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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.
Background: Tissue-resident macrophages have mixed developmental origins. They derive in variable extent from yolk sac (YS) hematopoiesis during embryonic development. Bone marrow (BM) hematopoietic progenitors give rise to tissue macrophages in postnatal life, and their contribution increases upon organ injury. Since the phenotype and functions of macrophages are modulated by the tissue of residence, the impact of their origin and developmental paths has remained incompletely understood. Methods: In order to decipher cell-intrinsic macrophage programs, we immortalized hematopoietic progenitors from YS and BM using conditional HoxB8, and carried out an in-depth functional and molecular analysis of differentiated macrophages. Results: While YS and BM macrophages demonstrate close similarities in terms of cellular growth, differentiation, cell death susceptibility and phagocytic properties, they display differences in cell metabolism, expression of inflammatory markers and inflammasome activation. Reduced abundance of PYCARD (ASC) and CASPASE-1 proteins in YS macrophages abrogated interleukin-1β production in response to canonical and non-canonical inflammasome activation. Conclusions: Macrophage ontogeny is associated with distinct cellular programs and immune response. Our findings contribute to the understanding of the regulation and programming of macrophage functions.
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.
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.
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.
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 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.
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 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.