No description available.

No description available.

The antimicrobial effects of silver nanoparticles were studied. Silver nanoparticles were synthesized through wet chemistry method, and were dispersed in aqueous suspension. With nanoscale silica particles served as heterogeneous nucleation sites, silver nanoparticles were formed anchoring on the silica surface. Suspensions were found to be stable at high silver concentrations as well as over a broad pH range. By varying the processing conditions, diameter of the silver nanoparticles could be controlled between ~2 nm to ~25 nm. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to reveal the formation and the corresponding morphology of the silver-silica coupling nanoparticles. Ultra-violet visible (UV-vis) scanning spectrophotometer was used to detecting the distinct absorption spectrum of silver nanoparticles. The antimicrobial activities of these silver-silica coupling nanoparticles were investigated. E. coli and S aureus were used as representatives of Gram-negative and Gram-positive bacteria, respectively. Bacteriological tests showed either bacterial growth inhibition or cell death occurred, corresponding to different concentrations of silver nanoparticles and the type of bacteria that were testing on. Fluorescent microscopic images were also provided to confirm the bacterial viability after several hours' treatment with silver nanoparticles. / Master of Science

Staphylococcus aureus is a Gram-positive bacterium and part of the human bacterial microflora but it also is an opportunistic pathogen and a notorious cause of hospital – acquired and epidemic infections. S. aureus shows a remarkable adaptation to a range of niches within its human host.Previously viewed as an exclusively extracellular pathogen, S. aureus has been demonstrated to replicate inside virtually all cell types after cell invasion. S. aureus thereby either multiplies within phagosomal compartments in professional phagocytes or escapes into the cytosol prior to replication in non-professional phagocytic cells such as epithelial cells. Besides α-type phenol soluble modulins(PSMα, an important role in phagosomal escape was attributed to the non-ribosomal peptide synthase (NRPS) AusAB. AusAB incorporates the aromatic amino acids (AAAs) phenylalanine or tyrosine, as well as the branched-chain amino acids (BCAAs) valine and leucine into three cyclic dipeptides collectively called aureusimines: Phevalin, Tyrvalin and Leuvalin. The role of AusAB in S. aureus infection is not entirely understood. BCAAs are essential amino acids for S. aureus and serve as protein building blocks,precursors for the biosynthesis of branched-chain fatty acids, as well as regulatory molecules for the transcriptional regulator CodY. Severe BCAA depletion triggers stringent response. It is therefore counterintuitive that AusAB would incorporate valine into aureusimines thereby depleting BCAAs in host niches where available nutrients are scarce. The present study therefore analysed the role of the AusAB NRPS and its BCAA-derived monoketopiperazine products, in the interaction of S. aureus with various host niches and with other bacterial species. By using genetic tools and metabolomic approaches, it could be established that the AusAB NRPS preferentially incorporates only the exogenous aromatic amino acids phenylalanine and tyrosine, while the source of valine can be either endogenous or exogenous. By using promoter reporter assays, an effect of the global SaeR regulator on ausAB expression was observed. Additionally, a possible role of the NRPS in modulatory metabolic processes of overflow metabolism emerged. Finally, while the role of AusAB in infection is still unclear, experiments using ex vivo human lung tissue suggest, for the first time, that AusAB NRPS might be involved in the staphylococcal virulence against human lung tissue. By employing bioluminescence and biofilm assays in both Gram-negative (Vibrio harveyi and Pseudomonas aeruginosa) and Gram-positive (CoNS Staphylococcus spp.) model species, a potential role as a Quorum Quenching molecule arose for Phevalin, but not Tyrvalin. While studying a potential connection of AusAB and stringent response, an unexpected role for the BrnQ1 BCAA transporter was revealed. BrnQ1 is known as the major BCAA import protein in S. aureus. The bacteria therefore rely on its activity in environments with low concentrations of available peptides or free BCAAs. The present study shows that BrnQ1 is necessary for efficient phagosomal escape of S. aureus in epithelial cells and that BrnQ1-mediated BCAA uptake is crucial for intracellular bacterial replication in epithelial cells, macrophages and whole human blood. Moreover, BrnQ1 loss of function allows bacteria to survive indefinitely inside macrophages without causing phenotypic changes in colony morphology, size and pigmentation as well as haemolysis after recovery. A dual RNA-sequencing approach further showed that, while no major host transcriptomic rearrangements occurred in human macrophages infected with brnQ1 mutants compared to wild-type S. aureus, intracellular brnQ1 mutants do not respond to local host iron depletion and thus fail to upregulate iron uptake systems. In summary, the present work elaborates on multiple functions of BCAA-based small molecules as well as BCAA-transporters in the S. aureus host and bacterial interactions. This work provides a consolidation of the role of the AusAB NRPS in S. aureus lung infection, gives an insight into the unique mode of action of the NRPS and attributes a novel function for the NRPS-product Phevalin in bacterial communication providing, to my knowledge, the first example of a natural monoketopiperazine involved in bacterial communication. Further, experiments using ex vivo human lung tissue suggest, for the first time, that AusAB NRPS might be involved in the staphylococcal virulence against human lung tissue. Additionally, this work describes a potential mechanism of S. aureus long-term survival in macrophages involving a BCAA-iron metabolism axis. / Staphylococcus aureus ist ein Gram-positives Bakterium und Teil der menschlichen bakteriellen Mikroflora, aber auch ein opportunistisches Pathogen und eine allgemein bekannte Ursache für Krankenhaus- und epidemische Infektionen. S. aureus zeigt eine bemerkenswerte Anpassung an eine Reihe von Nischen innerhalb seines menschlichen Wirtes. Früher wurde der Erreger ausschließlich als extrazelluläres Pathogen angesehen. Doch es hat sich gezeigt, dass S. aureus nach einer Zellinvasion in praktisch allen Zelltypen replizieren kann. Dabei vermehrt sich S. aureus in professionellen Phagozyten innerhalb phagosomaler Kompartimente, in nicht-professionellen phagozytischen Zellen wie Epithelzellen entweicht das Bakterium vor der Replikation in das Zytosol. Neben α-Typ Phenollöslichen Modulinen (PSMα) wurde der nicht-ribosomalen Peptidsynthase (NRPS) AusAB eine wichtige Rolle beim phagosomalen Ausbruch zugeschrieben. AusAB baut die aromatischen Aminosäuren (AAAs) Phenylalanin oder Tyrosin, sowie die verzweigtkettigen Aminosäuren (BCAAs) Valin und Leucin zu den drei zyklische Dipeptiden Phevalin, Tyrvalin und Leuvalin zusammen, die unter dem Namen Aureusimine zusammengefasst werden. Die Rolle von AusAB bei der S. aureus-Infektion ist nicht vollständig geklärt. BCAAs sind essentielle Aminosäuren für S. aureus und dienen als Proteinbausteine, Vorstufen für die Biosynthese von verzweigtkettigen Fettsäuren sowie als regulatorische Moleküle für den Transkriptionsregulator CodY. Eine starke BCAA-Depletion löst eine stringente Antwort aus. Es ist daher kontraintuitiv, dass AusAB Valin in Aureusimine einbaut und damit BCAAs in Wirtsnischen, in denen die verfügbaren Nährstoffe knapp sind, dezimiert. In der vorliegenden Studie wurde daher die Rolle der AusAB NRPS und seiner BCAA-abgeleiteten Monoketopiperazin-Produkte bei der Interaktion von S. aureus mit verschiedenen Wirtsnischen und mit anderen Bakterienarten analysiert. Durch den Einsatz von genetischen Werkzeugen und metabolomischen Ansätzen konnte festgestellt werden, dass die AusAB NRPS bevorzugt nur die exogenen aromatischen Aminosäuren Phenylalanin und Tyrosin einbaut, während die Quelle für Valin entweder endogen oder exogen sein kann. Mit Hilfe von Promotor-Reporter-Assays wurde ein Effekt des globalen Regulators SaeR auf die ausAB-Expression beobachtet. Zusätzlich zeigte sich eine mögliche Rolle des NRPS bei modulatorischen Stoffwechselvorgängen des Überlaufmetabolismus. Schließlich, während die Rolle von AusAB bei der Infektion noch unklar ist, deuten Experimente mit ex vivo menschlichem Lungengewebe zum ersten Mal darauf hin, dass das NRPS von AusAB an der Virulenz von Staphylokokken gegenüber menschlichem Lungengewebe beteiligt sein könnte. Durch den Einsatz von Biolumineszenz- und Biofilm-Experimenten bei repräsentativen Gramnegativen (Vibrio harveyi und Pseudomonas aeruginosa) als auch Gram-positiven (CoNS Staphylococcus spp.) Spezies ergab sich für Phevalin, nicht aber für Tyrvalin, eine mögliche Rolle als Quorum Quenching-Molekül. Bei Untersuchung einer möglichen Verbindung von AusAB und der stringenten Antwort wurde eine unerwartete Rolle für den BrnQ1 BCAA-Transporter entdeckt. BrnQ1 ist als das wichtigste BCAAImportprotein in S. aureus bekannt. Die Bakterien sind daher auf seine Aktivität in Umgebungen mit niedrigen Konzentrationen an verfügbaren Peptiden oder freien BCAAs angewiesen. Die vorliegende Studie zeigt, dass BrnQ1 für einen effizienten phagosomalen Ausbruch von S. aureus in Epithelzellen notwendig ist und dass die BrnQ1-vermittelte BCAA-Aufnahme für die intrazelluläre bakterielle Replikation in Epithelzellen, Makrophagen und menschlichem Vollblut entscheidend ist. Darüber hinaus ermöglicht der Funktionsverlust von BrnQ1 den Bakterien ein unbegrenztes Überleben im Inneren von Makrophagen ohne phänotypische Veränderungen in Koloniemorphologie, -größe und -pigmentierung sowie in Hämolyse nach der Rückgewinnung der Bakterien. Ein dualer RNASequenzierungsansatz zeigte darüber hinaus, dass in humanen Makrophagen, die mit brnQ1- Mutanten infiziert waren, im Vergleich zu Wildtyp S. aureus keine größeren Transkriptomänderungen im Wirt auftraten. Jedoch reagierten intrazelluläre brnQ1-Mutanten nicht auf eine lokale Eisenverarmung des Wirts und regulieren somit keine Eisenaufnahmesysteme hoch. Zusammenfassend zeigt die vorliegende Arbeit die vielfältigen Funktionen von BCAA-basierten kleinen Molekülen sowie von BCAA-Transportern in der Interaktion zwischen dem S. aureus-Wirt und -Bakterium auf. Diese Arbeit liefert eine Konsolidierung der Rolle des AusAB NRPS in der S. aureus Lungeninfektion, gibt einen Einblick in die einzigartige Wirkweise des NRPS und schreibt dem NRPSProdukt Phevalin eine neuartige Funktion in der bakteriellen Kommunikation zu, die meines Wissens das erste Beispiel eines an der bakteriellen Kommunikation beteiligten natürlichen Monoketopiperazins darstellt. Darüber hinaus deuten Experimente mit ex vivo menschlichem Lungengewebe zum ersten Mal darauf hin, dass das AusAB NRPS an der Virulenz von Staphylokokken gegenüber menschlichem Lungengewebe beteiligt sein könnte. Zusätzlich beschreibt diese Arbeit einen möglichen Mechanismus für das Langzeitüberleben von S. aureus in Makrophagen, der eine BCAA-Eisenmetabolismus-Achse beinhaltet.

Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis. / Ph. D.

<i>Staphylococcus aureus</i> is a versatile opportunistic pathogen causing a wide spectrum of diseases in both humans and animals. My research focused on characterization of the immune responses of monocyte derived dendritic cells (DC) to <i>S. aureus</i>. We initially evaluated the potential of circulating monocytes to serve as precursors for DC during <i>S. aureus</i> infection. The CD14⁺ monocytes, when stimulated with irradiated (ISA) or live <i>S. aureus</i> (LSA), differentiated into CD11c^high CD11b^high DC (MonoDC) in an autocrine fashion. This was associated with the up- regulation of granulocyte-macrophage colony stimulating factor (GMCSF) and tumor necrosis factor-α (TNF-α) gene transcription. We continued our studies to identify the role of TNF-α in the LSA induced differentiation of monocyte to MonoDC. Blocking TNF-α reduced the expression of CD11c and increased the expression of CD14 on LSA stimulated monocyte derived MonoDC. Stimulated monocytes were able to secrete monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes to the site of infection/injury and induces the expression of β₂ integrins on DC. Characterization of the response of DC derived from monocytes using GMCSF and IL-4 revealed that, intact <i>S. aureus</i> rather than its purified structural components were efficient in DC activation. In response to ISA or LSA stimulation, DC induced proliferation of T cells collected from the peripheral circulation of cows with a history of <i>S. aureus</i> mastitis. Subsequent characterization of the proliferating T cells identified the presence of memory T cells. Finally, we identified a unique population of DEC205⁺CD8^a+ in monocyte derived DC. We further elucidated the role of DC DEC205, a C-type lectin, in <i>S. aureus</i> uptake. Blocking of receptor mediated endocytosis resulted in reduced uptake of <i>S. aureus</i> by DC. Confocal microscopy confirmed a role for DEC205 in <i>S. aureus</i> internalization and delivery to endosomes. DEC205 DC upon stimulation with <i>S. aureus</i> displayed enhanced maturation and antigen presentation. In conclusion, monocyte derived DC can uptake <i>S. aureus</i> and elicit cell mediated immune responses. / Ph. D.

Influenza A virus (IAV) is a significant problem worldwide, and respiratory disease is further complicated by secondary bacterial infection. The emergence of highly pathogenic strains of IAV in conjunction with the increase of antibiotic-resistant bacteria threatens human health. A large-animal model effective for study of polymicrobial infection comparable to humans must therefore be developed. IAV has been studied extensively in small animals, including mice, rats and ferrets. However, these species frequently require IAV adaptation, reducing the capacity of these models to adequately represent human infection. Furthermore, species commonly used lack likeness to humans in both the presentation of symptoms and in lethality of infection. However, pigs are naturally susceptible to unadapted IAV and are considered to be the 'mixing vessel' for the recent pandemic IAV virus. Pigs are also susceptible to infection with Staphylococcus aureus, the most commonly isolated bacteria from IAV-infected human adults. Therefore, the use of pigs in the study of polymicrobial respiratory infections would be ideal for characterizing a host immune response comparable to humans, as well as for the development of diagnostics and therapeutics. Using this novel model, we determined that pigs are susceptible to Staphylococcus aureus, swine IAV, and polymicrobial infection. Furthermore, we showed that IAV infection predisposes pigs to Staphylococcus aureus pneumonia, and this susceptibility is dependent on day post-IAV infection. / Master of Science

Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The identification of differentially expressed proteins was carried out using a quantitative mass spectrometry (MS)-based proteomic approach, known as stable isotope labeling of amino acids in cell culture (SILAC). Four commensal and pathogenic strains each were grown in the SILAC minimal media (RPMI 1640), containing light (12C) and heavy (13C) form of lysine, respectively, until early stationary growth phase. Various protein fractions, including cell wall, membrane and secreted, were extracted from the bacterial cultures and mixed in a 1:1 ratio. The relative abundance of proteins present in light and heavy labeled samples was determined using MS analysis. From a total of 151 differentially expressed proteins, 58 were found to be upregulated in the pathogenic strains. These proteins are involved in a variety of cellular functions, including immune modulation, iron-binding, cellular transport, redox reactions, and metabolic enzymes. The differentially expressed proteins can serve as putative candidates to improve current approach towards development of a vaccine against S. aureus. / Master of Science

Staphylococcus aureus (S. aureus) is a gram-positive bacterium that causes mastitis in bovines and leads to financial losses to the dairy industry. Although antibody response plays a role in immune defense against S. aureus, cellular responses are of interest for vaccine development. A vaccine that stimulates both antibody and cellular responses could promote memory cell formation and provide effective protection against S. aureus. The superantigens and virulence factors secreted by live S. aureus (LSA) can interfere with immune responses and memory cell formation. Because irradiation reduces the metabolic activity and secretion of proteins, including S. aureus superantigens and hemolysins, we hypothesized the irradiated S. aureus (ISA) could drive immune cell responses. Dendritic cells (DC) were co-cultured with lymphocytes to study the cellular responses to ISA and LSA. Dendritic cells present antigens and polarize lymphocytes into different helper T (Th) cell types that drive cellular immune responses. The DC loaded with either ISA or LSA induced increased mRNA transcription of Th17-related cytokines and cytotoxic effector memory cell formation during antigen recall experiments. Lymphocytes co-cultured with LSA-loaded DC exhibited a higher fold-change in interferon (IFN) γ mRNA compared to ISA-loaded DC, suggesting the secreted antigens and the metabolic activity of S. aureus play a role in Th1 polarization. Th1 polarization can drive excessive inflammation and suppress beneficial Th17 responses. Bovine DC were stimulated with a mutant α-toxin deletion S. aureus strain to evaluate if α-toxin-mediated NOD2 receptor signaling activates Th1 polarization in response to S. aureus, which revealed that NOD2 mRNA transcription in DC was independent of α-toxin and that the deletion of α-toxin had no effect on the transcription of the cytokine IL-12 or the production of IFNγ by lymphocytes, events that drive Th1 polarization, in co-cultures. The deletion of accessory gene regulator (agr), which controls α-toxin production, reduced IFNγ production in lymphocytes co-cultured with the S. aureus-loaded DC, indicating that agr controlled the ability of S. aureus antigens to drive the Th1 polarization of lymphocytes. Overall, this thesis demonstrates that ISA is a promising source of antigens that stimulate memory cells formation and Th17 polarization in bovine immune cells. The reduced Th1 cytokine response to S. aureus was not dependent on α-toxin, but other virulence factors controlled by agr should be screened to determine the source of Th1 stimulation. / Ph. D. / Dairy cows’ health and productivity is negatively impacted by mastitis, infection starting at the mucosal surfaces of the udder. <i>Staphylococcus aureus</i> is a bacterium that can cause mastitis and there is no efficacious vaccine available. I explored the use of weakened <i>S. aureus</i> as a source of vaccine components and the α-toxins role in stimulating the immune cells like dendritic cells (DC) and lymphocytes. <i>S. aureus</i> was weakened using gamma irradiation to conserve the structural components of the bacterium and render it unable to secrete α-toxin. The DC were collected from dairy cows and stimulated with irradiated <i>S. aureus</i> and live <i>S. aureus</i> before lymphocytes were added to the cultures. The DC signaling, lymphocytes’ pro-inflammatory interferon gamma and mucosal immunity related interleukin responses were measured from RNA production. Memory cell formation and production of interferon gamma were measured from whole cells. The role of α-toxin in lymphocyte stimulation was further studied using a strain of bacterium that does not produce the toxin. Irradiated <i>S. aureus</i> induced low production of inflammatory interferon gamma compared to the live <i>S. aureus</i>. The α-toxin played no role in this, even if other components produced under the same regulatory element likely did, as shown by reduced interferon production in response to bacteria without the regulatory element. Irradiation of the bacterium did not reduce mucosal immunity related cytokine production or formation of memory cells. The irradiated <i>S. aureus</i> is a source for vaccine components that stimulate immune cells like DC and immunity to <i>S. aureus</i> on mucosal surfaces of the udder.

Bacteria adapt to stress conditions by altering their physiology, behavior, and gene expression in re- sponse to external challenges. They use evolved mechanisms to thrive in environments with various stress factors including temperature changes, nutrient shortages, and toxins. Specific stress-response pathways are activated upon encountering stress, regulating genes to cope and maintain cellular func- tions. This thesis focuses on a bioinformatical analysis of two bacterial pathogens: a) Staphylococcus aureus and b) Chlamydia trachomatis during stress. To understand the role of S. aureus proteins during infection, gene expression data from two strains (NewHG and NCTC 8325) were analyzed, and flux changes were examined during the middle and late exponential growth phases. The aim was to identify metabolic variations between the wild-type (WT) and various knockout mutants, including the Ser/Thr Kinase PknB, its phosphatase Stp, and the double knockout PknB/Stp. Both S. aureus strains were cultured in nutrient-poor medium to simulate infection conditions similar to those in an abscess of infected individuals. Subsequently, a comprehensive meta- bolic model was constructed using time-resolved transcriptome data, which was validated by qRT-PCR. This study highlights the critical role of PknB-mediated phosphorylation on Ser/Thr residues in regulat- ing amino acid catabolism and promoting gluconeogenesis, ensuring the cell's supply of essential com- ponents. Both PknB and Stp play crucial roles in multiple cellular processes, such as peptidoglycan, nucleotide, and aromatic amino acid synthesis, as well as aspartate transaminase catabolism. Deletion of stp signif- icantly impaired pyrimidine synthesis, while functional loss of PknB had a minor impact. In double knock-out experiments, genes responsible for synthesizing peptidoglycan, purines, and aromatic amino acids from glucose showed increased activity, while pyrimidine synthesis from glucose was less active compared to the WT. Furthermore, this thesis extensively explores the regulatory modules associated with the glmR/yvcK regulon and the cdaA-cdaR-glmM-glmS module. The study emphasizes the conservation of the glmR/yvcK regulon and its core genes, namely yvcJ, glmR/yvcK, and whiA, across various bacteria and cellular processes. Notably, the presence of structural and phosphorylation site similarities in glmR sequences among different bacteria suggests a complex interactome involving PknB/Stp and these regulatory modules, potentially leading to broader implica- tions for bacterial adaptation and virulence. The aim of the second part of this thesis was to investigate how Chlamydia trachomatis adapts to stress induced by the host and evades immune defenses by manipulating human cells. The study introduces an innovative in silico model that captures the dynamic regulatory processes of C. trachomatis and its in- teractions with host signaling proteins during infection. The network model gives particular emphasis to chlamydial chaperones like ClpB, ClpC, and Dnak, along with related proteins such as UhpC, PyrG, inclusion membrane proteins, and the host proteins associated with various signaling pathways. The analysis of multiple time points and subsequent statistical data analysis pinpointed significantly up- and down-regulated genes and proteins, which were used as the basis for dynamic modeling and interac- tome analysis. Network models of predicted and validated protein interactions were used to study the time course of pathogen-host interactions, particularly focusing on chlamydial membrane proteins and chaperones. Through transcriptomic data (GSE104166, GSE147538, and GSE165628) analysis and pathway enrichment, the study identifies key host proteins, including PI3K, MAP kinases, MDM2, c- Myc, and hexokinases, that play significant roles in C. trachomatis pathogenesis. The infection substan- tially modulates multiple signaling pathways, including Interferon-α and -γ response, TNF-α signaling via NFκB, and inflammatory responses. Gene set enrichment analysis provides valuable insights into the host's response to C. trachomatis in- fection, showing distinct gene enrichment patterns at different time points. The integration of pathways from multiple transcriptome datasets enhances the specificity of the network model. Additionally, the research underscores the importance of metabolic reprogramming, particularly involving glutamine uti- lization, for chlamydial survival and the transition between the two different morphological forms. Developed bioinformatic pipeline and systems biology model used in this study offer valuable tools for identification of essential genes/proteins for bacterial survival in the stress environment, virulence profiling, and understanding host-pathogen interactions, with potential applications in various research areas beyond the scope of this study. / Bakterien passen sich an Stressbedingungen an, indem sie ihre Physiologie, ihr Verhalten und ihre Genexpression als Reaktion auf externe Herausforderungen verändern. Sie nutzen weiterentwickelte Mechanismen, um in Umgebungen mit verschiedenen Stressfaktoren wie Temperaturschwankungen, Nährstoffmangel und Toxinen zu gedeihen. Beim Auftreten von Stress werden spezifische Stressreaktionswege aktiviert, die Gene regulieren, um mit dem Stress fertig zu werden und die zellulären Funktionen aufrechtzuerhalten. Diese Arbeit konzentriert sich auf eine bioinformatische Analyse zweier bakterieller Krankheitserreger: a) Staphylococcus aureus und b) Chlamydia trachomatis. Um die Rolle von S. aureus-Proteinen während der Infektion zu verstehen, wurden Genexpressionsdaten von zwei Stämmen (NewHG und NCTC 8325) analysiert und Flussänderungen während der mittleren und späten exponentiellen Wachstumsphase untersucht. Ziel war es, metabolische Variationen zwischen dem Wildtyp und verschiedenen Knockout-Mutanten zu identifizieren, nämlich Ser/Thr-Kinase PknB, ihre Phosphatase Stp und der Doppel-Knockout PknB/Stp. Beide S. aureus-Stämme wurden in nährstoffarmem Medium kultiviert, um Infektionsbedingungen zu simulieren, die denen in einem Abszess infizierter Personen ähneln. Anschließend wurde anhand von zeitaufgelösten Transkriptom-Daten ein umfassendes Stoffwechselmodell erstellt, das durch qRT-PCR Messungen validiert wurde. Diese Studie unterstreicht die kritische Rolle der PknB-vermittelten Phosphorylierung an Ser/Thr-Resten bei der Regulierung des Aminosäurekatabolismus und der Förderung der Gluconeogenese, wodurch die Versorgung der Zelle mit essentiellen Komponenten gewährleistet wird. Sowohl PknB als auch Stp spielen eine entscheidende Rolle bei zahlreichen zellulären Prozessen wie der Peptidoglykan-, Nukleotid- und aromatischen Aminosäuresynthese sowie dem Aspartat-Transaminase-Katabolismus. Die Deletion von stp beeinträchtigte die Pyrimidin-Synthese erheblich, während der Funktionsverlust von PknB nur geringe Auswirkungen hatte. In doppelten Knock-out-Experimenten zeigten die Gene, die für die Synthese von Peptidoglykan, Purinen und aromatischen Aminosäuren aus Glukose verantwortlich sind, eine erhöhte Aktivität, während die Pyrimidinsynthese aus Glukose im Vergleich zum Wildtyp weniger aktiv war. Darüber hinaus werden in dieser Arbeit die regulatorischen Module im Zusammenhang mit dem glmR/yvcK-Regulon und dem cdaA-cdaR-glmM-glmS-Modul eingehend untersucht. Die Studie unterstreicht die Konservierung des glmR/yvcK-Regulons und seiner Kerngene, nämlich yvcJ, glmR/yvcK und whiA, über verschiedene Bakterien und zelluläre Prozesse hinweg. Insbesondere das Vorhandensein von Struktur- und Phosphorylierungsstellen in glmR-Sequenzen zwischen verschiedenen Bakterien deutet auf ein komplexes Interaktionssystem hin, an dem PknB/Stp und diese regulatorischen Module beteiligt sind, was möglicherweise zu umfassenderen Auswirkungen auf die bakterielle Anpassung und Virulenz führt. Ziel des zweiten Teils dieser Arbeit war es, zu untersuchen, wie sich C. trachomatis an den vom Wirt ausgelösten Stress anpasst und der Immunabwehr entgeht, indem es menschliche Zellen manipuliert. In der Studie wird ein innovatives in silico Modell vorgestellt, das die dynamischen Regulationsprozesse von C. trachomatis und seine Interaktionen mit Wirtssignalproteinen während der Infektion abbildet. Das Netzwerkmodell legt besonderes Augenmerk auf Chlamydien-Chaperone wie ClpB, ClpC und Dnak sowie auf verwandte Proteine wie UhpC, PyrG, Einschlussmembranproteine und Wirtsproteine, die mit die mit verschiedenen Signalwegen assoziiert sind. Mehrere Zeitpunkte und statistische Datenanalysen ermittelten signifikant hoch- und herunterregulierte Gene und Proteine, die als Grundlage für die dynamische Modellierung und Interaktomanalyse verwendet wurden. Mit Hilfe von Netzwerkmodellen für vorhergesagte und validierte Proteininteraktionen wurde der zeitliche Verlauf von Pathogen-Wirt-Interaktionen untersucht, wobei der Schwerpunkt auf chlamydialen Membranproteinen und Chaperonen lag. Die Analyse von Transkriptom-Daten (GSE104166, GSE147538 und GSE165628) und die Anreicherung von Signalwegen identifizierte wichtige Wirtsproteine, darunter PI3K, MAP-Kinasen, MDM2, c-Myc und Hexokinasen, die eine wichtige Rolle in der Pathogenese von C. trachomatis spielen. Die Infektion moduliert im Wesentlichen mehrere Signalwege, darunter die Interferon-α- und -γ-Antwort, die TNF-α -Signalisierung über NFκB und Entzündungsreaktionen. Die Analyse der Anreicherung von Gensätzen liefert wertvolle Einblicke in die Reaktion des Wirts auf die Infektion mit C. trachomatis und zeigt unterschiedliche Muster der Genanreicherung zu verschiedenen Zeitpunkten. Die Integration von Signalwegen aus mehreren Transkriptom-Datensätzen erhöht die Spezifität des Netzwerkmodells. Darüber hinaus unterstreicht die Forschung die Bedeutung der metabolischen Reprogrammierung, insbesondere der Glutaminverwertung, für das Überleben von Chlamydien und den Übergang zwischen den beiden morphologisch unterschiedlichen Lebensformen. Sowohl die entwickelten bioinformatischen Pipelines als auch die systembiologischen Modelle bieten wertvolle Werkzeuge zur Identifizierung wesentlicher Gene/Proteine für das Überleben von Bakterien in der Stressumgebung, zur Erstellung von Virulenzprofilen und das Verständnis von Wirt-Pathogen-Interaktionen, mit potenziellen Anwendungen in verschiedenen Forschungsbereichen, die über den Rahmen dieser Studie hinausgehen.