@phdthesis{Raspe2011,
  author    = {Raspe, Matthias Eduard},
  title     = {Zellul{\"a}re Invasivit{\"a}t und molekulare Marker von kolonisierenden und Infektions-assoziierten Methicillin resistenten Staphylococcus aureus-Isolaten},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69444},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Hintergrund: Zunehmend wird der Eigenschaft von Staphylococcus aureus als fakultativ intrazellul{\"a}rem Erreger Bedeutung zugemessen. Ein direkter Nachweis der in vivo Relevanz von fakultativ intrazellul{\"a}rem S. aureus bleibt allerdings bisher aus. Der Mechanismus zellul{\"a}rer Invasivit{\"a}t ist bekannt und korreliert mit verschiedenen molekularen Markern (spa-Typ, SCCmec-Typ und pls/Pls). In dieser Studie wurde die Zuverl{\"a}ssigkeit und Ausweitbarkeit dieser Marker getestet. Des Weiteren wurde {\"u}berpr{\"u}ft, ob sich die zellul{\"a}re Invasivit{\"a}t von kolonisierenden und Infektions-assoziierten MRSA-Isolaten unterscheidet und, ob die alleinige Bestimmung molekularer Marker in vitro die Virulenz eines Isolats in vivo abzusch{\"a}tzen vermag. Methoden:Insgesamt wurden 109 MRSA-Isolate gesammelt, molekular charakterisiert (spa-Typ, BURP-Analyse, SCCmec-Typ, pls, agr-Typ, H{\"a}molyseverhalten) und das Potential zellul{\"a}rer Invasivit{\"a}t in vitro ermittelt. Die Assoziation eines Isolates mit einer Infektion in vivo wurde nachverfolgt (93 Kolonisierer versus 16 Infektions-assoziierte-Isolate). Zus{\"a}tzlich wurde eine Referenzgruppe aus 13 S. aureus-Isolaten etabliert, die klinisch mit vergleichsweise invasiven Infektionen assoziiert waren (12 Osteomyelitis-Isolate und 1 Endokarditis-Isolat). Ergebnisse: Die bekannten molekularen Marker zellul{\"a}rer Invasivit{\"a}t korrelieren zuverl{\"a}ssig in einer Population klinischer MRSA-Isolate und lassen sich auch auf bisher nicht bekannte (spa- und SCCmec-) Typen ausweiten. Das H{\"a}molyseverhalten korrelierte nicht mit der zellul{\"a}ren Invasivit{\"a}t. Der agr-Typ wurde als weiterer molekularer Marker identifiziert. Die zellul{\"a}re Invasivit{\"a}t war unabh{\"a}ngig von der Etablierung einer Infektion in vivo (mediane Invasivit{\"a}t der Kolonisierer 100\% versus 108\% der Infektions-assoziierten Studienisolate und 110\% der externen Referenzisolate). Des Weiteren waren die molekularen Marker spa- und agr-Typ nicht in der Lage, die Virulenz eines MRSA-Isolats in vivo abzusch{\"a}tzen. Diskussion: Die zellul{\"a}re Invasivit{\"a}t klinischer MRSA-Isolate korreliert zuverl{\"a}ssig mit molekularen Markern. Allerdings verm{\"o}gen weder die zellul{\"a}re Invasivit{\"a}t, noch mit ihr assoziierte molekulare Marker die Etablierung einer Infektion in vivo vorherzusagen. Beide scheinen also als Surrogat-Parameter zur Absch{\"a}tzung der klinischen Virulenz eines Isolats ungeeignet. Zur Kl{\"a}rung der Frage, ob molekulare Marker zellul{\"a}rer Invasivit{\"a}t in anderen Abschnitten der Pathogenese von S. aureus- Infektionen eine Rolle spielen, bedarf es weiterer Studien.},
  subject      = {MRSA},
  language  = {de}
}
@phdthesis{Hertlein2014,
  author    = {Hertlein, Tobias},
  title     = {Visualization of Staphylococcus aureus infections and antibiotic therapy by bioluminescence and 19F magnetic resonance imaging with perfluorocarbon emulsions},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-105349},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Staphylococcus aureus is a major threat to public health systems all over the globe. This second most cause of nosocomial infections is able to provoke a wide variety of different types of infection in humans and animals, ranging from superficial skin and skin structure infections to invasive disease like sepsis or pneumonia. But not enough, this pathogen is also notorious in acquiring and/or developing resistance to antimicrobial compounds, thus limiting available treatment options severely. Therefore, development of new compounds and strategies to fight S. aureus is of paramount importance. But since only 1 out of 5 compounds, which entered clinical trials, becomes a drug, the preclinical evaluation of promising compounds has to be reconsidered, too. The aim of this thesis was to address both sides of this problem: first, to improve preclinical testing by incorporating in vivo imaging technologies to the preclinical testing procedure in order to acquire additional and clearer data about efficacy of promising compounds and second, by evaluating lysostaphin, which is a promising, new option to fight S. aureus infections. The first aim of this thesis focused on the establishment of a dual modality in vivo imaging platform, consisting of Bioluminescence Imaging (BLI) and Magnetic Resonance Imaging (MRI), to offer detailed insights into the course and gravity of S. aureus infection in the murine thigh infection model. Since luciferase-expressing S. aureus strains were generated in former studies and enabled thus bioluminescence imaging of bacterial infection, this technology should be implemented into the compound evaluation platform in order to non-invasively track the bacterial burden over time. MRI, in contrast, was only rarely used in earlier studies to visualize and measure the course of infection or efficacy of anti-bacterial therapy. Thus, the first set of experiments was performed to identify benefits and drawbacks of visualizing S. aureus infections in the mouse model by different MR methods. Native, proton-based MR imaging showed in this regard increased T2 relaxation times in the infected thigh muscles, but it was not possible to define a clear border between infected and uninfected tissue. Iron oxide nanoparticles and perfluorocarbon emulsions, two MR contrast agents or tracer, in contrast, offered this distinction. Iron oxide particles were detected in this regard by their distortion of 1H signal in proton-based MRI, while perfluorocarbon emulsion was identified by 19F MRI. Mammals do not harbor sufficient intrinsic amounts of 19F to deliver specific signal and therefore, 19F MR imaging visualizes only the signal of administered perfluorocarbon emulsion. The in vivo accumulation of perfluorocarbon emulsion can be imaged by 19F MRI and overlayed on a simultaneously acquired 1H MR image, which shows the anatomical context in clear detail. Since this is advantageous compared to contrast agent based MR methods like iron oxide particle-based MRI, further experiments were performed with perfluorocarbon emulsions and 19F MRI. Experimental studies to elucidate the accumulation of perfluorocarbon emulsion at the site of infection showed robust 19F MR signals after administration between day 2 and at least day 8 p.i.. Perfluorocarbon emulsion accumulated in all investigated mice in the shape of a 'hollow sphere' at the rim of the abscess area and the signal remained stable as long as the infection prevailed. In order to identify the mechanism of accumulation, flow cytometry, cell sorting and histology studies were performed. Flow cytometry and cell sorting analysis of immune cells at the site of infection showed that neutrophils, monocytes, macrophages and dendritic cells carried contrast media at the site of infection with neutrophils accounting for the overwhelming portion of perfluorocarbon signal. In general, most of the signal was associated with immune cells, thus indicating specific immune cell dependent accumulation. Histology supported this observation since perfluorocarbon emulsion related fluorescence could only be visualized in close proximity to immune cell nuclei. After establishing and testing of 19F MRI with perfluorocarbon emulsions as infection imaging modality, the effects of antibiotic therapy upon MR signal was investigated in order to evaluate the capability of this modality for preclinical testing procedure. Thus, the efficacy of vancomycin and linezolid, two clinically highly relevant anti - S. aureus compounds, were tested in the murine thigh infection model. Both of them showed reduction of the colony forming units and bioluminescence signal, but also of perfluorocarbon emulsion accumulation strength and volume at the site of infection, which was visualized and quantified by 19F MRI. The efficacy pattern with linezolid being more efficient in clearing bacterial infection was shown similarly by all three methods. In consequence, 19F MRI with perfluorocarbon emulsion as MR tracer proved to be capable to visualize antibacterial therapy in preclinical testing models. The next step was consequently to evaluate a promising new compound against S. aureus infections. Thus, lysostaphin, an endo-peptidase that cleaves the cell wall of S. aureus, was tested in different concentrations alone or in combination with oxacillin for efficacy in murine thigh and catheter associated infection models. Lysostaphin only in the concentration of 5 mg/kg body weight or combined with oxacillin in the concentration of 2 mg/kg showed strong reduction of bacterial burden by colony forming unit determination and bioluminescence imaging in both models. The perfluorocarbon accumulation was investigated in the thigh infection model by 19F MRI and was strongly reduced in terms of volume and signal strength in both above-mentioned groups. In general, lysostaphin showed comparable or superior efficacy than vancomycin or oxacillin alone. Therefore, further development of lysostaphin for the treatment of S. aureus infections is recommended by these experiments. Overall, the antibiotic efficacy pattern of all applied antibiotic regimens was similar with all three applied methods, demonstrating the usefulness of MRI for antibiotic efficacy testing. Importantly, treatment with oxacillin either alone or in combination with lysostaphin resulted in stronger perfluorocarbon emulsion accumulation at the site of infection than expected compared to the results from bioluminescence imaging and colony forming unit determination. This might be an indication for immunomodulatory properties of oxacillin. Further murine infection experiments demonstrated in this context a differential release of cytokine and chemokines in the infected thigh muscle in dependence of the applied antibacterial therapy. Especially treatment with oxacillin, but to a less degree with minocycline or linezolid, too, exhibited high levels of various cytokines and chemokines, although they reduced the bacterial burden efficiently. In consequence, possible immunomodulatory effects of antibacterial compounds have to be taken into account for future applications of imaging platforms relying on the visualization of the immune response. However, this observation opens a new field for these imaging modalities since it might be extraordinary interesting to study the immunomodulatory effects of compounds or even bacterial factors in vivo. And finally, a two modality imaging platform which combines methods to visualize on the one hand the bacterial burden and on the other hand the immune response offers an innovative, new platform to study host-pathogen interaction in vivo in a non-invasive fashion. In summary, it could be shown that perfluorocarbon emulsions accumulate in immune cells at the site of infection in the murine S. aureus thigh infection model. The accumulation pattern shapes a 'hollow sphere' at the rim of the abscess area and its size and perfluorocarbon content is dependent on the severity of disease and/or efficacy of antibiotic therapy. Thus, 19F MRI with perfluorocarbon emulsions is a useful imaging modality to visualize sites and course of infection as well as to evaluate promising antibacterial drug candidates. Furthermore, since the accumulation of tracer depends on immune cells, it might be additionally interesting for studies regarding the immune response to infections, auto-immune diseases or cancer, but also to investigate the efficacy of immunomodulatory compounds and immunization.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@article{JakobHertleinSturmetal.2011,
  author    = {Jakob, Peter and Hertlein, Tobias and Sturm, Volker and Kircher, Stefan and Basse-L{\"u}sebrink, Thomas and Haddad, Daniel and Ohlsen, Knut},
  title     = {Visualization of Abscess Formation in a Murine Thigh Infection Model of Staphylococcus aureus by 19F-Magnetic Resonance Imaging (MRI)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74994},
  year      = {2011},
  abstract  = {Background: During the last years, 19F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings: In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance: We introduce 19F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Kaspar2018,
  author    = {Kaspar, Stefanie Maria},
  title     = {Untersuchungen zur Bedeutung des Toxins Panton - Valentine - Leukozidin bei ambulant erworbenen Hautinfektionen durch Staphylococcus aureus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161535},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {In der vorgelegten Promotionsarbeit wurden die typischen bakteriellen MSSA bzw. MRSA Hautinfektionen einer dermatologischen Klinik mit dem Einzugsgebiet Nordbayern auf krankheitsrelevante Faktoren von PVL untersucht. Interessanterweise fand sich bei der Pr{\"a}senz von PVL keine Korrelation mit Methicillinresistenz oder Krankheitsschwere. Weder atopische Diathese noch Rauchen oder K{\"o}rpergewicht scheinen das Auftreten des Pathogenit{\"a}tsfaktors zu beg{\"u}nstigen. Allerdings traten die PVL positiven S. aureus Hautinfektionen bevorzugt bei j{\"u}ngeren und weiblichen Patienten auf. Bei den untersuchten Hauterkrankungen zeigten S. aureus St{\"a}mme eine ausgepr{\"a}gte Vielfalt. Es konnte kein spezieller epidemiologischer Stamm identifiziert werden. Die Ergebnisse dieser Studie sind jedoch nur eingeschr{\"a}nkt auf ein großes Kollektiv projizierbar, da der Untersuchungszeitraum insgesamt nur 7 Jahre betrug und sich das Patientenkollektiv auf das Einzugsgebiet des Klinikums beschr{\"a}nkte.},
  subject      = {Staphylococcus aureus},
  language  = {de}
}
@phdthesis{Ibrahim2024,
  author    = {Ibrahim, Eslam Samir Ragab},
  title     = {Unraveling the function of the old yellow enzyme OfrA in \(Staphylococcus\) \(aureus\) stress response},
  doi       = {10.25972/OPUS-28960},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289600},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Biological systems are in dynamic interaction. Many responses reside in the core concepts of biological systems interplay (competition and cooperation). In infection situation, the competition between a bacterial system and a host is shaped by many stressors at spatial and temporal determinants. Reactive chemical species are universal stressors against all biological systems since they potentially damage the basic requirements of these systems (nucleic acids, proteins, carbohydrates, and lipids). Either produced endogenously or exogenously, reactive chemical species affect the survival of pathogens including the gram-positive Staphylococcus aureus (S. aureus). Therefore, bacteria developed strategies to overcome the toxicity of reactive species. S. aureus is a widely found opportunistic pathogen. In its niche, S. aureus is in permanent contact with surrounding microbes and host factors. Deciphering the deterministic factors in these interactions could facilitate pinpointing novel bacterial targets. Identifying the aforementioned targets is crucial to develop new strategies not only to kill the pathogenic organisms but also to enhance the normal flora to minimize the pathogenicity and virulence of potential pathogens. Moreover, targeting S. aureus stress response can be used to overcome bacterial resistance against host-derived factors. In this study, I identify a novel S. aureus stress response factor against reactive electrophilic, oxygen, and hypochlorite species to better understand its resilience as a pathogen. Although bacterial stress response is an active research field, gene function is a current bottleneck in characterizing the understudied bacterial strategies to mediate stress conditions. I aimed at understanding the function of a novel protein family integrated in many defense systems of several biological systems. In bacteria, fungi, and plants, old yellow enzymes (OYEs) are widely found. Since the first isolation of the yellow flavoprotein, OYEs are used as biocatalysts for decades to reduce activated C=C bonds in α,β-unsaturated carbonyl compounds. The promiscuity of the enzymatic catalysis is advantageous for industrial applications. However, the physiological function of OYEs, especially in bacteria, is still puzzling. Moreover, the relevance of the OYEs in infection conditions remained enigmatic.   Here, I show that there are two groups of OYEs (OYE flavin oxidoreductase, OfrA and OfrB) that are encoded in staphylococci and some firmicutes. OfrA (SAUSA300_0859) is more conserved than OfrB (SAUSA300_0322) in staphylococci and is a part of the staphylococcal core genome. A reporter system was established to report for ofrA in S. aureus background. The results showed that ofrA is induced under electrophilic, oxidative, and hypochlorite stress. OfrA protects S. aureus against quinone, methylglyoxal, hydrogen peroxide, and hypochlorite stress. Additionally, the results provide evidence that OfrA supports thiol-dependent redox homeostasis. At the host-pathogen interface, OfrA promotes S. aureus fitness in murine macrophage cell line. In whole human blood, OfrA is involved in S. aureus survival indicating a potential clinical relevance to bacteraemia. In addition, ofrA mutation affects the production of the virulence factor staphyloxanthin via the upper mevalonate pathway. In summary, decoding OfrA function and its proposed mechanism of action in S. aureus shed the light on a conserved stress response within multiple organisms.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Blaettner2016,
  author    = {Bl{\"a}ttner, Sebastian},
  title     = {The role of the non-ribosomal peptide synthetase AusAB and its product phevalin in intracellular virulence of Staphylococcus aureus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146662},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis. In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70\% of the CA infections. The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur. S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient. In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae. The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Krones2022,
  author    = {Krones, David},
  title     = {The Role of Acid Sphingomyelinase in \(Staphylococcus\) \(aureus\) Infection of Endothelial Cells},
  doi       = {10.25972/OPUS-29049},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290492},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Staphylococcus aureus is a human bacterial pathogen responsible for a variety of diseases including bacterial pneumonia and sepsis. Recent studies provided an explanation, how S. aureus and its exotoxins contribute to the degradation of endothelial junction proteins and damage lung tissue [4]. Previous findings were indicating an involvement of acid sphingomyelinase (ASM) activity in cell barrier degradation [5]. In the presented study the impact of singular virulence factors, such as staphylococcal α-toxin, on in vitro cell barrier integrity as well as their ability to elicit an activation of ASM were investigated. Experiments with bacterial supernatants performed on human endothelial cells demonstrated a rapid dissociation after treatment, whereas murine endothelial cells were rather resistant against cell barrier degradation. Furthermore, amongst all tested staphylococcal toxins it was found that only α-toxin had a significant impact on endothelial junction proteins and ASM activity. Ablation of this single toxin was sufficient to protect endothelial cells from cell barrier degradation and activation of ASM was absent. In this process it was verified, that α-toxin induces a recruitment of intracellular ASM, which is accompanied by rapid and oscillating changes in cytoplasmic Ca2+ concentration and an increased exposure of Lysosomal associated membrane protein 1 (LAMP1) on the cell surface. Recruitment of lysosomal ASM is associated, among other aspects, to plasma membrane repair and was previously described to be involved with distinct pathogens as well as other pore forming toxins (PFT). However, with these findings a novel feature for α-toxin has been revealed, indicating that the staphylococcal PFT is able to elicit a similar process to previously described plasma membrane repair mechanisms. Increased exposure and intake of surface membrane markers questioned the involvement of ASM activity in S. aureus internalization by non-professional phagocytes such as endothelial cells. By modifying ASM expression pattern as well as application of inhibitors it was possible to reduce the intracellular bacterial count. Thus, a direct connection between ASM activity and S. aureus infection mechanisms was observed, therefore this study exemplifies how S. aureus is able to exploit the host cell sphingolipid metabolism as well as benefit of it for invasion into non-professional phagocytic cells},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Wencker2022,
  author    = {Wencker, Freya Dorothea Ruth},
  title     = {The methionine biosynthesis operon in \(Staphylococcus\) \(aureus\): Role of concerted RNA decay in transcript stability and T-box riboswitch turnover},
  doi       = {10.25972/OPUS-20712},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207124},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Methionine is the first amino acid of every newly synthesised protein. In combination with its role as precursor for the vital methyl-group donor S-adenosylmethionine, methionine is essential for every living cell. The opportunistic human pathogen Staphylococcus aureus is capable of synthesising methionine de novo, when it becomes scarce in the environment. All genes required for the de novo biosynthesis are encoded by the metICFE-mdh operon, except for metX. Expression is controlled by a hierarchical network with a methionyl-tRNA-specific T-box riboswitch (MET-TBRS) as centrepiece, that is also referred to as met leader (RNA). T-box riboswitches (TBRS) are regulatory RNA elements located in the 5'-untranslated region (5'-UTR) of genes. The effector molecule of T-box riboswitches is uncharged cognate tRNA. The prevailing mechanism of action is premature termination of transcription of the nascent RNA in the absence of the effector (i.e. uncharged cognate tRNA) due to formation of a hairpin structure, the Terminator stem. In presence of the effector, a transient stabilisation of the alternative structure, the Antiterminator, enables transcription of the downstream genes ('read-through'). Albeit, after the read-through the thermodynamically more stable Terminator eventually forms. The Terminator and the Antiterminator are two mutually exclusive structures. Previous work of the research group showed that in staphylococci the MET-TBRS ensures strictly methionine-dependent control of met operon expression. Uncharged methionyl-tRNA that activates the system is only present in sufficient amounts under methionine-deprived conditions. In contrast to other bacterial TBRS, the staphylococcal MET-TBRS has some characteristic features regarding its length and predicted secondary structure whose relevance for the function are yet unkown. Aim of the present thesis was to experimentally determine the structure of the met leader RNA and to investigate the stability of the met operon-specific transcripts in the context of methionine biosynthesis control. Furthermore, the yet unknown function of the mdh gene within the met operon was to be determined. In the context of this thesis, the secondary structure of the met leader was determined employing in-line probing. The structural analysis revealed the presence of almost all highly conserved T-box riboswitch structural characteristics. Furthermore, three additional stems, absent in all T-box riboswitches analysed to date, could be identified. Particularly remarkable is the above average length of the Terminator stem which renders it a potential target of the double-strand-specific endoribonuclease III (RNase III). The RNase III-dependent cleavage of the met leader could be experimentally verified by the use of suitable mutants. Moreover, the exact cleavage site within the Terminator was determined. The unusual immediate separation of the met leader from the met operon mRNA via the RNase III cleavage within the Terminator stem induces the rapid degradation of the met leader RNA and, most likely, that of the 5'-region of the met mRNA. The met mRNA is degraded from its 5'-end by the exoribonuclease RNase J. The stability of the met mRNA was found to vary over the length of the transcript with an instable 5'-end (metI and metC) and a longer half-life towards the 3'-end (metE and mdh). The varying transcript stability is reflected by differences in the available cellular protein levels. The obtained data suggest that programmed mRNA degradation is another level of regulation in the complex network of staphylococcal de novo methionine biosynthesis control. In addition, the MET-TBRS was studied with regard to a future use as a drug target for novel antimicrobial agents. To this end, effects of a dysregulated methionine biosynthesis on bacterial growth and survival were investigated in met leader mutants that either caused permanent transcription of the met operon ('ON') or prevented operon transcription ('OFF'), irrespective of the methionine status in the cell. Methionine deprivation turned out to be a strong selection pressure, as 'OFF' mutants acquired adaptive mutations within the met leader to restore met operon expression that subsequently re-enabled growth. The second part of the thesis was dedicated to the characterisation of the Mdh protein that is encoded by the last gene of the met operon and whose function is unknown yet. At first, co-transcription and -expression with the met operon could be demonstrated. Next, the Mdh protein was overexpressed and purified and the crystal structure of Mdh was solved to high resolution by the Kisker research group (Rudolf-Virchow-Zentrum W{\"u}rzburg). Analysis of the structure revealed the amino acid residues crucial for catalytic activity, and zinc was identified as a co-factor of Mdh. Also, Mdh was shown to exist as a dimer. However, identification of the Mdh substrate was, in the context of this thesis, (still) unsuccessful. Nevertheless, interactions of Mdh with enzymes of the met operon could be demonstrated by employing the bacterial two-hybrid system. This fact and the high conservation of mdh/Mdh on nucleotide and amino acid level among numerous staphylococcal species suggests an important role of Mdh within the methionine metabolism that should be a worthwhile subject of future research.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Schiebel2013,
  author    = {Schiebel, Johannes},
  title     = {Structure-Based Drug Design on Enzymes of the Fatty Acid Biosynthesis Pathway},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69239},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {W{\"a}hrend die Wirkung der meisten gebr{\"a}uchlichen Antibiotika auf einer Beeintr{\"a}chtigung wichtiger bakterieller Prozesse beruht, wirken manche Substanzen durch die St{\"o}rung der Zellmembran-Struktur. Da Fetts{\"a}uren ein essentieller Bestandteil von Membran-Phospholipiden sind, stellt die bakterielle Fetts{\"a}urebiosynthese II (FAS-II) einen relativ wenig erforschten, aber dennoch vielversprechenden Angriffspunkt f{\"u}r die Entwicklung neuer Antibiotika dar. Das wichtige Antituberkulotikum Isoniazid blockiert die mykobakterielle Fetts{\"a}urebiosynthese und ruft dadurch morphologische {\"A}nderungen sowie letztlich die Lyse des Bakteriums hervor. Eine wichtige Erkenntnis war, dass Isoniazid den letzten Schritt des FAS-II Elongationszyklus inhibiert, der durch die Enoyl-ACP Reduktase katalysiert wird. Darauf aufbauend wurden mehrere Programme ins Leben gerufen, die sich zum Ziel gesetzt hatten, neue Molek{\"u}le zu entwickeln, welche dieses Protein verschiedener Pathogene hemmen. Die S. aureus Enoyl-ACP Reduktase (saFabI) ist von besonders großem Interesse, da drei vielversprechende Inhibitoren dieses Proteins entwickelt werden konnten, die momentan in klinischen Studien eingehend untersucht werden. Trotz dieser Erfolgsaussichten waren zum Zeitpunkt, als die vorliegenden Arbeiten aufgenommen wurden, keine Kristallstrukturen von saFabI {\"o}ffentlich verf{\"u}gbar. Daher war es eines der Hauptziele dieser Doktorarbeit, auf der Basis von kristallographischen Experimenten atomar aufgel{\"o}ste Modelle f{\"u}r dieses wichtige Protein zu erzeugen. Durch die Entwicklung einer verl{\"a}sslichen Methode zur Kristallisation von saFabI im Komplex mit NADP+ und Diphenylether-Inhibitoren konnten Kristallstrukturen von 17 verschiedenen tern{\"a}ren Komplexen gel{\"o}st werden. Weitere kristallographische Experimente ergaben zwei apo-Strukturen sowie zwei Strukturen von saFabI im Komplex mit NADPH und 2-Pyridon-Inhibitoren. Basierend auf der nun bekannten saFabI-Struktur konnten Molekulardynamik-Simulationen durchgef{\"u}hrt werden, um zus{\"a}tzliche Erkenntnisse {\"u}ber die Flexibilit{\"a}t dieses Proteins zu erhalten. Die so gewonnenen Informationen {\"u}ber die Struktur und Beweglichkeit des Enzyms dienten in Folge als ideale Grundlage daf{\"u}r, den Erkennungsprozess von Substrat und Inhibitor zu verstehen. Besonders bemerkenswert dabei ist, dass die verschiedenen saFabI Kristallstrukturen Momentaufnahmen entlang der Reaktionskoordinate der Ligandenbindung und des Hydrid-Transfers repr{\"a}sentieren. Dabei verschließt der so genannte Substratbindungsloop das aktive Zentrum des Enzyms allm{\"a}hlich. Die außergew{\"o}hnlich hohe Mobilit{\"a}t von saFabI konnte durch molekulardynamische Simulationen best{\"a}tigt werden. Dies legt nahe, dass die beobachteten {\"A}nderungen der Konformation tats{\"a}chlich an der Aufnahme und Umsetzung des Substrates beteiligt sind. Eine Kette von Wassermolek{\"u}len zwischen dem aktiven Zentrum und einer wassergef{\"u}llten Kavit{\"a}t im Inneren des Tetramers scheint f{\"u}r die Beweglichkeit des Substratbindungsloops und somit f{\"u}r die katalysierte Reaktion von entscheidender Bedeutung zu sein. Außerdem wurde die erstaunliche Beobachtung gemacht, dass der adaptive Substratbindungsprozess mit einem Dimer-Tetramer {\"U}bergang gekoppelt ist, welcher die beobachtete positive Kooperativit{\"a}t der Ligandenbindung erkl{\"a}ren kann. Alles in allem weist saFabI im Vergleich zu FabI Proteinen aus anderen Organismen mehrere außergew{\"o}hnliche Eigenschaften auf, die f{\"u}r die Synthese von verzweigten Fetts{\"a}uren n{\"o}tig sein k{\"o}nnten, welche wiederum f{\"u}r die {\"U}berlebensf{\"a}higkeit von S. aureus im Wirt von Bedeutung sind. Diese Erkenntnis k{\"o}nnte erkl{\"a}ren, warum S. aureus selbst bei Anwesenheit von exogenen Fetts{\"a}uren von FAS-II Inhibitoren abget{\"o}tet werden kann. Somit k{\"o}nnen die gewonnenen atomaren saFabI Modelle einen entscheidenden Beitrag zur Entwicklung neuer Hemmstoffe dieses validierten Angriffszieles leisten. Tats{\"a}chlich konnten die neuen Strukturen genutzt werden, um die Bindungsst{\"a}rken sowie die Verweilzeiten verschiedener saFabI Inhibitoren molekular zu erkl{\"a}ren. Die Struktur von saFabI im Komplex mit dem 2-Pyridon Inhibitor CG400549 hingegen enth{\"u}llte spezifische Wechselwirkungen in der geweiteten Bindetasche des S. aureus Enzyms, welche das geringe Aktivit{\"a}tsspektrum dieses derzeit klinisch erprobten Inhibitors erkl{\"a}ren. Diese Studien schaffen somit eine ideale Voraussetzung f{\"u}r die Entwicklung neuer wirksamer saFabI Inhibitoren, was am Beispiel des 4-Pyridons PT166 belegt werden kann. Im Rahmen der vorliegenden Dissertation konnten außerdem die Strukturen des Enzyms KasA im Komplex mit mehreren Derivaten des Naturstoffs Thiolactomycin gel{\"o}st werden.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Mietrach2020,
  author    = {Mietrach, Nicole Aline},
  title     = {Structural and functional elucidation of the Type VIIb secretion system from Staphylococcus aureus},
  doi       = {10.25972/OPUS-21482},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214824},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion. EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 {\AA} resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely. The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 {\AA}. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition.},
  subject      = {Secretion},
  language  = {en}
}