofilm zuträglich sind.
Fazit: Im Rahmen dieser Dissertation wurde ein vollständiger Metaproteom Arbeitsablauf von der Probensammlung, über die Probenpräparation bis hin zu Datenanalyse für das Speichel-, Zungen- und Plaque-Mikrobiom erarbeitet. In drei Studien konnten wir dessen Anwendbarkeit demonstrieren und erreichten vergleichbare Ergebnisse zu anderen Metaproteomstudien, beispielsweise bezüglich der Proteinidentifikation. Für die Sammlung von Speichelproben stellte sich der Paraffinkaugummi für Metaproteomstudien als die Methode der Wahl heraus. Für das Zungen-Mikrobiom veröffentlichten wir die ersten Metaproteomdaten. Darüber hinaus publizierten wir die erste Metaproteomstudie, welche die beiden Mikrobiome von Speichel und Zunge miteinander vergleicht. Hinsichtlich des Plaque-Mikrobioms handelte es sich ebenfalls um die erste Metaproteomstudie, die ein
anerkanntes und etabliertes zahnklinisches Modell mit den Vorzügen der Metaproteomiks verbindet. Die Ergebnisse liefern erste Daten, um (auf längere Sicht gesehen) ein Produkt zur täglichen Mundhygiene entwickeln zu können, welches die bakterielle Zusammensetzung des Plaque-Biofilms positiv beeinflusst.
Zerebrale kavernöse Malformationen (CCMs) sind Gefäßfehlbildungen im Gehirn oder Rückenmark und können sich klinisch aufgrund einer erhöhten Blutungsbereitschaft mit Kopfschmerzen, Gefühls- und Sprachstörungen bis hin zu Krampfanfällen äußern. Sie treten sporadisch oder im Rahmen einer autosomal-dominant erblichen Form auf. Kausale Sequenzveränderungen sind dabei in den drei Genen CCM1, CCM2 und CCM3 bekannt. Die Detektionsrate für pathogene Varianten ist mit bis zu 60 % für sporadische Fälle und mit weit über 90 % für familiäre Fälle sehr hoch. Während Genpanel-Analysen sehr verlässlich Einzelnukleotidveränderungen, kleine Insertions- und Deletionsvarianten sowie Kopienzahlveränderungen detektieren können, werden komplexe Strukturvarianten oder Veränderungen in nicht-kodierenden Regionen kaum erfasst. Diese rücken jedoch für die bisher genetisch unaufgeklärten Fälle immer mehr in den Fokus des Interesses. Diese Arbeit adressiert daher zum einen die Identifizierung neuer Strukturvarianten und deren funktionale Interpretation im Kontext der CCM-Erkrankung.
Im Rahmen der vorliegenden Arbeit ist der erstmalige Nachweis einer interchromosomalen Insertion bei einem CCM-Patienten gelungen. Die unbalancierte Insertion genomischen Materials von Chromosom 1 in die kodierende Region des CCM2-Gens konnte durch die Verbindung von bioinformatischen Auswertestrategien der Next Generation Sequencing-Genpanel-Daten, molekularzytogenetischen Analysen und einer molekularen Bruchpunktkartierung genau charakterisiert werden. Die Identifikation einer weiteren Strukturvariante, einer Deletion des Transkriptionsstarts von CCM1, verdeutlichte die Herausforderungen bei der Bewertung von Veränderungen in nicht-kodierenden Genbereichen. Für eine eindeutige Klassifikation der Variante wurden daher funktionale Analysen durchgeführt, die auf einer CRISPR/Cas9-vermittelten Nachbildung der Deletion in iPSCs und der anschließenden Differenzierung in Endothelzellen beruhte. Damit konnte gezeigt werden, dass die Deletion zu einem Verlust der CCM1 mRNA- und Proteinexpression führt. Zudem wurde in den differenzierten Endothelzellen eine für die CCM-Pathogenese charakteristische Deregulation von KLF2, THBS1, NOS3 und HEY2 beobachtet. Schließlich war es auf Basis dieser in vitro-Analysen möglich, die Variante entsprechend den ACMG-Richtlinien als wahrscheinlich pathogen zu bewerten und somit die molekulare CCM-Diagnose zu sichern.
Die Verbindung des CRISPR/Cas9-Systems mit iPSCs ist nicht nur für die Variantenbewertung von großem Nutzen, sondern bietet auch das Potential zum besseren Verständnis von Krankheitsmechanismen. Ein weiterer Fokus der vorliegenden Arbeit lag daher auf der Etablierung und Verwendung iPSC-basierter Zellkulturmodelle für die CCM-Modellierung. Zunächst ist es gelungen, mehrere iPSC-Linien mit einer kompletten CRISPR/Cas9-vermittelten CCM1-, CCM2- oder CCM3-Inaktivierung zu generieren. Diese wurden anschließend für die Differenzierung in hBMEC-ähnliche Zellen und innovative dreidimensionale vaskuläre Organoide verwendet. In diesen Systemen konnte beispielsweise eindrücklich eine tumorähnliche Proliferation CCM3-defizienter Endothelzellen nachvollzogen werden, die nur in Kontakt mit Wildtyp-Zellen auftrat. RNA-Sequenzierungen in einem CCM1-basierten Knockout-Modell konnten darüber hinaus die Rolle von CCM1 als Endothel-spezifisches Suppressorgen stärken. Die im Rahmen der Arbeit etablierten Systeme werden zukünftig für weitere Fragestellungen der CCM-Pathogenese wie der endothelialen Barrierestörung eingesetzt und stellen darüber hinaus sehr gut geeignete Plattformen für die effektive Entwicklung dringend benötigter therapeutischer Ansätze dar.
Influenza A Virus (IAV), Staphylococcus aureus (staphylococci), and Streptococcus pneumoniae (pneumococci) are leading viral and bacterial causes of pneumonia. Dendritic cells (DCs) are present in the lower respiratory tract. They are characterized by low expression of co-stimulatory molecules, including CD80 and CD86 and high capacity of antigen uptake. Subsequently, DCs upregulate co-stimulatory signals and cytokine secretion to effectively induce T-cell priming. Here, we investigated these processes in response to bacterial and viral single as well as coinfections using human monocyte-derived (mo)DCs. Irrespective of single or coinfections, moDCs matured in response to IAV and/or staphylococcal infections, secreted a wide range of cytokines, and activated CD4+, CD8+ as well as double-negative T cells. In contrast, pneumococcal single and coinfections impaired moDC maturation, which was characterized by low expression of CD80 and CD86, downregulated expression of CD40, and a mild cytokine release resulting in abrogated CD4+ T-cell activation. These actions were attributed to the cholesterol-dependent cytotoxin pneumolysin (Ply). Infections with a ply-deficient mutant resulted in restored moDC maturation and exclusive CD4+ T-cell activation. These findings show that Ply has important immunomodulatory functions, supporting further investigations in specific modalities of Ply-DC interplay.
Staphylococcus aureus is one of the commonly encountered bacteria of the human microbiome. Although mostly a seemingly harmless commensal microbe, S. aureus can act as an invasive pathogen with seriously devastating effects on its host’s health and wellbeing. A wide range of infections caused by this bacterium has been reported to affect diverse parts of the human body, including the skin, soft tissues and bones, as well as important organs like the heart, kidneys and lungs. Particularly, S. aureus is infamous for being a major causative agent of respiratory tract infections that may escalate up to necrotizing pneumonia. Due to its clinical relevance, this pathogen has been intensively studied for many years. Nonetheless, further research in this field is still needed, because of the high capacity of S. aureus to evolve drug resistance, its high genomic plasticity and adaptability and, not in the last place, the plethora of niches within the human body where it can thrive and survive. In this regard, there are still many uncertainties concerning the specific adaptations carried out by S. aureus during colonization and infection of the human body, the transition between both stages, and upon the invasion of different types of host cells. To shed more light on some of these adaptations, the research described in this thesis has employed in vitro models of infection that mimic particular conditions during the infectious process with special focus on the lung epithelium. The adaptations displayed by S. aureus were monitored using advanced proteomics. Furthermore, the analyses documented in this thesis included S. aureus strains with diverse backgrounds and epidemiology to take into account the genetic diversity encountered in this species.
The Membrane Transporter OAT7 (SLC22A9) Is Not a Susceptibility Factor for Osteoporosis in Europeans
(2020)
Bone production, maintenance, and modeling are a well-balanced process involving mineralization by osteoblasts and resorption by osteoclasts. Sex steroid hormones, including their conjugated forms, contribute majorly to maintaining this balance. Recently, variants in the SLC22A9 gene have been associated with osteoporosis in Korean females. We had recently shown that SLC22A9, encoding organic anion transporter 7 (OAT7), is an uptake transporter of estrone sulfate and identified several genetic variants in Europeans leading to functional consequences in vitro. We therefore hypothesized that SLC22A9 genetic variants may contribute to the pathophysiology of osteoporosis in Europeans. To test this hypothesis, we examined the associations of SLC22A9 variants with bone quality, fractures, and bone turnover markers. We genotyped SLC22A9 variants in 5,701 (2,930 female) subjects (age range, 20–93 years) extracted from the population-based Study of Health in Pomerania (SHIP and SHIP-TREND) covered by the Illumina Infinium HumanExome BeadChip version v1.0 (Exome Chip). Descriptive data (e.g., history of fractures), ultrasonography of the calcaneus, as well as serum concentrations of carboxy-terminal telopeptide of type I collagen, amino-terminal propeptide of type I procollagen, and vitamin D were determined. Comprehensive statistical analyses revealed no association between low-frequency and rare SLC22A9 variants and bone quality, fractures, and bone turnover markers. Our results indicate that single genetic SLC22A9 variants do not have a major impact on osteoporosis risk prediction in Europeans, yet findings need to be replicated in larger-scale studies.
Rabies virus (RABV) is an ancient, highly neurotropic rhabdovirus that causes lethal encephalitis. Most RABV pathogenesis determinants have been identified with laboratory-adapted or attenuated RABVs, but details of natural RABV pathogenesis and attenuation mechanisms are still poorly understood. To provide a deeper insight in the cellular mechanism of pathogenies of field RABV, this work was performed to assess virus strain specific differences in intra-neuronal virus transport, to identify cell culture adaptive mutations in recombinant field viruses and to explore shRNA-expressing RABVs as research tools for targeted host manipulation in infected cells.
Comparison of chimeric RABVs with glycoprotein (G) ecto-domains of different lyssaviruses, together with field RABVs from dog and fox in dorsal root ganglion (DRG) neurons revealed no detectable differences in the axonal accumulation of the viruses. This indicates that previously described G-dependent transport of newly formed RABV in axons can occur both in laboratory-adapted and field RABV. Moreover, partial overlap of nucleoprotein (N) and G protein particles in field virus infected DRG axons supported the hypothesis of the “separate model” for anterograde RABV transport.
Serial passages of recombinant dog and fox field clones in different cell lines led to the identification of general (D266N) and cell line specific (K444N) adaptive mutations in the G ecto-domain of both viruses. In BHK cells, synergistic effects of D226N, K444N and A417T on field dog virus G protein surface localization led to the loss of endoplasmic reticulum (ER) retention of G and increased virus titers in the supernatant, indicating that limited virus release by ER retention is a major bottleneck in cell culture adaptation. In addition, selection of mutations within the C-terminus of the RABV phosphoprotein (P) (R293H and R293C in fox and dog viruses, respectively) led to the hypothesis of altered binding affinities to nucleoprotein and RNP complexes. Identification of the above mentioned amino acid substitutions together with alterations in a suboptimal transcription stop signal in the P/M gene border indicated that adaptation to cell culture replication occurs on both levels, RNA transcription/replication and virus release.
To evaluate the possibility of an expression of a functional microRNA-adapted short-hairpin RNAs (miR-shRNA) expressing RABV, recombinant RABVs encoding miR-shRNAs against cellular Dynein Light Chain 1 (DYNLL1) and Acidic Nuclear Phosphoprotein 32 family member B (ANP32B) were generated. In spite of cytoplasmic transcription of the respective mRNAs, downregulation of DYNLL1 and ANP32B mRNA and respective protein levels in infected cells revealed correct processing to functional shRNAs. Specific downregulation of the cellular genes at 2, 3 and 4 days post infection further demonstrated feasibility of the approach in standard cell lines. However, it remained open whether miR-shRNA expressing RABV can be used to study neuro-infection in vivo. Since first attempts in primary rat neuron cultures failed, it has to be clarified in further experiments whether this strategy can be used in mature, non-dividing neurons or whether breakdown of the nucleus in the course of cell division is a requirement for the processing of cytoplasmically expressed miR-RNA by nuclear RNases.
By providing novel insights in axonal RABV transport and cell culture adaptive mutations this work extends the current understanding of RABV pathogenesis in natural and non-natural cell environments. Moreover, it provides a basis for further pathogenicity studies in which the impact of cell culture adaptation through increased virus release on RABV virulence can be investigated. With successful expression of functional miR-shRNAs from RABV vectors, this work also provides a tool for RABV gene targeting in infected cell lines and thus may contribute to the further investigation of RABV-host-cell-interactions.
Staphylococcus aureus ist ein Gram-positives pathogenes Bakterium, welches bei ca. 30 % der gesunden Bevölkerung zur kommensalen Flora der Nasenschleimhaut gehört. Jedoch zählt S. aureus auch zu den häufigsten Erregern bakterieller Infektionen beim Menschen. Aus diesem Grund wurden S. aureus-Stämme in zahlreichen Studien untersucht, um die Pathophysiologie und Virulenz der Bakterien sowie die zugrundeliegenden Regulationsmechanismen zu verstehen. Die Expression von Virulenzfaktoren wird direkt oder indirekt durch verschiedene Regulatoren beeinflusst. Zu diesen zählen beispielsweise das Quorum-Sensing-System Agr, der alternative Sigma-Faktor SigB und das Zweikomponentensystem SaeRS. Bei der Regulation der Genexpression spielt neben Mechanismen, die die Transkriptionsinitiation beeinflussen, auch die Transkriptions-termination eine Rolle. Bei Bakterien unterscheidet man zwischen der Rho-unabhängigen und der Rho-abhängigen Transkriptionstermination. In bisherigen Studien wurde die Rolle des Transkriptionsterminationsfaktors Rho in Escherichia coli, Bacillus subtilis und Mycobacterium tuberculosis untersucht. Hierzu zählt unter anderem das Silencing von horizontal erworbenen Genen, die Verhinderung von DNA-Doppelstrangbrüchen und die Unterdrückung der persistierenden Antisense-Transkripten. Besonders die erhöhte Antisense-Transkription konnte auch in einer Tiling Array-Studie des S. aureus Wildtypstammes HG001 und einer isogenen Δrho-Mutante ST1258 festgestellt werden. In dieser Transkriptom-Analyse wurden die S. aureus-Stämme in RPMI- und TSB-Medium in der exponentiellen und stationären Wachstumsphase untersucht. Es konnten insgesamt 416 chromosomale Regionen identifiziert werden, deren Transkriptmenge in einer der vier Bedingungen in der Δrho-Mutante im Vergleich zum Wildtyp wenigstens 4-fach erhöht waren. Von diesen Regionen ließen sich nur 11 % annotierten Genen zuordnen, während eine massive Erhöhung der Menge solcher Transkripte festgestellt wurde, die vom Gegenstrang kodierender Gene stammen.
Ausgehend von diesen Befunden wurde in dieser Studie das zelluläre und extrazelluläre Proteom des S. aureus Wildtyps HG001 und der Δrho-Mutante ST1258 verglichen, um die Auswirkungen der Abwesenheit von Rho auf das Proteom zu untersuchen. Dabei lag die Mehrheit der relativ quantifizierten Proteine in erhöhten Mengen in der Δrho-Mutante im Vergleich zum Wildtyp vor. Viele dieser Proteine konnten dem SaeRS-Zweikomponentensystem von S. aureus zugeordnet werden. In der Proteomanalyse konnten 34 von 39 Proteinen, die durch SaeR reguliert werden, quantifiziert werden. Von diesen wiesen 29 erhöhte Proteinmengen in der Δrho-Mutante auf. Durch das Sae-System werden Gene reguliert, von denen die meisten für Virulenzfaktoren, wie Adhäsine, Toxine und immune evasion-Proteine, kodieren. Die Daten der Proteomanalyse zeigen, dass in S. aureus-Zellen, denen die Aktivität von Rho fehlt, das Sae-System aktiviert wird und dadurch die Induktion des SaeR-Regulons zu beobachten ist.
Die Relevanz dieser Ergebnisse wurde durch ein in vivo-Infektionsexperiment untersucht. In einem Bakteriämie-Modell führte die Inaktivierung von Rho zu einer signifikant erhöhten Virulenz von S. aureus, welche sich in einer signifikant reduzierten Überlebensrate der Mäuse äußerte. Zwischen dem Wildtyp und dem Komplementationsstamm konnte kein signifikanter Unterschied in der Überlebensrate der infizierten Mäuse gezeigt werden.
Es ist bekannt, dass SaeRS-abhängige Virulenzfaktoren auch für die Invasion in Epithel- und Endothelzellen entscheidend sind. Anhand der Zahl der internalisierten S. aureus-Bakterien nach Infektion von humanen Lungenepithelzellen konnten in dieser Arbeit keine Unterschiede zwischen dem Wildtyp HG001 und ∆rho-Mutante ST1258 im zeitlichen Verlauf festgestellt werden. Dabei konnten weder Unterschiede im Überleben in 16HBE14o- Zellen noch in der Internalisierungsrate in A549-Zellen zwischen den beiden Stämmen gezeigt werden.
Das Antibiotikum Bicyclomycin ist ein spezifischer Inhibitor des Transkriptionsterminationsfaktors Rho und wird in Studien zur Rho-abhängigen Transkriptionstermination in Gram-negativen Bakterien eingesetzt, da in diesen Rho ein essentielles Protein ist. In Transkriptom- und Proteomanalysen konnten vergleichbare Effekte durch die Behandlung des Wildtyps mit Bicyclomycin wie in der ∆rho-Mutante hervorgerufen werden. Die Antisense-Transkription und die Expression SaeRS-abhängigen Gene waren im Wildtyp nach Gabe von Bicyclomycin deutlich erhöht. Es konnte gezeigt werden, dass die Aktivierung des Sae-Systems unter Rho-defizienten Bedingungen direkt mit der Transkriptionsterminationsaktivität von Rho verbunden ist und eine neue Verbindung zwischen antibiotischer Wirkung und schädlicher Virulenzgenexpression in S. aureus herstellt werden konnte. In anderen Studien konnten Effekte von Antibiotika auf die Expression von Virulenzfaktoren in S. aureus gezeigt werden, jedoch wurden in diesen Studien die Effekte von Anti-Staphylokokken-Wirkstoffen untersucht. Im Gegensatz dazu ist im Fall von Bicyclomycin ein Antibiotikum verwendet worden, das gegen Gram-negative Bakterien wirksam ist und dennoch die Expression von Virulenzfaktoren in S. aureus beeinflusst. Diese Untersuchungen haben damit auch klinische Relevanz, nicht nur für Patienten, die an gemischten Infektionen mit verschiedenen Bakterienarten leiden, sondern auch für Patienten mit einer Gram-negativen bakteriellen Infektion, die jedoch Träger von S. aureus sind.
Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the ‘uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis.
Zerebrale kavernöse Malformationen (CCM) sind Fehlbildungen im neurovaskulären System, welche aufgrund von Krampfanfällen und hämorrhagischen Schlaganfällen zu einer stark eingeschränkten Lebensqualität führen können. Die Prävalenz von symptomatischen, autosomal-dominant erblichen CCMs auf Basis einer pathogenen Keimbahnvariante in einem der drei Gene CCM1, CCM2 oder CCM3 wird auf 1:5400 bis 1:6200 geschätzt. Durch Genpanelanalysen können sowohl Punktmutationen als auch kleine Indels und Kopienzahlveränderungen in einem parallelen Ansatz nachgewiesen werden.
Zur weiteren Aufklärung der molekularen Pathogenese hereditärer Kavernome und zur Suche nach potenziellen Therapieansätzen wurde im Rahmen dieser Arbeit erstmals ein Patienten-spezifisches in vitro Zellmodell der Kavernomatose etabliert. Hierzu wurden zirkulierende endotheliale Vorläuferzellen (Blood Outgrowth Endothelial Cells, BOECs) aus dem Blut eines CCM-Patienten mit krankheitsverursachender heterozygoter CCM1-Keimbahnveränderung isoliert. Im Sinne der Knudson´schen Zweischrittinaktivierung zur Entstehung von Kavernomen wurde das zweite CCM1-Allel in Zellkulturen mit Hilfe der CRISPR/Cas9-Technologie ausgeschaltet. In klonal expandierten Zellkolonien konnte das Vorliegen einer Compound-Heterozygotie für die CCM1-Keimbahnvariante und eine zweite CRISPR/Cas9-induzierte loss-of-function Variante bestätigt werden.
Durch die zweite Mutation zeigten sich morphologische Veränderungen, die Störung endothelialer Zell-Zell-Verbindungen und eine vermehrte Aktin-Stressfaserbildung sowie eine deutliche Hochregulation des Transkriptionsfaktors KLF2. Zudem führte die komplette Inaktivierung des CCM1-Gens zu einer Akkumulation des von-Willebrand-Faktors (vWF). Die in der vorgelegten Arbeit erstmals beschriebene starke Anreicherung des vWF konnte in immortalisierten humanen Endothelzellen und zudem im Kavernomgewebe von Trägern pathogener CCM1-, CCM2- oder CCM3-Veränderungen bestätigt werden. Es ist daher denkbar, dass der erhöhte vWF-Spiegel im Endothel des kavernösen Gefäßkonvoluts zum lokalen hämostatischen Ungleichgewicht beiträgt.
Im Patienten-spezifischen BOEC-Modell konnte zudem erstmals in einem nicht-viralen und Plasmid-freien Ribonukleoprotein-basierten CRISPR/Cas9-Ansatz eine Mutationskorrektur der vorliegenden CCM1-Keimbahnveränderung erreicht werden. Die Ergebnisse der weiteren Kultivierungen und Amplikon-Tiefensequenzierungen zeigten jedoch sehr eindrücklich, dass der klonogene Überlebensvorteil von verbleibenden CCM1-inaktivierten BOECs zu einem deutlichen Überwachsen der korrigierten BOECs im Zellgemisch führt und eine CRISPR/Cas9-vermittelte Mutationskorrektur damit für CCM-Patient*innen keine
realistische Therapieoption darstellt. Diese Ergebnisse fließen in neue Ansätze der Therapieentwicklung ein, welche vor allem den klonalen Überlebensvorteil und die Apoptoseresistenz CCM-defizienter Zellen adressieren.
Autosomal dominant cerebral cavernous malformation (CCM) represents a genetic disorder with a high mutation detection rate given that stringent inclusion criteria are used and copy number variation analyses are part of the diagnostic workflow. Pathogenic variants in either CCM1 (KRIT1), CCM2 or CCM3 (PDCD10) can be identified in 87–98% of CCM families with at least two affected individuals. However, the interpretation of novel sequence variants in the 5′-region of CCM2 remains challenging as there are various alternatively spliced transcripts and different transcription start sites. Comprehensive genetic and clinical data of CCM2 patients with variants in cassette exons that are either skipped or included into alternative CCM2 transcripts in the splicing process can significantly facilitate clinical variant interpretation. We here report novel pathogenic CCM2 variants in exon 3 and the adjacent donor splice site, describe the natural history of CCM disease in mutation carriers and provide further evidence for the classification of the amino acids encoded by the nucleotides of this cassette exon as a critical region within CCM2. Finally, we illustrate the advantage of a combined single nucleotide and copy number variation detection approach in NGS-based CCM1/CCM2/CCM3 gene panel analyses which can significantly reduce diagnostic turnaround time.
Summary
Streptococcus pneumoniae (the pneumococcus), a bacterium belonging to the normal flora in the human respiratory tract, continues to be an important pathogen due to its contribution to morbidity and mortality among children, the elderly, and immunocompromised persons. Global estimates of pneumococcal deaths among children declined by 51% between 2000 and 2015. This achievement was mainly due to the introduction of pneumococcal conjugate vaccines (PCVs) in countries with the highest pneumococcal burden. Since May 2012, children in Ghana have been receiving PCV vaccination as part of routine immunization. The continuous monitoring of the pneumococcus after PCV introduction is essential to understand the changing epidemiology of the pathogen in the population.
This study therefore, aims to determine the (1) prevalence, serotypes, and sequence types of pneumococcal isolates, (2) antibiotic susceptibility patterns and the genetic basis for the antibiotic resistance among these pneumococcal isolates, and (3) prevalence of selected virulence genes that have been identified as potential vaccine candidates. Nasopharyngeal swabs were obtained from vaccinated children under five years of age in Cape Coast, Ghana. Six years after PCV implementation, we provide data on the epidemiology of pneumococcal strains circulating among children in Cape Coast Ghana. Standard microbiological and molecular techniques were used to identify and characterize the pneumococcal strains.
Overall, pneumococcal carriage prevalence was 29.4% (151/513). All participating children were fully vaccinated. Of the 26 different serotypes identified, the top five PCV13 serotypes (VT) were 6B, 23F, 19F, 3, 6A and non-PCV13 vaccine serotypes (NVT) were 23B, 13, 11A, 15B, and 34. PCV13 coverage was 38.4%, however, more than half of the isolates were NVT with a coverage rate of 61.6%. The isolates were highly susceptible to levofloxacin, ceftriaxone, vancomycin, and erythromycin. However, marked resistance to cotrimoxazole and tetracycline was observed. The reduction in penicillin resistance (35.8%) as compared to pre-vaccination data (45% - 63%) suggests an attributable effect from PCV13 vaccination. However, penicillin resistance was also detected in some NVT serotypes. Overall, 28.5% of the isolates resistant to three or more different classes of antibiotics were classified as multidrug-resistant (MDR). To analyze the genetic basis for resistance to penicillin, erythromycin and tetracycline, pbp2b, ermB, mefA, and tetM genes were amplified.
Thirty-eight (70%) out of the 54 penicillin-resistant isolates contained the pbp2b resistance gene. Out of the 11 erythromycin-resistant isolates, 7 (63.6) and 4 (36.4%) were positive for the ermB and mefA genes, respectively. The tetM gene was detected in 85 (98.8%) of the 86 tetracycline resistance isolates.
To determine the extent to which potential protein-based vaccines could be protective in Ghanaian children, we sought to determine the prevalence of selected virulence genes among the isolates. The lytA, pavB, and cpsA genes were present in all the carrier isolates. However, psrP, pcpA, pilus islet (PI) PI-1, and PI-2 were present in 62.7%, 87.5%, 11.8%, and 6.5% of the strains, respectively. The psrP and pcpA virulence genes were evenly distributed among all the serotypes. However, the pilus islets were detected in only seven serotypes namely 19F, 6B, 9V, 6A, 13, 11A, and 23B. Five serotype 19F isolates possessed both PI-1 and PI-2. Furthermore, the pilus islets were associated with multidrug resistance.
The predominant NVT serotype 23B and isolates resistant to ≥ 4 antibiotics were analysed by multilocus sequence typing (MLST). Nine known sequence types (STs) and 10 novel STs were identified. Seven out of the 10 new STs belonged to serotype 23B, while the remaining 3 were VTs 6B and 19F. A capsular switch was identified among isolates of ST802, which comprised of both serotype 23F and 19F. The majority of serotype 23B strains belonged to ST172. The ST172 is associated with serotype 23F and a single locus variant (SLV) of internationally disseminated clone ST338 (Colombia23F-26). Consequently, ST172 was characterised with marked antibiotic resistance and with traits of capsular switching. One serotype 6B strain was identified as a SLV of ST273 (Greece6B-22) while two serotype 9V strains belonged to the internationally disseminated clone ST156 (Spain9V-3).
In conclusion, this study showed a marginal decline in overall pneumococcal carriage prevalence, persistence of VTs despite the increase in NVTs, and the occurrence of serotype replacement and capsular switching. In addition, sequence types related to internationally disseminated clones are circulating in Ghana. With the high pcpA and psrP coverage detected,including these genes in protein-based vaccines could provide adequate protection for Ghanaian Children.
Antibiotic resistance in pneumococci contributes to the high pneumococcal deaths in children. We assessed the molecular characteristics of multidrug-resistant (MDR) pneumococci isolated from healthy vaccinated children under five years of age in Cape Coast, Ghana. A total of 43 MDR isolates were selected from 151 pneumococcal strains obtained from nasopharyngeal carriage. All isolates were previously serotyped by multiplex PCR and Quellung reaction. Susceptibility testing was performed using either the E-test or disk diffusion method. Virulence and antibiotic resistance genes were identified by PCR. Molecular epidemiology was analyzed using multilocus sequence typing (MLST). Vaccine-serotypes 23F and 19F were predominant. The lytA and pavB virulence genes were present in all isolates, whiles 14–86% of the isolates carried pilus-islets 1 and 2, pcpA, and psrP genes. Penicillin, tetracycline, and cotrimoxazole resistance were evident in >90% of the isolates. The ermB, mefA, and tetM genes were detected in (n = 7, 16.3%), (n = 4, 9.3%) and (n = 43, 100%) of the isolates, respectively. However, >60% showed alteration in the pbp2b gene. MLST revealed five novel and six known sequence types (STs). ST156 (Spain9V-3) and ST802 were identified as international antibiotic-resistant clones. The emergence of international-MDR clones in Ghana requires continuous monitoring of the pneumococcus through a robust surveillance system.
Preventive strategies involving the use of pneumococcal conjugate vaccines (PCVs) are known to drastically reduce pneumococcal disease. However, PCV vaccination has been plagued with serotype replacement by non-PCV serotypes. In this study, we describe the prevalence and molecular characteristics of non-PCV13 serotypes (non-vaccine serotypes, NVTs) from pneumococcal carriage isolates obtained from children < 5 years old in Cape Coast, Ghana, after PCV introduction. The isolates were subjected to antibiotic susceptibility testing and multilocus sequence typing (MLST), and molecular techniques were used to detect the presence of virulence genes. Serotypes 11A, 13, 15B, 23B, and 34 formed the top five of the 93 NVT isolates. As such, 20 (21.5%), 49 (48.4%), and 70 (74.3%) isolates were non-susceptible to penicillin, tetracycline, and cotrimoxazole, respectively. Sixteen (17.2%) multidrug-resistant isolates were identified. However, non-susceptibility to ceftriaxone and erythromycin was low and all isolates were fully susceptible to levofloxacin, linezolid, and vancomycin. Whereas pcpA, pavB, lytA, and psrP genes were detected in nearly all serotypes, pilus islet genes were limited to serotypes 11A, 13, and 23B. MLST for predominant serotype 23B isolates revealed three known and seven novel sequence types (STs). ST172 and novel ST15111 were the most dominant and both STs were related to PMEN clone Columbia23F-26 (ST338). In conclusion, non-PCV13 serotype 23B was the most prevalent, with characteristics of rapid clonal expansion of ST172 and ST15111, which are related to international clones of the pneumococcus. Continuous monitoring of NVTs in Ghana is, therefore, essential, as they have the potential to cause invasive disease, show high antibiotic resistance, and attenuate the effects of PCV vaccination.
Staphylococcus aureus (S. aureus) endocarditis is still one of the most fatal heart diseases, with a mortality rate of 20-45%. In recent years, the importance of endothelial cells (ECs) in the context of endocarditis has become more evident. The vascular endothelium forms a selective barrier between blood and the adjacent tissue by maintaining an anti-inflammatory and anti-thrombogenic phenotype. However, in case of insertion of cardiac implants, an injury of the endothelium can occur which promotes platelet aggregation followed by S. aureus adherence to the platelets, especially in areas with low hemodynamic shear stress. This process is considered as a key event in the development of infective endocarditis (IE) and allows bacteria to colonize the heart valves. Despite extensive research, the pathogenesis of IE is still not completely understood. Therefore, further investigations are needed to enable an effective prevention of this life-threatening disease.
In order to study the infection process of S. aureus, internalization experiments with two different S. aureus strains, one control strain (HG001) and one strain isolated from an endocarditis patient (T-72949) were performed in human coronary artery endothelial cells (HCAEC). Subsequently, an extensive proteome analysis of the host cells was carried out. More specific analyses were performed using peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, which causes a pro-inflammatory response in ECs. In this context, the focus remained on the analysis of cellular changes in terms of cell stiffness, wound healing, and additionally platelet aggregation.
The analysis of the HCAEC host proteome revealed a time-related difference depending on the infecting bacterial strain. Several proteins involved in host cell signaling pathways exhibited a higher abundance at earlier time points in host cells infected with endocarditis strain T-72949 compared to those infected with HG001. Further proteome analysis uncovered several adaptations on the cellular side that enable internalization and replication of both S. aureus strains as well as the activation of pathways that promote cellular recovery. Furthermore, it could be shown that PGN reduced cellular stiffness which could lead to an increased bacterial uptake and would thereby promote the development of a chronic S. aureus infection. Additionally, PGN prevented effective wound healing which promotes a pro-thrombotic and pro-inflammatory condition. This status could facilitate the bacterial infection of further cells. Apart from that, PGN induced platelet aggregation which could ease bacterial adhesion to thrombotic surfaces (e.g., dysfunctional endothelium). The following formation of a mature vegetation might protect the bacteria from the immune system and antibiotics.
The results of the present work emphasize the central role of ECs in the context of IE. It could be demonstrated that a healthy monolayer of ECs enables a beneficial cell response and may prevent the development of vascular diseases. Moreover, the comprehensive proteome dataset which was generated in this project provides a valuable source of information for future studies to unravel further molecular mechanisms of endocarditis and possible therapeutic approaches.
Transcriptional corepressors Sin3, Cyc8 and Tup1 are important for downregulation of gene expression by recruiting various histone deacetylases once they gain access to defined genomic locations by interaction with pathway-specific repressor proteins. In this work we systematically investigated whether 17 yeast repressor proteins (Cti6, Dal80, Fkh1, Gal80, Mig1, Mot3, Nrg1, Opi1, Rdr1, Rox1, Sko1, Ume6, Ure2, Xbp1, Yhp1, Yox1 and Whi5) representing several unrelated regulatory pathways are able to bind to Sin3, Cyc8 and Tup1. Our results show that paired amphipathic helices 1 and 2 (PAH1 and PAH2) of Sin3 are functionally redundant for some regulatory pathways. WD40 domains of Tup1 proved to be sufficient for interaction with repressor proteins. Using length variants of selected repressors, we mapped corepressor interaction domains (CIDs) in vitro and assayed gene repression in vivo. Systematic comparison of CID minimal sequences allowed us to define several related positional patterns of hydrophobic amino acids some of which could be confirmed as functionally supported by site-directed mutagenesis. Although structural predictions indicated that certain CIDs may be α-helical, most repression domains appear to be randomly structured and must be considered as intrinsically disordered regions (IDR) adopting a defined conformation only by interaction with a corepressor.
Zur Untersuchung molekularer Prozesse sind die Wechselwirkungen der beteiligten Faktoren von zentraler Bedeutung, was besonders für die präzise Steuerung der eukaryotischen Genregulation zutrifft, die im Mittelpunkt dieser Arbeit steht. Die Transkription wird durch den Zusammenbau des Präinitiationskomplexes (PIC) am Promotor der Zielgene initiiert. Neben der RNA-Polymerase II, dem Mediatorkomplex und mehreren generellen Transkriptionsfaktoren sind daran Aktivatorproteine beteiligt, welche an UAS-Elemente (upstream activation site) im Promotor binden. Daneben können aber auch Repressoren an URS-Elemente (upstream repression site) binden oder mit Promotor- gebundenen Aktivatoren interagieren und durch Rekrutierung sog. Corepressoren (z. B. Sin3, Cyc8 und Tup1) die Transkription hemmen. Diese Corepressoren können dann über assoziierte Histon- deacetylasen (z. B. Rpd3) die Chromatinstruktur im Promotorbereich spezifischer Gene verdichten und damit eine Bindung der Transkriptionsmaschinerie verhindern. In der Regel führt dies zu einer reduzierten Expression des jeweiligen Gens.
Untersuchungen zu den Wechselwirkungen zwischen genspezifischen Repressoren und pleiotropen Corepressoren haben in der Vergangenheit bereits zur Identifizierung einzelner Sequenzmotive und individueller Strukturen geführt. Um dieses Netzwerk zu ergänzen, wurden in dieser Arbeit zahlreiche Repressor-Corepressor-Interaktionen in der Hefe Saccharomyces cerevisiae in vitro und in vivo charakterisiert und durch Verkürzung der interagierenden Proteine (Dal80, Mot3, Sko1, Ure2, Xbp1 und Yox1) hierfür relevante Aminosäuresequenzen ermittelt. Durch systematische Vergleiche solcher Repressorsequenzen konnten Varianten eines hydrophob-amphipathischen Konsensusmotivs identifiziert und z. T. durch gerichtete Mutagenese als funktionell wichtig nachgewiesen werden. Sekundärstrukturvorhersagen zeigten oft die Beteiligung α-helikaler, aber auch β-Faltblatt- oder ungeordneter Strukturen. Diese strukturelle Varianz lässt die Vermutung zu, dass es sich bei solchen Corepressor-Interaktionsdomänen (CID) um IDRs (intrinsically disorderd regions) handeln könnte, die erst durch Kontaktherstellung zum Corepressor eine definierte Konformation annehmen.
Ein in dieser Arbeit intensiv untersuchter Repressor war Gal80, der bekanntermaßen das GAL-System der Hefe solange abschaltet, bis Galactose als induzierender Zucker verfügbar ist. Man unterscheidet hierbei drei Zustände: Die Glucoserepression beschreibt das Abschalten der GAL-Gene durch den Repressor Mig1 bei Glucoseverfügbarkeit. Bei Glucosemangel und Verfügbarkeit einer alternativen Kohlenstoffquelle (z. B. Lactat oder Ethanol) wird der Aktivator Gal4 synthetisiert und bindet an UASGAL- Motive in Promotoren der GAL-Gene. Unter diesen Derepressionsbedingungen wird die Transkriptionsaktivierungsdomäne von Gal4 noch durch den Gal80-Repressor maskiert. In dieser Arbeit wurde gezeigt, dass Gal80 zusätzlich in der Lage ist, den Corepressorkomplex Cyc8/Tup1 zu rekrutieren und die Transkription der Strukturgene dadurch zu reprimieren. Chromatinimmunopräzi- pitationsstudien belegten die Gal80-abhängige Präsenz der Corepressoren Cyc8 und Tup1 am GAL1 Promotor. Außerdem stellte sich bei der Charakterisierung von cyc8 und tup1 Mutantenstämmen heraus, dass Corepressoren durchaus auch aktivierende Wirkungen entfalten können. So fiel die Expression eines GAL1-lacZ Reportergens in einer cyc8 Nullmutante unter allen getesteten Bedin- gungen geringer aus als im Wildtyp. Die duale Wirkung solcher Transkriptionsfaktoren wurde in der Vergangenheit immer wieder beobachtet und steht auch im Einklang mit den Befunden dieser Arbeit.
While ionizing radiation (IR) is a powerful tool in medical diagnostics, nuclear medicine,and radiology, it also is a serious threat to the integrity of genetic material. Mutagenic effects ofIR to the human genome have long been the subject of research, yet still comparatively little isknown about the genome-wide effects of IR exposure on the DNA-sequence level. In this study,we employed high throughput sequencing technologies to investigate IR-induced DNA alterationsin human gingiva fibroblasts (HGF) that were acutely exposed to 0.5, 2, and 10 Gy of 240 kVX-radiation followed by repair times of 16 h or 7 days before whole-genome sequencing (WGS).Our analysis of the obtained WGS datasets revealed patterns of IR-induced variant (SNV and InDel)accumulation across the genome, within chromosomes as well as around the borders of topologicallyassociating domains (TADs). Chromosome 19 consistently accumulated the highest SNVs andInDels events. Translocations showed variable patterns but with recurrent chromosomes of origin(e.g., Chr7 andChr16). IR-induced InDels showed a relative increase in number relative to SNVs anda characteristic signature with respect to the frequency of triplet deletions in areas without repetitiveor microhomology features. Overall experimental conditions and datasets the majority of SNVs pergenome had no or little predicted functional impact with a maximum of 62, showing damagingpotential. A dose-dependent effect of IR was surprisingly not apparent. We also observed a significantreduction in transition/transversion (Ti/Tv) ratios for IR-dependent SNVs, which could point to acontribution of the mismatch repair (MMR) system that strongly favors the repair of transitions overtransversions, to the IR-induced DNA-damage response in human cells. Taken together, our resultsshow the presence of distinguishable characteristic patterns of IR-induced DNA-alterations on agenome-wide level and implicate DNA-repair mechanisms in the formation of these signatures
Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells
(2016)
Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during bacterial uptake. The participation of essential host cell signaling kinases in pneumococcal phagocytosis was deciphered for the kinase Akt, ERK1/2, and p38 and phosphoimmunoblots showed an increased phosphorylation and thus activation upon infection with pneumococci. Taken together, this study deciphers host cell kinases in innate immune cells that are induced upon infection with pneumococci and interfere with bacterial clearance after phagocytosis.
Deciphering the influence of Streptococcus pneumoniae global regulators on fitness and virulence
(2019)
Streptococcus pneumoniae (S. pneumoniae; the pneumococcus) is a Gram-positive, aerotolerant, and opportunistic bacteria, which colonizes the upper respiratory tract of human. S. pneumoniae can further migrate to other sterile parts of the body, and causes local as well as fatal infections like, pneumonia, septicaemia and meningitis. Due to incomplete amino acid pathways, pneumococci are auxotrophic for eight different amino acids including glutamine and arginine. The pneumococcus has adapted to the various host environmental conditions and a number of systems are dedicated for the transport and utilization of nutrients such as monosaccharides, amino acids and oligopeptides.
In this study the amino acid metabolism was characterised by 15N-isotopologue profiling in two different pneumococcal strains, D39 and TIGR4. Efficient uptake of a labelled amino acids mixture of 15N-labelled amino acids showed that S. pneumoniae has a preference for the amino acids transport instead of a de novo biosynthesis. It is known that glutamine (Gln) serves as main nitrogen source for S. pneumoniae. The 15N-labelled Gln used in this study demonstrated an efficient 15N-enrichment of Glu, Ala, Pro and Thr. Minor enrichment was seen for the amino acids Asp, Ile, Leu, Phe, Tyr, and Val. Remarkably, labelled Gly and Ser could be determined in strain TIGR4, whereas for strain D39 these two labelled amino acids were not detected. This confirms earlier studies with 13C-labelled glucose, which showed the biosynthesis of Ser out of Gly. Strain TIGR4 was able to grow in chemically-defined medium depleted of Gly confirming that Gly can be synthesized out of serine by the action of the enzyme serine hydroxymethyltransferase (SHMT).
The transcriptional regulator GlnR controls the Gln and Glu metabolism in S. pneumoniae. Hence, the impact of the repressor GlnR on amino acids metabolism was also studied. An increased 15N-enrichment was determined for Ala and Glu in both used pneumococcal strains, while an increased level of Pro was only measured in the isogenic glnR-mutant of non-encapsulated D39.
Arginine can also serve as nitrogen source in strain TIGR4. The arginine deiminase system metabolizes Arg into ornithine, carbamoyl phosphate and CO2 by the generation of 1 ATP and 2 mol NH3. Because of the truncation of the arcA gene strain D39 lacks arginine deiminase activity and has thus no functional ADS system. When 15N-Arg was added for growth, only in strain TIGR4, thirteen (13) labelled amino acids were detected with the highest enrichment for Ala, Glu and Thr. Genes coding for the enzymes of the arginine metabolism and for arginine uptake are regulated by the activator ArgR2 in strain TIGR4. Inactivation of ArgR2 was not accompanied by an enrichment of labelled amino acids, when the argR2-mutant was grown with 15N-labelled Arg indicative of the important role of ArgR2.
The bicistronic operon arcDT encoding the arginine/ornithine transporter ArcD and a putative peptidase ArcT belong to the peptidase family M20. The in silico comparison of structures revealed a significant homology of ArcT to PepV of L. delbrueckii and to Sapep of S. aureus known as carboxypeptidase. ArcT was heterologously expressed in E. coli and purified under reducing conditions. An enzymatic reaction was established and several dipeptides like Ala-Arg, Arg-Ala, and Ala-Asp were used as substrates. In addition, the dependency on divalent cations was analysed. Cleavage of the dipeptide Ala-Arg was detected in the presence of Mn2+ as cofactor under reducing conditions. Reduced peptidase activity was observed when Zn2+ was added. No cleavage of the tripeptide Ala-Ala-Arg could be shown indicating that ArcT acts as dipeptidase with the preference to the Arg residue at the C-terminal end.
Bacterial meningitis caused by S. pneumoniae was studied in an in vivo proteomic analysis. In a mouse meningitis model S. pneumoniae was isolated from the cerebrospinal fluid (CSF) by a filter extraction step. The MS analysis identified AliB and ComDE only from CSF isolated pneumococci indicating that these proteins are expressed under infection conditions. Mice infected with D39 wild-type and isogenic aliB, comDE and aliB-comDE double knockout mutants showed significantly less number of pleocytosis in the CSF and lower bacterial load in the blood compared to the wild-type. The results indicate that AliB and ComDE play an important role during meningitis.
Phenotypic characterization was carried out to identify differences between the wild-type and the aliB-, comDE- and aliB-comDE double mutants. Oxidative stress conditions were induced by the application of hydrogen peroxide or paraquat during growth in a chemically-defined medium similar to the CSF. No alteration in growth and survival of these mutants compared to the wild-type was observed suggesting that oxygen radicals play not an important role during the progression of meningitis. In addition, no differences of AliB expression was detected in the ComDE deficient D39. No impact of aliB and comDE-mutation on the expression of different virulence factors like pneumolysin or proteins involved in capsular biosynthesis was detected.
In vitro proteome analysis was performed to compare the wild-type to the AliB, and ComDE deficient D39 in the early and mid logarithmic growth phase. More than 70 % of theoretically expressed proteins were identified. In the aliB-mutant 33 proteins were differentally expressed in the early growth phase and 50 proteins differed during mid log growth. For the comDE mutant 24 and 11 proteins differed in expression in these two growth phases. Interestingly, high level of AliA expression was identified in all samples. The aliB-mutant had a decreased abundance of the proteins resembling an oligopeptide ABC transporter (AmiA, AmiC, AmiD, AmiE). In addition, another ABC transporter for iron transport encoded by spd_1607 to spd_ 1610 was higher expressed in the aliB-mutant. In the ComDE deficient mutant lower abundance of the Ami transporter sytem was identified. An increased abundance of proteins involved in the pyrimidine metabolism (PyrF, PyrE, PyrDb, PyrB and PyrR) was recognized only in the early growth phase of the comDE-mutant. These analyses demonstrate the marginal changes in protein synthesis during growth of S. pneumoniae. These studies demonstrated the adaptation of the proteome of S. pneumoniae to different growth conditions and the impact of regulatory proteins on the availability of carbon and nitrogen sources.
To enable control of African swine fever (ASF) in Eastern and Southern Africa, prototype live vaccine candidates were generated by targeted gene deletions from a Kenyan genotype IX ASF virus (ASFV). It was attempted to delete known nonessential genes involved in virulence (encoding TK, dUTPase, CD2v, 9GL), possibly essential genes (p12, pA104R, ribonucleotide reductase), and genes with widely unknown functions (pK145R). Isolation of the desired virus recombinants by plaque assays or limiting dilutions on a wild boar lung cell line (WSL-HP) was facilitated by substitutive reporter gene insertions encoding fluorescent proteins (GFP, DsRed), or the human membrane protein CD4. The latter protein permitted enrichment of recombinant virus particles by magnetic activated cell sorting (MACS). The isolated ASFV recombinants were characterized by PCR and sequencing of the mutated genome parts, and replication kinetics and virus spread in cell culture were investigated. Deletion of TK, CD2v, or pK145R had no detectable effect on in vitro growth of ASFV Kenya. Interestingly, virus mutants lacking the DNA binding protein pA104R which has been considered to be essential for DNA replication, also exhibited almost wild type-like growth properties.
In contrast, ASFV mutants lacking ribonucleotide reductase or p12 could not be purified to homogeneity on WSL-HP cells, indicating these proteins are essential for virus replication in cell culture. Therefore, trans-complementing cells lines stably expressing ASFV p12 have been prepared which can now be used for mutant virus purification. If this approach is successful the resulting defective mutant ASFV Kenya-p12 might be suitable as a safe “disabled in second cycle” (DISC) live vaccine in swine.
In a novel approach to improve reverse genetics of ASFV the CRISPR/Cas9 cell line WSL-gRp30 (Hübner et al., 2018a) was co-transfected with genomic DNA of ASFV-KenyaCD2vDsRed, sgRNA plasmids targeting K145R or 9GL, and GFP-expressing recombination plasmids for homology-directed repair. For booting up of the noninfectious virus genome the cells were infected with phylogenetically distant helper virus (genotype II ASFV Armenia, 84% identity) which was selectively inhibited on the used cell line. The desired double-fluorescent double-deletion mutants could be isolated after few plaque purification steps on selective WSL-gRp30 cells. Next generation sequence (NGS) analyses of reconstituted ASFV Kenya genomes showed that no unwanted recombination with the helper virus occurred, indicating that the method might be also suitable for booting of synthetic ASFV genomes cloned and mutagenized in E. coli or yeast.
The modified CRISPR/Cas9 system of S. pyogenes might be also usable for generation of ASFV resistant pigs. To evaluate this alternative control measure WSL cell clones stably expressing Cas9 nuclease and single or multiple sgRNAs against essential ASFV proteins were prepared and tested for their susceptibility to infection. Strain specific sgRNAs targeting the p30 gene of ASFV Kenya or Armenia selectively inhibited the respective viruses, and a p12 gene-specific sgRNA abrogated replication of both genotypes almost completely. Interestingly, coexpression of four ASFV-specific sgRNAs did not enhance virus inhibition, but might help to reduce the frequency of escape mutants which were occasionally isolated from the single sgRNA-expressing cells, and exhibited silent base substitutions or in-frame deletions within the target genes. First attempts to express the in vitro tested CRISPR/Cas9 constructs in transgenic pigs are in progress.
CRISPR/Cas9 supported rescue of a defective BAC clone of pseudorabies virus (PrV) vaccine strain Bartha (Hübner et al., 2018b) was used to develop putative vectored vaccines against ASFV. In the present study expression cassettes for the codon-optimized p12 and p54 genes of ASFV were successfully inserted into the PrV genome. The insertions did not significantly affect PrV recombination in cell culture, and the transgenes were expressed at similar levels as in ASFV-infected cells. It has to be tested whether coinfection with vector constructs for these and other immunogenic ASFV proteins is able to protect pigs against a lethal challenge.
For characterization of the generated ASFV mutants and PrV vector constructs, monospecific antisera against several ASFV gene products (p11.5, p12, p54, pK145R, p285L) were prepared by immunization of rabbits with bacterial GST fusion proteins. The anti-p12 serum showed only weak and strain-specific reactions with the ASFV Kenya protein, but was nevertheless useful for identification of p12-expressing PrV recombinants and WSL cell lines. All other sera showed satisfying reactions in Western blot and mostly immunofluorescence analyses, and allowed i.a. precise localization of the pK145R and p285L proteins in ASFV-infected cells and virions (Hübner et al., 2019).
Group B streptococci (GBS) cause a range of invasive maternal–fetal diseases during pregnancy and post-partum. However, invasive infections in non-pregnant adults are constantly increasing. These include sepsis and streptococcal toxic shock syndrome, which are often complicated by systemic coagulation and thrombocytopenia. GBS express a hyper-hemolytic ornithine rhamnolipid pigment toxin with cytolytic and coagulatory activity. Here, we investigated the effects of GBS pigment on human platelets. Infections of platelets with pigmented GBS resulted initially in platelet activation, followed by necrotic cell death. Thus, this study shows that GBS pigment kills human platelets.
Abstract
Background
Toxins are key virulence determinants of pathogens and can impair the function of host immune cells, including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections.
Materials and Methods
In this study, we deciphered the effects of Staphylococcus aureus toxins α‐hemolysin, LukAB, LukDE, and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. Activation of platelets and loss of platelet function were investigated by flow cytometry, aggregometry, platelet viability, fluorescence microscopy, and intracellular calcium release. Thrombus formation was assessed in whole blood.
Results
α‐hemolysin (Hla) is known to be a pore‐forming toxin. Hla‐induced calcium influx initially activates platelets as indicated by CD62P and αIIbβ3 integrin activation, but also induces finally alterations in the phenotype of platelets. In contrast to Hla and pneumolysin, S. aureus bicomponent pore‐forming leukocidins LukAB, LukED, and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. The presence of small amounts of Hla (0.2 µg/ml) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. Damage of platelets by Hla was not neutralized by intravenous immune globulins.
Conclusion
Our findings might be of clinical relevance for S. aureus induced endocarditis. Stabilizing the aortic‐valve thrombi by inhibiting Hla‐induced impairment of platelets might reduce the risk for septic (micro‐)embolization.
Streptococcus pneumoniae (S. pneumoniae, pneumococci) and Staphylococcus aureus (S. aureus) belong to the Gram-positive, facultative pathogenic bacteria. They are typical commensals of the human upper respiratory tract and most people get colonized at least once during their life. Nevertheless, these potentially pathogenic bacteria are able to spread from the site of colonization to invade into deeper tissues and the blood circulation. Thereby, severe local and invasive infections like bacteremia and life-threatening sepsis can be caused. Once reaching the bloodstream, bacteria get in contact with platelets. Platelets are small, anucleated cells and the second most abundant cell type in the circulation. The role of platelets in hemostasis is well known. Circulating resting platelets sense vessel injury independent of its cause. Platelets bind to injured endothelium and exposed molecules of the underlying extracellular matrix, get activated and release intracellular adhesion proteins and different modulatory molecules. This in turn initiates activation and binding of nearby platelets resulting in closure of vascular injury by formation of small thrombi. Despite being pivotal in maintenance of the endothelial barrier they got increasingly recognized as cells with important immune functions. Platelets excert functions of the immune response by either, i) interacting with immune cells of different pathways of the immune response, ii) releasing immunomodulatory molecules stored in their granules or iii) interacting with invading pathogens via direct or indirect binding.
The basis for this study were results demonstrating direct binding of different S. aureus proteins to platelets resulting in platelet activation. The identified proteins in the mentioned study are the S. aureus proteins Eap, AtlA-1, CHIPS and FlipR. Severe invasive infections with S. pneumoniae are quite often associated with development of thrombocytopenia or disseminated vascular dissemination. This frequent observation hints towards either a direct or indirect interplay of platelets with pneumococci. Hence, this study aims to analyze potential interactions and aims to decipher involved factors on both the platelet- and bacterial site.
A screening of recombinant pneumococcal surface proteins identified proteins belonging to the group of lipoproteins, sortase-anchored proteins and choline-binding proteins to directly activate human platelets. Besides these surface proteins also the intracellular pneumococcal pneumolysin (Ply) induced highly increased values for the platelet activation marker P-selectin. Since Ply is a major virulence factor of
S. pneumoniae the primary focus was set on involvement of this pore forming toxin on platelet activation. Surprisingly, our data revealed Ply induced platelet activation to be a false positive result based on formation of large Ply pores in the platelet membrane. In fact, it was clearly demonstrated that Ply lyses platelets even at low concentrations and thereby rendering them non-functional. Lysis of platelets could be inhibited by the addition of pharmaceutical immunoglobulin preparations as well as antibodies specifically targeting Ply. Inhibition of Ply also resulted in fully rescued platelet function either in washed platelets or in whole blood as shown by thrombus formation. Next to pneumococci also S. aureus expresses pore forming toxins, namely α-hemolysin (Hla) and different pairs of bicomponent pore forming leukocidins. Whereas the different tested leukocidins did not affect platelets, Hla acted in a two-step mechanism on human platelets. The results confirm previous data on Hla induced platelet activation via Hla resulting in e.g., reversible platelet aggregation or surface expression of activation markers. Nevertheless, platelet activation by Hla is followed by dose- and time-dependent lysis of platelets resulting in loss of platelet function and abrogated thrombus formation. Platelet lysis by Hla could neither be rescued with specific monoclonal anti-Hla antibodies nor with pharmaceutical IgG preparations containing anti-Hla IgGs. Taken together, the presented data reveal new pathomechanisms involving disturbance of platelets by bacterial pore forming toxins. Platelet lysis as well as impaired platelet function play an important role in development of severe complications during invasive infections. In life threatening infections caused by S. pneumoniae the usage of antibody formulations containing antibodies targeting Ply might be a promising approach for the prevention or even intervention and improvement of clinical outcome.
Human donor milk (HDM) provides appropriate nutrition and offers protective functionsin preterm infants. The aim of the study is to examine the impact of different storage conditions onthe stability of the human breast milk peptidome. HDM was directly frozen at−80◦C or stored at−20◦C (120 h), 4◦C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiledby mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storagetemperature and time and varied for different peptides. The highest impact was observed whensamples were stored at RT. Smaller but significant effects were still observed in samples stored at4◦C, while samples showed highest similarity to those immediately frozen at−80◦C when storedat−20◦C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombinand other proteases cleaving proteins at lysine/arginine. The results point to an ongoing proteindegradation/peptide production by milk-derived proteases. They underline the need for immediatefreezing of HDM at−20◦C or−80◦C to prevent degradation of peptides and enable reproducibleinvestigation of prospectively collected samples.
New World arenaviruses represent an important group of zoonotic pathogens that pose a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and neurological symptoms, other closely related family members exhibit little or no pathogenicity. For instance, Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, while the closely related Tacaribe virus (TCRV) is avirulent in humans. Little is known about host cell responses to infection, or how they contribute to virulence; however, TCRV strongly induces caspase-dependent apoptosis (i.e. non-inflammatory programmed cell death) in infected cells, whereas JUNV does not.
In order to better understand the connection between apoptosis and pathogenesis, we sought to unravel the regulation of pro- and anti-apoptotic signaling in response to arenavirus infection. We demonstrated that apoptosis induced by TCRV proceeds over the mitochondrial-regulated intrinsic pathway and involves activation of p53 (accumulation and phosphorylation), activation of the pro-apoptotic BH3-only factors Puma and Noxa (accumulation), as well as inactivation of another pro-apoptotic factor called Bad (phosphorylation). The regulation of these factors in response to TCRV infection is accompanied by other classical hallmarks of intrinsic apoptosis, such as disorganization of the mitochondrial network, cytochrome c release, PS flipping, caspase cleavage and nuclear condensation. The involvement of the BH3-only factors as key players in regulating TCRV-induced apoptosis could also be validated in knockout cells, which showed either suppressed or increased apoptosis depending on the respective activation (i.e. Puma and Noxa) or inactivation (i.e. Bad) status of the respective BH3 protein. Interestingly, while JUNV does not trigger late stages of apoptosis induction (i.e. caspase activation, nuclear condensation and cell death), we could show that it activates similar upstream pro-apoptotic signaling events including activation of p53, Puma and Noxa. This supports the current hypothesis that JUNV actively evades the induction of apoptosis through the involvement of a mechanism targeting late steps in the apoptotic cascade. Specifically, this model proposes that intrinsic activation is suppressed at the level of caspase activation by JUNV NP, which serves as an alternative substrate for caspase cleavage.
Additionally, in order to identify viral factors associated with the induction of apoptosis, a full genome sequencing of TCRV was performed and contributed to the validation and correction of substantial errors reported in existing sequences for TCRV. With the help of this sequence, correct expression plasmids containing the viral genes for NP, GP and Z were constructed and tested for their ability to induce apoptosis in vitro. This revealed that both TCRV and JUNV Z are triggers for apoptosis, which further supports our finding that JUNV also induces activation of pro-apoptotic factors. Again, consistent with a model where JUNV NP blocks caspase activation directly, co-expression of JUNV Z and NP abrogated caspase activation, while simultaneous expression of TCRV NP and Z still resulted in cell death.
Finally, identification of the specific apoptotic factors involved in regulating TCRV-induced apoptosis (i.e. Bad, Puma and Noxa) and the generation of the respective knockout cell lines allowed us to investigate what influence apoptosis induction has on virus infection. Interestingly, knockout of these factors showed no direct impact on virus growth in Vero cells. However, TCRV particles produced in cells with the individual pro-apoptotic (i.e. Puma and Noxa) or anti-apoptotic (i.e. Bad) factors knocked out showed altered infectivity in primary human monocytes and macrophages, which represent important target cells for arenaviruses. Since TCRV particles that originate from the different knockout cells would be expected to contain different amounts of PS in their envelope (depending on the level of apoptosis taking place), this suggests a role of apoptosis in facilitating PS-receptor-mediated entry and/or PS-receptor signaling through downstream kinases, either of which could be contributing to successful infection in professional phagocytic cells. In particular, phosphorylation of some of the identified factors involved in regulating TCRV-induced apoptosis indicates the involvement of upstream kinases from diverse signaling pathways, some of which also play a role in regulating cytokine production – another host cell reaction that differs significantly between TCRV- and JUNV-infected monocytes and macrophages. As such, these findings represent an exciting basis for a possible connection between apoptotic responses and the regulation of pro- and anti-inflammatory cytokine responses via their associated upstream signaling processes and provide a starting point for future studies that will help us to better understand how these processes contribute to arenavirus pathogenicity.
The Two-Component System 09 Regulates Pneumococcal Carbohydrate Metabolism and Capsule Expression
(2021)
Streptococcus pneumoniae colonizes asymptomatically the upper respiratory tract as a commensal, but has also a high virulence potential and can leave this ecological niche, thereby spreading to the lungs and blood. During this process, pneumococci must adapt to changing external environmental conditions and parameters such as nutrient availability, temperature, or oxygen levels. The transmission of these signals into the bacterial cell interior occurs via the process of signal transduction, which ultimately results in controlled differential gene expression. The most commonly strategy for signal transduction is the use of two-component regulatory systems (TCS), consisting of a membrane-bound histidine kinase as a sensor and a cytoplasmic response regulator that binds to the promoter region of its target genes and interferes with gene expression.
In this study the regulatory impact and influence of the TCS08 and TCS09 on the phenotype and pathophysiology of S. pneumoniae were investigated using two different serotypes
(serotype 2: D39 and serotype 4: TIGR4). For all functional assays, single (Δrr08/Δrr09 or Δhk08/Δhk09) and double (Δtcs08 or Δtcs09) mutants that were constructed by insertion-deletion mutagenesis, were applied.
In the first study a comparative transcriptome analysis using RNA-sequencing was conducted with our tcs09-mutants and the parental wild-type D39. The data indicated upregulation of the aga operon, which is related to galactose metabolism, and downregulation of the regulator AgaR, particularly in the absence of HK09. Interestingly, encapsulated and nonencapsulated hk09-mutants in D39 showed significant growth defects when galactose was used as sole carbohydrate source. Electron microscopy revealed morphological changes such as an increased number of membrane vesicles and cell wall degradation for the nonencapsulated hk09- and tcs09-mutants of strain D39. An increased capsule production was indicated for the encapsulated hk09- and tcs09-mutants in D39. The latter two mutants as well as the encapsulated rr09-mutant also showed altered colony morphology. While D39Δhk09 formed only opaque colonies, the mutants D39Δrr09 and D39Δtcs09 showed increased numbers of transparent colonies. In a Triton X-100 induced autolysis assay and in the presence of oxidative stress, a negative effect of the morphological changes of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 on their survivability was demonstrated. In conclusion, we observed that TCS09 in S. pneumoniae D39 is important for its fitness through regulation of carbohydrate metabolism. This indirectly influences cell wall integrity and capsular polysaccharide amount via other regulatory mechanisms, which ultimately affects stress tolerance.
In a second study, we investigated the virulence potential of TCS09 in pneumococcal strain TIGR4. In vitro growth analyses in complex medium showed no effect after loss of function of TCS09 on pneumococcal fitness. In contrast, using the disaccharides lactose and sucrose in chemically defined medium, an extended lag phase of tcs09-mutants was monitored. To assess changes of virulence factor expression, immunoblots were applied to demonstrate the abundance of various essential virulence factors of S. pneumoniae. The results revealed a decreased amount for RrgB, which is the backbone pilus component of type 1 pili, in the hk09-mutant. Field emission scanning electron microscopy and transmission electron microscopy images were applied to study alterations of the bacterial cell shape. The illustrations by FESEM and TEM showed no effect of TCS09-deletion on pneumococcal cell morphology. Cell culture-based infection analyses revealed a similar adhesion capacity of the parental strain and isogenic mutants to lung epithelial cells. However, phagocytosis assays indicated a significantly increased killing rate of intracellular TIGR4ΔcpsΔtcs09, when compared to the isogenic parental strain. In experimental mouse infection models of acute pneumonia and systemic infection the tcs09-mutants were not attenuated. However, to decipher in more detail differences between the wild-type and tcs09-mutants, in vivo co-infection were performed, which highlighted a significantly lower bacterial load of TIGR4luxΔhk09 and TIGR4luxΔtcs09 especially in the lungs, blood, and brain after 48 h. In conclusion, the TCS09 in TIGR4 is necessary for maintaining metabolic fitness, which in turn contributes to dissemination in the host.
In the third study, the influence of TCS08 on gene expression and metabolic and pathophysiological processes of S. pneumoniae was analyzed. In particular, differential gene expression in the hk08-mutant of TIGR4 was detected using microarray and qPCR. The transcriptome analysis revealed a downregulation of cellobiose specific phosphotransferase systems as well as an upregulation of the fab operon, arc operon, and psa operon. These operons encode proteins involved in fatty acid biosynthesis, arginine catabolism, and manganese uptake, respectively. Furthermore, we measured a downregulation of pilus 1 genes in TIGR4ΔcpsΔtcs08 and an increased expression of pavB in TIGR4ΔcpsΔhk08. These data were confirmed by immunoblotting and surface localization studies. Using in silico analysis, a SaeR-like binding motif was identified in the promoter region of pavB. Furthermore, the impact of TCS08 on pneumococcal virulence was investigated in vivo using the acute pneumonia and sepsis models. These models showed a strain-dependent effect of the single TCS08 component deletions between D39 and TIGR4 pneumococci. Whereas loss of HK08 or TCS08 in D39 attenuated the mutants in the pneumonia model, loss of RR08 in TIGR4 was responsible for a similar effect. In contrast, loss of HK08 in TIGR4 promoted increased virulence in the pneumonia and sepsis model. Overall, these data indicate that TCS08 is involved as key player in bacterial fitness during host colonization.
Teichonsäuren (TA) sind ein wesentlicher Bestandteil von Gram-positiven Bakterien und damit auch der Zellwand von Streptococcus pneumoniae. Die Glykopolymere werden in Peptidoglykan-verbundene Wandteichonsäuren (WTA) und Glykolipid-verbundene Lipoteichonsäuren (LTA) unterschieden. Anders als in anderen Gram-positiven Spezies weisen WTA und LTA von S. pneumoniae einen gemeinsamen zytoplasmatischen Biosyntheseweg auf, der in einer identischen Struktur beider Glykopolymere resultiert. Erst der finale Transfer der TA-Ketten an das Peptidoglykan oder den Glykolipid-Anker unterscheidet sich im Biosyntheseweg.
In der vorliegenden Arbeit wurde SPD_1672 von Stamm S. pneumoniae D39 (TIGR4: SP_1893) als putative Lipoteichonsäure Ligase (Teichoic acid Ligase = TacL) identifiziert. In experimentellen Maus-Infektionsmodellen der akuten Pneumonie und systemischen Infektion zeigte die tacL-Mutante eine verringerte Virulenz und in in vitro Zellkultur-basierten Infektionsanalysen eine abgeschwächte Adhäsion an Lungenepithelzellen. Obwohl die TacL-Defizienz einen signifikanten pathophysiologischen Einfluss in vivo zeigte, konnte bei in vitro Wachstumsanalysen in einem Komplexmedium sowie in chemisch definiertem Medium nur ein sehr geringer Effekt auf die Wachstumsrate gemessen werden. Die Feldemissions-Rasterelektronenmikroskopie (FESEM) und Transmissionselektronenmikroskopie (TEM) Aufnahmen zur Untersuchung der bakteriellen Morphologie sowie die Analyse der durchflusszytometrischen Bestimmungen des Kapselgehalts der tacL-Mutante zeigten, dass die Bakterienmorphologie mit dem typischen Erscheinungsbild als Diplokokken unbeeinflusst waren. Das an die TAs substituierte Phosphorylcholin (P-Cho) dient als Anker für sogenannte Cholin-Bindungproteine (CBP), die wiederum unter anderem eine Rolle in der Autolyse, Kompetenz und Virulenz von S. pneumoniae spielen. Unter Verwendung polyklonaler Antikörper gegen verschiedener CBPs wurde lediglich bei PspC, Pce und CbpJ eine veränderte Menge festgestellt, wohingegen bei PspA, CbpL, LytA und CbpG kein Unterschied in der Abundanz auf der Bakterienoberfläche in der tacL-Mutante im Vergleich zum isogenen Wildtyp gemessen werden konnte. Darüber hinaus gelang es im Rahmen dieser Arbeit weitere Deletionsmutanten in Genen zu generieren, die für Proteine kodieren, denen eine hypothetische Rolle in der Teichonsäurebiosynthese zugewiesen wird und deren Funktionen in einer nachfolgenden Arbeit charakterisiert werden sollte.
Im Rahmen dieser Arbeit konnte Ligase zu Assemblierung der LTA in S. pneumoniae identifiziert werden. Die beeinträchtigte Virulenz der tacL-Mutante im in vivo Maus-Infektionsmodell macht TacL zu einem potentiellen Ziel für antimikrobielle Substanzen.
Respiratory infection caused by Streptococcus pneumoniae is a leading cause of morbidity and mortality in older adults. Acquired CD4+ T cell mechanism are essential for the protection against colonization and subsequent development of infections by S. pneumoniae. In this study, we hypothesized that age-related changes within the CD4+ T-cell population compromise CD4+ T-cell specific responses to S. pneumoniae, thereby contributing to increased susceptibility at older age. To this end, we interrogated the CD4+ T-cell response against the immunogenic pneumococcal protein AliB, part of the unique oligopeptide ABC transporter system responsible for the uptake of nutrients for the bacterium and crucial for the development of pneumococcal meningitis, in healthy young and older adults. Specifically, proliferation of CD4+ T cells as well as concomitant cytokine profiles and phenotypic markers implied in immunosenescence were studied. Older adults showed decreased AliB-induced CD4+ T-cell proliferation that is associated with an increased frequency of regulatory T cells and lower levels of active CD25+CD127+CTLA-4−TIGIT-CD4+T cells. Additionally, levels of pro-inflammatory cytokines IFNy and IL-17F were decreased at older age. Our findings indicate that key features of a pneumococcal-specific CD4+ T-cell immune response are altered at older age, which may contribute to enhanced susceptibility for pneumococcal infections.
Streptococcus pneumoniae is a commensal of the human upper respiratory tract and
the etiological agent of several life-threatening diseases. This pathogen is the model bacterium
for natural competence. Furthermore, the pneumococci played an important role in the
identification of DNA as the main molecule involved in bacterial transformation. As a result,
studies on the pneumococcal genome provided an initial overview of the genetic potential of
this pathogen. The pneumococcus is a highly versatile bacterium possessing a high rate of
uptake and recombination of exogenous DNA from neighboring bacteria. As such, a significant
diversity in the genome content among the different pneumococcal strains has been reported.
The capsular polysaccharide, an important pneumococcal virulence factor, is the best example
on the pneumococcal diversity. There are over 98 serotypes characterized to date presenting
differences in their capsule (cps) locus. Additional to the cps locus, the pneumococcus also
presents 13 genomic islets annotated as regions of diversity (RD) encoded in the auxiliary
genome. Remarkably, 8 of the pneumococcal RD studied so far have been associated with
virulence. Furthermore, the ongoing sequencing of over 4000 pneumococcal genomes have
shed light on the conservation level of well-known pneumococcal virulence factors.
Interestingly, important pneumococcal virulence determinants show variations in the gene and
protein sequence among the different strains. Prototypes are for example the pneumococcal
surface protein C (PspC) and pneumococcal adherence and virulence factor B (PavB).
Conversely, gene regulation in S. pneumoniae is carried out by highly conserved and genome-
wide distributed transcriptional factors. Overall, the pneumococci interplays with its
environment with 4 major regulatory systems: quorum sensing (QS), stand-alone
transcriptional regulators, small RNAs (sRNAs) and two-component regulatory systems (TCS).
Some of these systems are multifaceted and share more than one feature. Furthermore, there
is crosstalk among the different systems, requiring the activation of a signaling cascade to
function properly.
A comprehensive analysis of the distribution and conservation of pneumococcal
virulence factors and TCS was obtained in this study. The results are summarized as a
simplified variome in which 25 pneumococcal strains with a complete sequenced genome were
analyzed. Interestingly, the genes encoding the glycolytic protein enolase and the toxin
pneumolysin were the most conserved virulence determinants. Additionally, the high level of
conservation was confirmed for the pneumococcal TCS regulators, especially for WalKR,
CiaRH and TCS08.
The main focus of this study was on the regulatory functions of pneumococcal TCS.
With this in mind, an extensive and detailed systematic review of the 13 pneumococcal TCS
and its orphan RR was undertaken. For this purpose, every pneumococcal TCS was analyzed
for its reported functional and structural information along with its contribution to the main
pathophysiology of the pneumococci. In brief, S. pneumoniae can utilize its TCS for the
regulation of important cellular processes and the sensing of detectable signals in the
environment. Additionally, the role of TCS in pneumococcal processes and signal sensing can
be divided further. In the first place, pneumococcal TCS regulate competence and fratricide,
the production of bacteriocins and host-pathogen interaction processes, while the detectable
signals include cell-wall perturbations, environmental stress, and nutrients. As a conclusion
from this section, it is possible to analyze the pneumococcal TCS in a comprehensive manner.
There is a complex network among the different pneumococcal regulators and the TCS play
an important role. Moreover, these systems are highly conserved and essential for the proper
functioning of the pneumococcus as a pathogen.
Following up on pneumococcal TCS, this study focused especially on the TCS08.
Interestingly, the pneumococcal TCS08 has been previously associated with the regulation of the cellobiose metabolism. Furthermore, this system has also been reported to regulate the
expression of genes encoded in the RD4 (Pilus-1). Remarkably, the pneumococcal TCS08
was shown to be highly homologous to the SaeRS system of Staphylococcus aureus. Initially,
mutant strains lacking a single (Δrr08 or Δhk08) or both components (Δtcs08) of the TCS08
were generated in pneumococcal D39 and TIGR4 strains. Transcriptomics and functional
assays showed a downregulation of the PI-1 in the absence of the complete tcs08, while PavB
presented an upregulation in the Δhk08 knockout. Moreover, an important number of genes
coding for intermediary metabolism proteins were also found to be differentially expressed by
microarray analysis. As such, the TIGR4Δhk08 strain presented a downregulation for the
cellobiose operon (cel). In contrast, an upregulation was reported for the fatty acid biosynthesis
(fab) and arginine catabolism (arc) operons. Conversely, a decrease in gene expression was
seen in the TIGR4Δrr08 strain for the arc operon. Finally, in vivo murine pneumonia and sepsis
models highlighted an involvement of TCS08 in pneumococcal virulence. Remarkably, the
different TCS08 mutants presented a strain dependent effect on their virulence severity. The
TIGR4Δrr08, and all TCS08 mutants in D39 showed a decrease in virulence in the pneumonia
model, with no changes in sepsis. Conversely, the absence of HK08 in TIGR4 presented a
highly virulent phenotype in both pneumonia and sepsis models. To sum up, the pneumococcal
TCS08 influenced the expression of genes involved in fitness and colonization. Specifically,
those coding for the adhesins PavB and PI-1 and fitness proteins from the cel, arc and fab
operons. Remarkably, the highest changes in expression were observed in the strains lacking
the HK08. Additionally, TCS08 has a strain dependent impact on pneumococcal virulence as
showed by murine pneumonia and sepsis models when comparing the effects in D39 and
TIGR4.
Die klassischen Schilddrüsenhormone (TH) Triiodthyronin (T3) und Thyroxin (T4) sind für die Regulation zahlreicher Stoffwechselprozesse von Bedeutung. Dabei beeinflussen sie unter anderem maßgeblich den hepatischen Energie- und Lipidstoffwechsel. In den letzten Jahren haben die beiden Schilddrüsenhormon-Metaboliten 3-Iodthyronamin (3-T1AM) und 3,5-Diiodthyronin (3,5-T2) an Aufmerksamkeit gewonnen, da sie in diversen Studien als endogene, biologisch aktive Substanzen beschrieben wurden.
Die durch 3-T1AM-vermittelten metabolischen Effekte sind dabei denen der klassischen TH teilweise entgegengesetzt. Zudem konnte eine Interferenz mit der Hypothalamus-Hypophysen-Schilddrüsen (HPT)-Achse demonstriert werden. In dieser Arbeit sollte die Hypothese einer direkten 3-T1AM-Wirkung auf die Schilddrüse überprüft werden. Dazu wurden in einem in vitro-Modell 3-T1AM-behandelte Thyreozyten der Zelllinie PCCL3 mittels Transkriptomanalysen untersucht.
Für den TH-Metaboliten 3,5-T2 konnten in früheren Arbeiten anti-steatotische, anti-lipidemische und kalorigene Effekte demonstriert werden. Aufgrund fehlender thyreotoxischer Nebenwirkungen, wie sie für die klassischen TH typisch sind, liegt ein therapeutisches Potenzial von 3,5-T2 zur Behandlung der mit steigender Inzidenz auftretenden Adipositas und der damit assoziierten Fettleber (Steatosis hepatis) nahe. Im Rahmen dieser Arbeit wurden die 3,5-T2-vermittelten Effekte auf die hepatischen Transkriptions- und Proteinmuster von Mäusen unter Standard- (SD) und Hochfettdiät (HFD) in komplementären Analysen charakterisiert.
Die TH-Homöostase wird durch den negativen Rückkopplungsmechanismus der HPT-Achse reguliert. Im klinischen Alltag wird jedoch häufig eine Störung dieses Gleichgewichts in Form einer Hypo- oder Hyperthyreose beobachtet. Um die physiologischen Auswirkungen dieser Erkrankungen zu untersuchen, wurden in dieser Arbeit Proteomanalysen der Lebern von Mäusen mit induzierter Hypo- und Hyperthyreose durchgeführt, um bereits vorliegende korrespondierende Transkriptomdaten zu ergänzen.
In den Transkriptomanalysen der 3-T1AM-behandelten Thyreozyten konnten keine Genexpressionsänderungen nachgewiesen werden. Um diese Diskrepanz zu den in anderen Studien demonstrierten metabolischen Effekten zu beheben, könnte eine Optimierung des experimentellen Designs sinnvoll sein. Alternativ könnte gefolgert werden, dass vor allem post-transkriptionelle Prozesse die Wirkungen von 3-T1AM vermitteln.
Die komplementären Transkriptom- und Proteomdaten der 3,5-T2-behandelten Mäuse deuteten auf eine Stimulation der hepatischen Cholesterol-, Gallensäure- und lokalen Sexualhormon-Biosynthese in Tieren unter HFD hin. Außerdem konnten in Mäusen unter HFD erhöhte hepatische Spiegel von Sexualhormonen nachgewiesen werden. Weiterhin zeigten zahlreiche Transkripte und Proteine, welche in den Lipidstoffwechsel und Citratzyklus involviert sind, signifikante Mengenveränderungen nach 3,5-T2-Behandlung unter SD und HFD. Die in dieser Arbeit unter beiden Diäten beobachteten 3,5-T2-vermittelten Effekte auf Xenobiotika-metabolisierende Proteine könnten dabei unter anderem auf unerwünschte thyreomimetische Nebeneffekte hindeuten. Daher sollte der therapeutische Einsatz von 3,5-T2 als ein potenzielles anti-steatotisches Agens, wie es diverse vorangegangene Studien propagiert haben, kritisch betrachtet werden.
Die ersten Ergebnisse der hepatischen Proteomanalysen hyperthyreoter Mäuse deuteten auf eine Reduktion von oxidativem Stress und eine Induktion der Proteinbiosynthese hin, während unter hypothyreoten Bedingungen entgegengesetzte Effekte beobachtet wurden.
Im Rahmen dieser Arbeit konnten umfangreiche globale und komplementäre Datensätze mit Hilfe der Omics-Technologien Transkriptomics und Proteomics, für die Microarray- und Massenspektrometrie-basierte Analysen zum Einsatz kamen, generiert werden. Diese ermöglichten die Gewinnung neuer Erkenntnisse über die physiologischen Effekte und Wirkungsweisen der TH-Metabolite 3-T1AM und 3,5-T2 sowie die Krankheitsbilder Hypo- und Hyperthyreose.
Mast cells reside on and near the cerebral vasculature, the predominant site of pneumococcal entry into the central nervous system (CNS). Although mast cells have been reported to be crucial in protecting from systemic bacterial infections, their role in bacterial infections of the CNS remained elusive. Here, we assessed the role of mast cells in pneumococcal infection in vitro and in vivo. In introductory experiments using mouse bone marrow-derived mast cells (BMMC), we found that (i) BMMC degranulate and release selected cytokines upon exposure to Streptococcus pneumoniae, (ii) the response of BMMC varies between different pneumococcal serotypes and (iii) is dependent on pneumolysin. Intriguingly though, apart from a slight enhancement of cerebrospinal fluid (CSF) pleocytosis, neither two different mast cell-deficient Kit mutant mouse strains (WBB6F1-KitW/Wv and C57BL/6 KitW-sh/W-sh mice) nor pharmacologic mast cell stabilization with cromoglycate had any significant impact on the disease phenotype of experimental pneumococcal meningitis. The incomplete reversal of the enhanced CSF pleocytosis by local mast cell engraftment suggests that this phenomenon is caused by other c-Kit mutation-related mechanisms than mast cell deficiency. In conclusion, our study suggests that mast cells can be activated by S. pneumoniae in vitro. However, mast cells do not play a significant role as sentinels of pneumococcal CSF invasion and initiators of innate immunity in vivo.
The thyroid as the largest endocrine gland mainly produces and secretes the thyroid hormones (TH): 3,3’,5-triiodo-L-thyronine (T3) and its pro-hormone L-thyroxine (T4). Besides the impact on growth, normal development, bone marrow structure, the cardiovascular system, body weight and thermogenesis, TH play a vivid role in many metabolic regulatory mechanisms in almost all tissues. Thyroid diseases are relatively prevalent and cause, due to the resulting TH imbalances, a broad spectrum of effects. Many of them manifest in pathologically increased or decreased TH levels defined as hyperthyroidism or hypothyroidism, respectively. Routinely, determination of the thyroid state is based on the assessment of the classical markers TSH and free T4. However, this practice has several drawbacks. Moreover, elucidation of the pleiotropic effects of TH on multiple molecular pathways is mostly based on cell culture, tissue and rodent models. Analysis of animal biofluids like serum and urine using metabolomics approaches demonstrated the extensive impact of TH on other body compartments. In contrast, proteome profiling has not been exploited for the comprehensive characterization of the general metabolic effects of TH. Plasma as a large and diverse compartment of the human proteome provides a great opportunity to identify novel protein markers of thyroid function as well as to characterize metabolic effects of TH in humans.
Therefore, a study of experimental thyrotoxicosis was performed with 16 male volunteers treated with 0.25 mg/d levothyroxine (L-T4) for 8 weeks to induce a hyperthyroid state. Plasma samples were collected before the L-T4 application started, two times during the treatment and additionally two times after withdrawal. Proteome analysis revealed remarkable alterations including increased levels of two known proteins known to correlate with TH levels (sex hormone-binding globulin and cystatin C). The correlation with free T4 levels revealed 76 out of 437 detected proteins with a Pearson correlation coefficient of r ≥ |0.9|. One prominent signature included 10 coagulation cascade proteins exhibiting significantly increased plasma levels during thyrotoxicosis, thereby revealing a trend towards a hypercoagulative state in hyperthyroidism. To overcome the statistical drawbacks of the Pearson correlation analysis, additionally a mixed-effect linear regression model using serum free T4 concentrations as exposure and protein abundances as outcome while controlling for age, BMI, and batch was implemented. Application of this model resulted in the detection of 63 proteins with significant associations to free T4 levels. Besides the already mentioned augmented coagulation, a significant drop in the amounts of three apolipoproteins (ApoD, ApoB-100 and ApoC3) was observed. Furthermore, an increased abundance of proteins assigned to the complement system was detected.
Experimental studies in humans were complemented by corresponding analyses in murine models. In the current work, plasma samples of two murine studies including male C57BL/6 wildtype mice were analyzed to elucidate the impact of thyroid dysfunction on the plasma proteome. The first study was similarly designed as the human model of experimentally induced thyrotoxicosis and assigned the animals to three groups: a control group, a T4 treatment group, and a T4 recovery group, whereupon the latter first received T4 followed by a subsequent TH normalization period. A high proportion of plasma proteins exhibited significantly different protein levels during T4 application (n = 120), where 90 of these also showed a corresponding reverse trend after T4 withdrawal (T4 recovery vs. T4), thereby displaying transient alterations. The molecular pattern of hyperthyroidism in the murine model indicated, as in the human study, a pronounced decrease in apolipoproteins. However, in clear contrast to the human data, the levels of proteins related to the coagulation cascade and complement system were also transiently decreased in mice, while being increased in humans.
The second murine analysis focused on the impact of hyper- and hypothyroidism caused by T3 or T4 treatment and MMI/KClO4 application, respectively. In general, compared to the first murine study less clear alterations of protein levels were detected. Proteins related to the complement system revealed fewer changes in the T3 group and only marginal changes after T4 induction. Unexpectedly, the MMI/KClO4-induced hypothyroidism caused a reduction of the levels of several proteins assigned to the complement system, although different components and factors were affected.
Generally, rodent studies partially provided a divergent picture of TH action as compared to human studies. However, in spite of inconsistent results in studies regarding the effects of TH that are possibly due to species-specific differences, an important role of TH on several metabolic and other pathways, e.g. in the process of blood coagulation and apolipoprotein regulation, is evident. The results from both murine and human studies presented here provide novel insights into changes in the plasma proteome in the context of thyroid diseases which might contribute to a better understanding of TH action on metabolism and other pathways.
Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID, among them the multifunctional Taf1 protein. In contrast to mammalian Taf1, yeast Taf1 lacks bromodomains which are instead encoded by separate proteins Bdf1 and Bdf2. In this work, we show that Bdf1 not only binds to acetylated histone H4 but can also be recruited by Ino2 and unrelated activators such as Gal4, Rap1, Leu3 and Flo8. An activator-binding domain was mapped in the N-terminus of Bdf1. Subunits Toa1 and Toa2 of yeast TFIIA directly contact sequences of basal promoters and TFIID subunit TBP but may also mediate the influence of activators. Indeed, Ino2 efficiently binds to two separate structural domains of Toa1, specifically with its N-terminal four-helix bundle structure required for dimerization with Toa2 and its C-terminal β-barrel domain contacting TBP and sequences of the TATA element. These findings complete the functional analysis of yeast general transcription factors Bdf1 and Toa1 and identify them as targets of activator proteins.
The success of pregnancy depends on precisely adjusted, local immune mechanisms. In early pregnancy, fetal trophoblast cells implant into the endometrium to build and anchor the placenta. Simultaneously, they mediate fetal tolerance and defense against infections. To cover these versatile requirements, local immune factors must be in balance. A too tolerogenic milieu can lead to an inadequate placentation; while a too inflammatory milieu can cause rejection of the semi-allogenic fetus. Bacterial infections can provoke these inflammatory pregnancy complications as well. Therefore, the pregnant uterus was long thought to be sterile. Descriptions of a placental microbiome opened a scientific discourse, which is unsolved due to contrary studies. The colonization of the non-pregnant endometrium is, however, confirmed. It is supposed to affect both, uterine pathologies and fertility. Precise data are lacking. Aim of this work was to assess if and under which circumstances a bacterial colonization would be tolerable.
One of the described species in placental and endometrial samples is Fusobacterium nucleatum. It is an opportunistic bacterium, which is known from the human oral cavity and associated with the development of colon carcinomas. F. nucleatum supports tumorigenesis by the induction of epithelial proliferation, survival, migration and invasion as well as angiogenesis and tumor tolerance. Since similar processes are required for implantation and placentation, F. nucleatum might support these as well. In this work, the effects of F. nucleatum on leukocyte-trophoblast-interactions, especially of macrophages and innate lymphoid cells type 3 (ILC3), were assessed.
The monocytic cells (THP-1) were differentiated into inflammatory M1 (IFN-γ) or tissue-repairing and tolerogenic M2a (IL-4) and M2c (TGF-β) macrophages. Inactivated F. nucleatum, LPS or E. coli was added. Only small concentrations of inactivated bacteria were used (bacteria:leukocyte ratio of 0.1 or 1), since it was not the aim to analyze infections. Conditioned medium of treated leukocytes was added to trophoblastic cells (HTR-8/SVneo). Migratory, invasive and tube formation behavior of trophoblastic cells was quantified.
Treated M1 macrophages impaired trophoblast function, whereas M2a macrophages induced trophoblast invasion. M2c macrophages supported trophoblast migration and tube formation if treated with the smaller, but not with the higher concentration of F. nucleatum. This treatment induced the accumulation of HIF-1α and the secretion of VEGF-A in M2c macrophages as well. Moreover, the higher concentration of F. nucleatum caused rather inflammatory responses (NF-κB activation and cytokine expression). The activation of the HIF-1α-VEGF-A axis under the influence of TGF-β might serve as a mild immune stimulation by low abundant commensal bacteria supporting placentation.
In contrast to macrophages, the function of ILC3s during pregnancy is still unknown. In general, ILC3s are located in mucosal tissue, such as the gut. They participate in tolerance mechanisms and form the local micromilieu by the secretion of cytokines and the presentation of antigens. In order to characterize local, uterine ILC3s, murine ILC3s were compared to peripheral, splenic ILC3s. Uterine ILC3s were more activated and produced higher levels of IL-17 compared to splenic ILC3s. However, uterine ILC3s barely expressed MHCII on their surface. A reduced antigen presentation potential was confirmed in human ILC3s differentiated from cord blood stem cells by the addition of TGF-β or hCG. The treatment with bacteria increased MHCII expression, but not to the initial level. The higher bacterial concentration induced IL-8 secretion and led to an increased trophoblast invasion. ILC3s were less sensitive to bacterial stimulation than macrophages.
Recent studies on the uterine or placental presence of bacteria during pregnancy are discrepant. The results of this project indicate that bacteria or bacterial residues might serve as a mild stimulus under certain circumstances to support implantation without negative effects. The current discussion must therefore not only be expanded by additional studies, but especially include differentiated local conditions. In this context, the sheer presence of bacteria or bacterial components must not be equated with an infection representing a known hazard.
To induce an appropriate immune response, pathogen- or damage-associated molecular
patterns (PAMPs and DAMPs) are sensed by innate immune cells. One of the first
mechanisms after recognition is the assembly of an inflammasome leading to the cleavage of
pro-IL-1β and pro-IL-18 into the active cytokines IL-1β and IL-18, respectively. Hyperinflammatory
conditions and excessive activation of the inflammasome, however, are drivers
of inflammatory and autoimmune diseases. The NLR family pyrin domain-containing 3
(NLRP3) inflammasome is suggested to be involved in the development of gout,
artherosclerosis, type II diabetes, cryopyrin-associated periodic syndrome (CAPS), various
types of cancer, and inflammatory bowel disease (IBD). To tackle these diseases, drugs
interfering with IL-1 signaling are of great value. Anakinra, an IL-1 receptor antagonist,
inhibits binding of both IL-1α and IL-1β. Rilonacept consists of the Fc portion of human IgG1
with two IL-1 receptors grafted onto it. This allows neutralization of IL-1α and IL-1β. The
monoclonal IgG1 antibody Canakinumab specifically blocks IL-1β. Although well tolerated,
patients undergoing a therapy with these drugs are at a higher risk of developing bacterial
respiratory tract infections.
The human upper respiratory tract (URT) is commonly colonized asymptomatically by
bacteria like Staphylococcus aureus and Streptococcus pneumoniae. Only a limited number of
studies have so far addressed the role of the NLRP3 inflammasome during pneumococcal
infections. Imbalances in the immune system enhance the dissemination of these bacteria into
the lungs thereby causing life-threatening infections. Asides from immune-compromising
therapies such as IL-1 inhibition, viral infections are common factors leading to the
development of a secondary, bacterial infection. Influenza A virus infection of C57BL/6J
mice was characterized by a bi-phasic disease progression with a complete remission around
day 16 irrespectively of a previous Streptococcus pneumoniae 19F colonization. At this time
point, low amounts of bacteria were recovered from the lungs of 50% of co-infected mice.
This co-infection model was used for in-depth analyses of the innate immune responses.
Bioactive lipids, such as eicosanoids, are host-derived metabolites, which can heavily impact
the cellular immune response and skew it towards a pro- or anti-inflammatory phenotype.
Eicosanoid analyses revealed increased levels of different hydroxydocosahexaenoic acids
(HDHAs) and hydroxyeicosatetraenoic acids (HETEs) in the lung after single viral infection.
Staphylococcal pneumonia mainly resulted in high expression of 20-HETE. During a coinfection
with S. pneumoniae and influenza A virus (IAV), reduction of HETEs and HDHAs
was measured. Furthermore, ceramide-1-phosphate (C1P) was elevated in the lung, whereas
sphingosine-1-phosphate (S1P) was reduced. The tight control of an inflammatory response
by bioactive lipid mediators might be protective during later stages of an infection.
Cytokine analyses revealed high expression of inflammatory cytokines in lungs and plasma of
single bacteria- and virus infected as well as co-infected mice at early stages of an infection.
In all three infections monocyte chemoattractant protein-1 (MCP-1) dependent monocyte
recruitment was noted. The elevated expression of CC chemokine receptor 2 (CCR2) and
major histocompatibility complex II (MHCII) in the majority of analysed myeloid cells
indicated an activation of these cells. In single virus and co-infections, we further observed a
mainly neutrophil-independent immune response. During pneumococcal pneumonia, an influx
of neutrophils was observed.
Neutrophils, as first recruited responders during an inflammation, can release their granule
contents after activation in order to eliminate extracellular pathogens. However, excessive
neutrophil activation correlates with tissue damage and detrimental outcome of an infection.
As a result of excessive cell death, large amounts of ATP are released into the extracellular
space. Pneumolysin (Ply), a pore-forming toxin and major virulence factor of S. pneumoniae,
is a potent activator of human neutrophils. Stimulation of neutrophils with sublytic
concentrations of Ply readily resulted in the release of resistin and other granule proteins into
the supernatant. Microscale Thermophoresis (MST) analyses revealed that ATP neutralizes
Ply by binding it in the extracellular space. Co-stimulation of neutrophils with both lytic and
sublytic amounts of Ply and physiologically relevant concentrations of ATP resulted in
diminished neutrophil activation. We speculate that during hyper-inflammation (caused by
excessive neutrophil recruitment and activation), tissue injury results in the release of large
amounts of extracellular ATP which, in turn, will bind to Ply. Ultimately, binding of ATP to
Ply inhibits further neutrophil activation and might mitigate immunopathology.
The transcriptome of non-coding RNA (ncRNA) species is increasingly focused in Alzheimer’s disease (AD) research. NcRNAs comprise, among others, transfer RNAs, long non-coding RNAs and microRNAs (miRs), each with their own specific biological function. We used smallRNASeq to assess miR expression in the hippocampus of young (3 month old) and aged (8 month old) Tg4-42 mice, a model system for sporadic AD, as well as age-matched wildtype controls. Tg4-42 mice express N-truncated Aβ4–42, develop age-related neuron loss, reduced neurogenesis and behavioral deficits. Our results do not only confirm known miR-AD associations in Tg4-42 mice, but more importantly pinpoint 22 additional miRs associated to the disease. Twenty-five miRs were differentially expressed in both aged Tg4-42 and aged wildtype mice while eight miRs were differentially expressed only in aged wildtype mice, and 33 only in aged Tg4-42 mice. No significant alteration in the miRNome was detected in young mice, which indicates that the changes observed in aged mice are down-stream effects of Aβ-induced pathology in the Tg4-42 mouse model for AD. Targets of those miRs were predicted using miRWalk. For miRs that were differentially expressed only in the Tg4-42 model, 128 targets could be identified, whereas 18 genes were targeted by miRs only differentially expressed in wildtype mice and 85 genes were targeted by miRs differentially expressed in both mouse models. Genes targeted by differentially expressed miRs in the Tg4-42 model were enriched for negative regulation of long-term synaptic potentiation, learning or memory, regulation of trans-synaptic signaling and modulation of chemical synaptic transmission obtained. This untargeted miR sequencing approach supports previous reports on the Tg4-42 mice as a valuable model for AD. Furthermore, it revealed miRs involved in AD, which can serve as biomarkers or therapeutic targets.
Abstract
Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid‐phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram‐positive and Gram‐negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain‐specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species‐specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.
A successful colonization of different compartments of the human host requires multifactorial contacts between bacterial surface proteins and host factors. Extracellular matrix proteins and matricellular proteins such as thrombospondin-1 play a pivotal role as adhesive substrates to ensure a strong interaction with pathobionts like the Gram-positive Streptococcus pneumoniae and Staphylococcus aureus. The human glycoprotein thrombospondin-1 is a component of the extracellular matrix and is highly abundant in the bloodstream during bacteremia. Human platelets secrete thrombospondin-1, which is then acquired by invading pathogens to facilitate colonization and immune evasion. Gram-positive bacteria express a broad spectrum of surface-exposed proteins, some of which also recognize thrombospondin-1. This review highlights the importance of thrombospondin-1 as an adhesion substrate to facilitate colonization, and we summarize the variety of thrombospondin-1-binding proteins of S. pneumoniae and S. aureus.
The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.
Kardiovaskuläre Erkrankungen gehören trotz zahlreicher medikamentöser und apparativer Therapiemaßnahmen noch immer zu den häufigsten Todesursachen in den Industrienationen. Die Herzinsuffizienz (HI) stellt dabei das Endstadium vieler Herzerkrankungen dar und beschreibt das Unvermögen des Herzens, die Blutzirkulation im Organismus bei normalem Ventrikeldruck konstant zu halten. Unabhängig von ihrer Ätiologie, wie Koronarerkrankungen, langjähriger Hypertonie oder auch Kardiomyopathien ist die HI neben der Funktionsreduktion des linken und/oder rechten Ventrikels gleichzeitig durch strukturelle Veränderungen (Remodeling) mit Gefäßverengung (Vasokonstriktion), endotheliale Dysfunktion mit Vasokonstriktion, sowie eine generalisierte neurohumorale Aktivierung gekennzeichnet. Die Suche nach neuen und alternativen Therapieverfahren zur Verbesserung der Symptomatik und Prognose der betroffenen Patienten ist daher notwendig. Einer der wichtigsten Mediatoren für die Regulation des Gefäßwiderstandes ist Stickstoffmonoxid (NO, nitric oxide), welches durch NO-Synthasen synthetisiert wird. NO aktiviert die lösliche Guanylatzyklase (sGC, soluble guanylate cyclase), wodurch es zu einer erhöhten Produktion des second messengers cGMP (cyclic guanosine monophosphate) kommt. Eine Beeinträchtigung des NO-sGC-cGMP-Signalweges und der dadurch bedingte Mangel an cGMP trägt zu den Prozessen der myokardialen und endothelialen Dysfunktion bei der Entwicklung und Progression einer HI bei. Die Entwicklung pharmakologisch aktiver Moleküle, die die sGC direkt stimulieren können, ist dabei von besonderem Interesse, da z.B. keine Toleranzentwicklung bei längerer Medikation oder andere negative Nebenwirkungen wie bei der Gabe von NO-Donatoren als Vasodilatatoren entstehen.
Im Rahmen dieser Arbeit sollte der Einfluss einer sGC-Stimulation mittels Riociguat (RIO), einem bereits für die Behandlung der pulmonal arteriellen Hypertonie (PAH) und der chronisch thromboembolischen pulmonalen Hypertonie (CTEPH) zugelassenen Medikament, auf die experimentelle HI untersucht werden. Neben Echokardiographie und histologischen Analysen zur Charakterisierung des Krankheitsphänotyps und der Auswirkung einer Behandlung darauf wurde ebenfalls auf Multi-Omics-Ansätze wie Proteomics und Transcriptomics zurückgegriffen, um detaillierte Einblicke in die molekularen Veränderungen auf Genexpressionsebene, Proteinebene und microRNA-Expressionsebene zu erlangen. Als Modell wurde die transverse Aortenkonstriktion (TAC) an C57BL/6N Mäusen verwendet, welche einen permanenten hämodynamischen Stressreiz auf das Herz ausübt, der schließlich zum Herzversagen führt. Im Hinblick auf die Pathogenese der HI simuliert TAC dabei auf elegante Weise eine arterielle Hypertonie, die unter anderem zu einer progressiven linksventrikulären Hypertrophie und einer reduzierten Herzfunktion unter chronischen Bedingungen führt. Für die medikamentöse Behandlung mit RIO wurde eine experimentelle Strategie gewählt, die der klinischen Situation entspricht. Dementsprechend wurde mit der Medikation zu einem Zeitpunkt begonnen, als die Herzfunktion bereits verschlechtert war und eine pathologische Hypertrophie und interstitielle Fibrose ausgebildet bzw. nachweisbar war.
TAC führte zu einer kontinuierlichen Abnahme der linksventrikulären Ejektionsfraktionsfraktion (LVEF) und einer kontinuierlichen Zunahme der linksventrikulären Masse (LVM). Eine fünfwöchige Behandlung mit RIO (3 mg/kg/d) ab der vierten postoperativen Woche führte zu einer Verbesserung der LVEF und zu einer Verringerung des Verhältnisses von LVM zu Gesamtkörpergewicht (LVM/BW), myokardialer Fibrose und Myozytenquerschnittsflächen. RNA-Sequenzierungsanalysen der linken Ventrikel ergaben, dass RIO die Expression von myokardialen Stress- und Remodeling-Genen, wie z.B. Nppa, Nppb, Myh7 und Kollagen, verringerte und die Aktivierung biologischer Signalwege abschwächte, die mit kardialer Hypertrophie und HI in Verbindung stehen. Diese protektiven Effekte einer RIO-Behandlung konnten auch auf Proteinebene beobachtet werden und spiegelten sich in einer deutlichen Reduktion der TAC-induzierten Veränderungen des linksventrikulären Proteoms wider. Durch die Aortenkonstriktion betroffene Signalwege, die mit kardiovaskulären Erkrankungen assoziiert sind, wie gewebe- und zellstrukturspezifische Signalwege, besonders aber Signalwege des Energiemetabolismus, zeigten eine Verbesserung nach einer RIO-Behandlung. Zudem schwächte RIO auch die TAC-induzierten Veränderungen auf microRNA-Ebene in den linken Ventrikeln ab.
Mit dieser Arbeit konnte gezeigt werden, dass eine Behandlung mit RIO positive Auswirkungen auf die kardiale Struktur bzw. das pathologische kardiale Remodeling und die Funktion in einem murinen Modell der chronischen Nachlasterhöhung/Drucküberlastung hat, was mit einer Umkehrung bzw. Abschwächung der TAC-induzierten Veränderungen des kardialen linksventrikulären Genexpressions-, Proteom- und microRNA-Profils einhergeht. Die vorliegenden Ergebnisse unterstützen die bisherigen Vermutungen und Erkenntnisse zum Potential von RIO als neuartigem HI-Therapeutikum. Des Weiteren wurden große Omics-Datensätze generiert, die als Informationsquelle zukünftigen Untersuchungen helfen können, die molekularen Mechanismen der chronischen HI und möglicher therapeutischer, medikamentöser Interventionen besser zu verstehen und weiter zu entschlüsseln.
Streptococcus pneumoniae (the pneumococcus) is an opportunistic human pathogen that causes life-threatening diseases including pneumonia, sepsis, meningitis but also non-invasive local infections such as otitis media. Pneumococci have evolved versatile strategies to colonize the upper respiratory tract (URT) of humans. Binding to epithelial surfaces is thereby mediated through direct interactions with host cell receptors or indirectly via binding to components of the extracellular matrix (ECM). However, successful colonization and subsequent infection require S. pneumoniae to cross tissue barriers protected by the immune system of the host. Pneumococci have therefore evolved a wide range of mechanisms to circumvent the antibacterial activity of the immune system such as the acquisition or expression of serine protease activity. Serine protease enzymes have emerged during evolution as one of the most abundant and functionally diverse groups of proteins in eukaryotic and prokaryotic organisms. However, the epithelial barriers, integrins, and other cell surface receptors are often initially inaccessible for pneumococci colonizing the nasopharyngeal cavity. Therefore, pneumococci recruit host-derived extracellular serine proteases such as plasmin(ogen) for extracellular matrix and mucus degradation, which results in enhanced binding to epithelial and endothelial cells. S. pneumoniae expresses four surface-anchored or surface-associated serine proteases depending on the serotype: HtrA, SFP, PrtA, and CbpG. These enzymes belong to the category of trypsin-like or subtilisin-like family proteins, which are characterized by the presence of three-conserved amino acid residues, Ser-His-Asp. The catalytic triads are critical for the cleavage of peptide bonds. Studies focusing on the deletion of single pneumococcal serine proteases are difficult to interpret due to the compensatory effects of the other serine proteases.
Initially, a comprehensive in silico analysis of the distribution and genes organization of pneumococcal serine proteases was carried out in this study. Interestingly, the genes encoding PrtA, HtrA, and CbpG were highly conserved among the 11 analyzed strains. Surprisingly, the gene encoding the subtilisin-like protein SFP was not present in some of the strains and seems to be strain-dependent. Therefore, pneumococci have at least three serine proteases as shown e.g., for serotype 19F_EF3030 strain. Computer-assisted analyses of the structure of pneumococcal serine proteases showed high similarities in the catalytic domains between HtrA and CbpG or between PrtA and SFP in 3D structural models.
The focus of this study lies on the impact of single extracellular pneumococcal serine proteases on pneumococcal pathogenesis during adherence, colonization, virulence and biofilm formation. Therefore, double and triple deletion mutants were generated in the colonizing S. pneumoniae serotype 19F strain EF3030 and the more invasive serotype 4 strain TIGR4, respectively. In adherence studies with human Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F_EF3030 mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. In a mouse colonization model, the inactivation of serine proteases in strain 19F_EF3030 strongly reduced nasopharyngeal colonization in CD-1 mice. The bacterial load in the nasopharynx was thereby monitored for a period of 14 days. Mutant strains showed significantly lower bacterial numbers in the nasopharynx on days 2, 3, 7, and 14 post-inoculations.
Following up on pneumococcal pathogenesis, an in vivo acute pneumonia mouse infection model and in vitro phagocytosis was used to analyze the impact of single serine proteases during infection and phagocytosis. Mice were intranasally infected with the bioluminescent TIGR4lux wild-type or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. The acute lung infection was monitored in real-time by using an IVIS®-Spectrum in vivo imaging system. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. Phagocytosis assays were conducted using murine J77A.1 macrophages. The number of triple serine protease mutants internalized by macrophages were significantly reduced in comparison to the isogenic wild-type.
Finally, two different experimental biofilm models were used to study the influence of serine proteases on biofilm formation grown on an abiotic surface (glass) and a biological surface. Biofilm development on living epithelial cells was stronger after 48 and 72h than on the glass surface. On epithelial substratum, the serine protease mutant with only CbpG+ showed higher and denser biofilm development after 48h and 72h of incubation compared to the parental strains and other serine protease mutants. Moreover, the bacterial dispersal from biofilms was significantly more in the mutant strains lacking serine proteases than in the wild type.
In conclusion, nasopharyngeal colonization is a prerequisite for invasive diseases and transmission. Pneumococcal serine proteases are indispensable for nasopharyngeal colonization and facilitate access to eukaryotic cell-surface receptors by the cleavage of ECM proteins. Thus, serine proteases could be promising candidates for developing antimicrobials to reduce pneumococcal colonization and transmission.
The pathobiont Streptococcus pneumoniae causes life-threatening diseases, including pneumonia, sepsis, meningitis, or non-invasive infections such as otitis media. Serine proteases are enzymes that have been emerged during evolution as one of the most abundant and functionally diverse group of proteins in eukaryotic and prokaryotic organisms. S. pneumoniae expresses up to four extracellular serine proteases belonging to the category of trypsin-like or subtilisin-like family proteins: HtrA, SFP, PrtA, and CbpG. These serine proteases have recently received increasing attention because of their immunogenicity and pivotal role in the interaction with host proteins. This review is summarizing and focusing on the molecular and functional analysis of pneumococcal serine proteases, thereby discussing their contribution to pathogenesis.
Streptococcus pneumoniae has evolved versatile strategies to colonize the nasopharynx of humans. Colonization is facilitated by direct interactions with host cell receptors or via binding to components of the extracellular matrix. In addition, pneumococci hijack host-derived extracellular proteases such as the serine protease plasmin(ogen) for ECM and mucus degradation as well as colonization. S. pneumoniae expresses strain-dependent up to four serine proteases. In this study, we assessed the role of secreted or cell-bound serine proteases HtrA, PrtA, SFP, and CbpG, in adherence assays and in a mouse colonization model. We hypothesized that the redundancy of serine proteases compensates for the deficiency of a single enzyme. Therefore, double and triple mutants were generated in serotype 19F strain EF3030 and serotype 4 strain TIGR4. Strain EF3030 produces only three serine proteases and lacks the SFP encoding gene. In adherence studies using Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. Consistent with these results, we show that the mutants of strain 19F, which preferentially colonizes mice, abrogate nasopharyngeal colonization in CD-1 mice after intranasal infection. The bacterial load in the nasopharynx was monitored for 14 days. Importantly, mutants showed significantly lower bacterial numbers in the nasopharynx two days after infection. Similarly, we detected a significantly reduced pneumococcal colonization on days 3, 7, and 14 post-inoculations. To assess the impact of pneumococcal serine proteases on acute infection, we infected mice intranasally with bioluminescent and invasive TIGR4 or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. We imaged the acute lung infection in real-time and determined the survival of the mice. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. In conclusion, our results showed that pneumococcal serine proteases contributed significantly to pneumococcal colonization but played only a minor role in pneumonia and invasive diseases. Because colonization is a prerequisite for invasive diseases and transmission, these enzymes could be promising candidates for the development of antimicrobials to reduce pneumococcal transmission.