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Streptococcus pneumoniae (pneumococci) and Staphylococcus aureus (S. aureus) are human-specific commensals of the upper respiratory tract. Every individual is asymptomatically colonized with both bacteria at least once in their life-time. The opportunistic pathogens can affect further organs and invade into deeper tissue. The occupation of normally sterile niches of the human body with the bacteria can lead to local infections such as sinusitis, otitis media and abscesses, or to life-threatening diseases like pneumonia, meningitis or sepsis. A strong interaction between the bacterium and the respiratory epithelial cells is a prerequisite for a successful colonization. This interaction is ensured by bacterial surface proteins, so called adhesins. The binding of the adhesins to the epithelial lineage occurs predominantly indirectly via components of the extracellular matrix (ECM), but also directly to cellular receptors. Pneumococci and S. aureus bind to various ECM glycoproteins, amongst others: fibronectin, fibrinogen, vitronectin, and collagen. Also binding of both pathogens to human thrombospondin-1 has been described. Thrombospondin-1 is mainly stored in the α-granula of thrombocytes (platelets) and released into the circulation upon activation. However, thrombospondin-1 is also produced and secreted by other cell types like endothelial cells, macrophages, and fibroblasts, which gets subsequently incorporated as component into the ECM. So far, no thrombosponin-1-binding adhesins of pneumococci were identified. PspC, Hic, and PavB are important surface-localized virulence factors, which were shown to interact with human ECM and plasma proteins. PspC and Hic bind to vitronectin and factor H, which inhibits the complement cascade of the human immune system. PavB interacts with fibronectin and plasminogen, and a pavB-deficient mutant of S. pneumoniae showed diminished capacity in colonization in a mouse model. Among the surface proteins of S. aureus, only Eap was identified as thrombospondin-1-binding adhesin. Beyond colonization, pneumococci and S. aureus can enter the blood circulation, interact with platelets, and cause their activation. The aggregation of platelets, especially initiated by S. aureus, plays an important role in the clinic, because most of the septic patients develop thrombocytopenia. Surface localized factors of
S. pneumoniae triggering platelet activation are unknown to date. In contrast, few proteins of S. aureus with potential to activate platelets, including Eap, were identified previously.
This study identified the surface proteins PavB, PspC, and Hic of S. pneumoniae as specific ligands of the human thrombospondin-1. Flow cytometric, surface plasmon resonance spectroscopic and immunological analyses revealed interactions between the pneumococcal proteins and soluble as well as immobilized thrombospondin-1. The use of specific pneumococcal deletion mutants verified the importance of the three virulence factors as binding partners of soluble thrombospondin-1. The results suggest that pneumococci are capable of acquiring soluble thrombospondin-1 from blood as well as utilizing immobilized glycoprotein of the ECM as substrate for adhesion. Furthermore, the thrombospondin-1-binding domain within the pneumococcal proteins was analyzed by use of recombinant fragments of PavB, PspC, and Hic. The binding capacity of thrombospondin-1 increased proportionally with the amount of repetitive sequences in PavB and PspC, and the length of the α-helical region within the Hic molecule. The binding behavior of thrombospondin-1 towards PavB and PspC is comparable with that of the ECM proteins vitronectin and fibronectin, but is unique towards Hic.
The localization of the binding domain of the adhesins within the thrompospondin-1 molecule occurred via use of glycosaminoglycans as competitive inhibitors for the interaction. The results suggest that the pneumococcal proteins Hic and PspC target the identical binding region within thrombospondin-1, which differs from the binding domain for PavB. However, all three virulence factors seem to bind in the N-terminal part of thrombospondin-1.
Two-dimensional gel electrophoresis, thrombospondin-1 overlay assay and subsequent mass spectrometric analysis identified AtlA of S. aureus as a surface localized interaction partner of human thrombospondin-1. Moreover, a vitronectin binding activity for AtlA was determined. Immunological and surface plasmon resonance binding studies with recombinant AtlA fragments revealed that interactions with both matrix proteins is mediated via the C-terminal located repeats R1R2 of the AtlA amidase domain. Binding of thrombospondin-1 and vitronectin occurred not simultaneously, due to a competitive inhibition.
The second part of the study focused on the activation of human platelets by recombinant pneumococcal and staphylococcal proteins. In total, 28 proteins of S. pneumoniae and 52 proteins of S. aureus were incubated with human platelets. The activation of the cells was detected by flow cytometry using the activation markers P-selectin and the dimerization of the integrin αIIbβIII. The proteins CbpL, PsaA, PavA, and SP_0899 of S. pneumoniae induced platelet activation, however, the detailed mechanism has to be deciphered in further studies. Furthermore, the secreted proteins CHIPS, FLIPr, and AtlA of S. aureus were discovered as inductors for the activation of platelets. In addition, the domains of AtlA and Eap, crucial for platelet activation, were narrowed down. Interestingly, CHIPS, FLIPr, and Eap were described as inhibitors of neutrophil recruitment. Platelets are recently recognized as immune cells, due to the expression of immune receptors. The data obtained in this study highlight a comprehensive spectrum of effects of the S. aureus proteins towards different type of immune cells. Besides the activation of platelets in suspension buffer and plasma, the aggregation of platelets in whole blood was triggered by the proteins CHIPS, AtlA, and Eap. These results suggest a contribution of the proteins during the S. aureus-induced infectious endocarditis. Secretion of the platelet activating virulence factors, which were identified within this study, might represent a pathogenic strategy during S. aureus infection in which a direct contact between S. aureus and platelets is not required or even avoided.
In conclusion, PavB, PspC, and Hic of S. pneumoniae and AtlA of S. aureus were identified as interaction partners of human thrombospondin-1. Furthermore, CHIPS, FLIPr, AtlA, and Eap were characterized as platelet activators. This study provides candidates for the development of protein-based vaccines, to prevent bacterial colonization and to neutralize secreted pathogenic factors.
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.
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.
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.
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.
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.
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.
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.
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.
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.