@phdthesis{Lerch2018,
  author    = {Lerch, Maike Franziska},
  title     = {Characterisation of a novel non-coding RNA and its involvement in polysaccharide intercellular adhesin (PIA)-mediated biofilm formation of \(Staphylococcus\) \(epidermidis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155777},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis. The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds. To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations. In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future.},
  subject      = {Biofilm},
  language  = {en}
}
@phdthesis{Bury2018,
  author    = {Bury, Susanne},
  title     = {Molekularbiologische Untersuchungen der antagonistischen Effekte des probiotischen \(Escherichia\) \(coli\) Stamms Nissle 1917 auf Shiga-Toxin produzierende \(Escherichia\) \(coli\) St{\"a}mme},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163401},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Shiga toxin produzierende E. coli (STEC) stellen mit einer Infektionsdosis von gerade einmal 100 Bakterien ein großes Risiko f{\"u}r unsere Gesundheit dar. Betroffene Patienten k{\"o}nnen milde Krankheitssymptome wie w{\"a}ssrigen Durchfall aufweisen, welcher sich allerdings zu blutigem Durchfall oder dem h{\"a}molytisch ur{\"a}mischen Syndrom (HUS) weiterentwickeln kann. Die Ursache f{\"u}r das Krankheitsbild ist das zytotoxische Protein Shiga-Toxin (Stx), welches von STEC St{\"a}mmen produziert wird, eukaryotischen Zellen angreift und den apoptotischen Zelltod induziert. Es konnte gezeigt werden, dass infizierte Patienten in ihrem Krankheitsverlauf stark variieren, was unter anderem auf die Zusammensetzung ihrer Mikrobiota zur{\"u}ckzuf{\"u}hren sein k{\"o}nnte. Diesbez{\"u}glich k{\"o}nnen zum Beispiel einige Bakterien bereits die Darmbesiedlung von STEC St{\"a}mmen unterbinden, wohingegen andere die Toxin Produktion der pathogenen St{\"a}mme beeinflussen und wieder andere von den stx tragenden Phagen infiziert werden k{\"o}nnen und daraufhin selbst zu Toxin produzierenden St{\"a}mmen werden. Da die genetischen Informationen f{\"u}r das Toxin auf einem Prophagen im Genom der STEC St{\"a}mme kodiert ist, f{\"u}hrt eine Antibiotika Behandlung von infizierten Patienten zwar zum Tod der Bakterien, hat allerdings auch einen Wechsel vom lysogenen zum lytischen Phagen Zyklus und damit einen enormen Anstieg an freigesetztem Stx zur Folge. In den letzten Jahrzehnten kam es immer wieder zu Epidemien mit STEC St{\"a}mmen, welche auch einige Todesopfer forderten. Die Behandlung von Patienten erfolgt auf Grund von mangelnden Behandlungsm{\"o}glichkeiten meist nur symptomatisch, weswegen neue Strategien f{\"u}r die Behandlung einer STEC Infektion dringend ben{\"o}tigt werden. Der probiotische E. coli Stamm Nissle 1917 (EcN) z{\"a}hlt bereits seit mehr als 100 Jahren als Medikament f{\"u}r Behandlungen von Darmentz{\"u}ndungen. In vitro und in vivo Studien mit dem probiotischen Stamm und STEC St{\"a}mmen konnten zeigen, dass EcN die Produktion von Stx unterdr{\"u}ckt und gleichzeitig die STEC Zellzahl reduziert. Diese Ergebnisse waren der Anlass f{\"u}r diese Studie in der die Auswirkungen von EcN auf STEC St{\"a}mme genauer untersucht wurden, um eine m{\"o}gliche Behandlung von STEC Infektionen mit dem Probiotikum zu gew{\"a}hrleisten. Eines der Hauptziele dieser Studie war es, herauszufinden, ob EcN von stx-Phagen infiziert werden kann und damit selbst zu einem Toxin Produzenten wird. In diesem Falle w{\"a}re eine Behandlung mit dem E. coli Stamm ausgeschlossen, da es den Krankheitsverlauf verschlimmern k{\"o}nnte. Verschiedene experimentelle Ans{\"a}tze in denen versucht wurde den YaeT stx-Phagen Rezeptor tragenden Stamm zu infizieren schlugen fehl. Weder mittels PCR Analysen, Phagen Plaque Assays oder der Phagen Anreicherung konnte eine Lyse oder eine Prophagen Integration nachgewiesen werden. Transkriptom Analysen konnten zeigen, dass Gene eines lambdoiden Prophagen in EcN in Anwesenheit von stx-Phagen stark reguliert sind. Auch andere E. coli St{\"a}mme, welche sich ebenfalls durch eine Resistenz gegen{\"u}ber einer stx-Phagen Infektion auswiesen, wurden positiv auf lambdoide Prophagen untersucht. Einzig dem stx-Phagen sensitiven K-12 Stamm MG1655 fehlt ein kompletter lambdoider Prophage, weswegen die Vermutung nahe liegt, dass ein intakter lambdoider Prophage vor der Superinfektion mit stx-Phagen sch{\"u}tzten kann. In weiteren Experimenten wurde der Einfluss der Mikrozin-negativen EcN Mutante SK22D auf STEC St{\"a}mme untersucht. Es konnte gezeigt werden, dass SK22D nicht nur die Produktion des zytotoxischen Proteins unterdr{\"u}ckt, sondern auch mit der Produktion der stx-Phagen von allen getesteten STEC St{\"a}mmen interferiert (O157:H7, O26:H11, O145:H25, O103:H2, O111:H- und zwei O104:H4 Isolate vom STEC Ausbruch in Deutschland im Jahr 2011). Transwell Studien konnten zeigen, dass der Faktor, welcher die Transkription des Prophagen unterdr{\"u}ckt, von SK22D sekretiert wird. Die Ergebnisse lassen vermuten, dass die Pr{\"a}senz von SK22D den lysogenen Zustand des Prophagen st{\"u}tzt und somit den lytischen Zyklus unterdr{\"u}ckt. Da stx-Phagen eine große Gefahr darstellen andere E. coli St{\"a}mme zu infizieren, haben wir uns in weiteren Studien dem Einfluss von EcN auf isolierte Phagen gewidmet. Die Kultivierungsexperimente von EcN mit Phagen zeigten, dass der probiotische Stamm in der Lage war die stx-Phagen in ihrer Effizienz der Lyse des K 12 Stammes MG1655 von~ 1e7 pfus/ml auf 0 pfus/ml nach einer 44 st{\"u}ndigen Inkubation zu inaktivieren. Diese Inaktivierung konnte auf die Aktivit{\"a}t eines hitzestabilen Proteins, welches in der station{\"a}ren Wachstumsphase synthetisiert wird, zur{\"u}ckgef{\"u}hrt werden. Studien welche einen Anstieg der Biofilmmasse zur Folge hatten zeigten eine gesteigerte Effizienz in der Phagen Inaktivierung, weswegen Komponenten des Biofilms m{\"o}glicherweise die Phagen Inaktivierung herbeif{\"u}hren. Neben dem direkten Einfluss auf die Phagen wurde auch ein Schutzeffekt von SK22D gegen{\"u}ber dem stx-Phagen empf{\"a}nglichen K 12 St{\"a}mmen untersucht. Lysogene K 12 St{\"a}mme zeichneten sich durch eine enorme Stx und stx-Phagen Produktion aus. Die Pr{\"a}senz von SK22D konnte den K 12 vermittelten Anstieg der pathogenen Faktoren unterbinden. Transwell Ergebnisse und Kinetik Studien lassen vermuten, dass SK22D eher die Phagen Infektion von K-12 St{\"a}mmen unterbindet als die Lyse von lysogenen K-12 St{\"a}mmen zu st{\"o}ren. Eine m{\"o}gliche Erkl{\"a}rung f{\"u}r den Schutz der K-12 St{\"a}mme vor einer stx-Phagen Infektion k{\"o}nnte darin liegen, dass die K-12 St{\"a}mme innerhalb der SK22D Kultur wachsen und dadurch von den infekti{\"o}sen Phagen abgeschirmt werden. Zusammenfassend konnte in dieser Studie gezeigt werden, dass der probiotische Stamm EcN sowohl die Lyse von STEC St{\"a}mmen unterdr{\"u}ckt als auch die infekti{\"o}sen stx-Phagen inaktiviert und sensitive E. coli St{\"a}mme vor der Phagen Infektion sch{\"u}tzen kann. Diese Ergebnisse sollten als Grundlage f{\"u}r in vivo Studien herangezogen werden, um eine m{\"o}gliche Behandlung von STEC infizierten Patienten mit dem Probiotikum zu gew{\"a}hrleisten.},
  subject      = {EHEC},
  language  = {en}
}
@phdthesis{Das2018,
  author    = {Das, Sudip},
  title     = {Genome-wide identification of virulence-associated genes in Staphylococcus aureus using Transposon insertion-site deep sequencing},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143362},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Staphylococcus aureus asymptomatically colonises one third of the healthy human population, finding its niche in the nose and on skin. Apart from being a commensal, it is also an important opportunistic human pathogen capable of destructing tissue, invading host cells and killing them from within. This eventually contributes to severe hospital- and community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA), resistant to commonly used antibiotics are protected when residing within the host cell. This doctoral thesis is focused on the investigation of staphylococcal factors governing intracellular virulence and subsequent host cell death. To initiate an unbiased approach to conduct this study, complex S. aureus mutant pools were generated using transposon insertional mutagenesis. Genome-wide infection screens were performed using these S. aureus transposon mutant pools in vitro and in vivo, followed by analysis using Transposon insertion site deep sequencing (Tn-seq) technology. Amongst several other factors, this study identified a novel regulatory system in S. aureus that controls pathogen-induced host cytotoxicity and intra-host survival. The primary components of this system are an AraC-family transcription regulator called Repressor of surface proteins (Rsp) and a virulence associated non-coding RNA, SSR42. Mutants within rsp exhibit enhanced intra-host survival in human epithelial cells and delayed host cytotoxicity. Global gene-expression profiling by RNA-seq demonstrated that Rsp controls the expression of SSR42, several cytotoxins and other bacterial factors directed against the host immune system. Rsp enhances S. aureus toxin response when triggered by hydrogen peroxide, an antimicrobial substance employed by neutrophils to destroy pathogens. Absence of rsp reduces S. aureus-induced neutrophil damage and early lethality during mouse pneumonia, but still permits blood stream infection. Intriguingly, S. aureus lacking rsp exhibited enhanced survival in human macrophages, which hints towards a Trojan horse-like phenomenon and could facilitate dissemination within the host. Hence, Rsp emerged as a global regulator of bacterial virulence, which has an impact on disease progression with prolonged intra-cellular survival, delayed-lethality but allows disseminated manifestation of disease. Moreover, this study exemplifies the use of genome-wide approaches as useful resources for identifying bacterial factors and deduction of its pathogenesis.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{GarciaBetancur2018,
  author    = {Garcia Betancur, Juan Carlos},
  title     = {Divergence of cell-fates in multicellular aggregates of \(Staphylococcus\) \(aureus\) defines acute and chronic infection cell types},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148059},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Staphylococcus aureus is a versatile human pathogen that normally develops acute or chronic infections. The broad range of diseases caused by this bacterium facilitates the escape from the host's immune response as well as from target-specific antimicrobial therapies. Nevertheless, the underlying cellular and molecular mechanisms that enable S. aureus to cause these disparate types of infections are largely unknown. In this work, we depicted a novel genetic program involved in the development of cell-fate decision, which promotes the differentiation of the staphylococcal cells into two genetically identical but differently heritable cell lines capable of defining the course of an infection, by simultaneously progressing to (i) a biofilm-associated chronic infection or (ii) a disperse acute bacteremia. Here, S. aureus growing in architecturally complex multicellular communities harbored different cell types that followed an exclusive developmental plan, resulting in a clonal heterogeneous population. We found that these cell types are physiologically specialized and that, this specialization impacts the collective behavior within the multicellular aggregates. Whereas one cell line that we named BRcells, promotes biofilm formation that engenders chronic infections, the second cell line, which we termed DRcells is planktonic and synthetizes virulence factors, such as toxins that can drive acute bacteremia. We identified that the positive feedback loop present in Agr quorum sensing system of S. aureus acts a bimodal switch able to antagonistically control the divergence of these two physiologically distinct, heritable cell lines. Also, we found that this bimodal switch was triggered in response to environmental signals particularly extracellular Mg2+, affecting the size of the subpopulations in specific colonization environments. Specifically, Mg2+-enriched environments enhanced the binding of this cation to the staphylococcal teichoic acids, increasing the rigidity of the cell wall and triggering a genetic program involving the alternative sigma factor σB that downregulated the Agr bimodal switch, favoring the enrichment of the BRcells type. Therefore, colonization environments with different Mg2+ content favored different outcomes in the bimodal system, defining distinct ratio in the BRcells/DRcells subpopulations and the S. aureus outcome in our in vitro model of development of multicellular aggregates and, the infection outcome in an in vivo mice infection model. In this prime human pathogen cell-fate decision-making generates a conserved pattern of heritable, physiological heterogeneity that actively contributes to determine the course of an infection through the emergence and spatio-temporal dynamics of distinct and specialized cell types. In conclusion, this work demonstrates that cell differentiation in pathogenic bacteria is a fundamental phenomenon and its understanding, is central to understand nosocomial infections and to designing new anti-infective strategies},
  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{Jarick2020,
  author    = {Jarick, Marcel},
  title     = {Molekulare und funktionelle Charakterisierung der Serin/Threonin-Proteinkinase Stk und -Proteinphosphatase Stp von \(Staphylococcus\) \(aureus\)},
  doi       = {10.25972/OPUS-17654},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176542},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Staphylococcus aureus ist ein Kommensale, der die menschliche Haut und Schleimhaut der Nase und des Rachens besiedelt. Der Keim verursacht aufgrund zahlreicher Virulenzfaktoren leichte aber auch schwere Infektionen wie Pneumonie, Endokarditis oder Sepsis. Die Behandlung von S. aureus-Infektionen gestaltet sich heutzutage schwierig, da der Keim Resistenzen gegen verschiedenste Antibiotika ausgebildet hat. Zur Bek{\"a}mpfung dieser Resistenzen werden neue Antibiotika ben{\"o}tigt, die u.a. mit der Zellphysiologie und der Zellwandwandsynthese der Bakterien interferieren. Die Zellphysiologie und Zellwandsynthese wird abh{\"a}ngig von der Wachstumsphase und Umwelt-einfl{\"u}ssen in den Bakterien streng reguliert. Neben den Zweikomponentensystemen sind Serin/Threonin-Proteinkinasen und -Phosphatasen wesentliche Sensoren und Regulatoren der Bakterien. Durch Phosphorylierung und Dephosphorylierung bewirken diese beiden Systeme eine Hemmung oder Aktivierung der entsprechenden Zielproteine. Dadurch kann sich die Bakterienzelle an innere und {\"a}ußere Reize anpassen. In dieser Arbeit wurde die konservierte Serin/Threonin-Proteinkinase Stk und die Serin/Threonin-Phosphatase Stp von S. aureus untersucht. Die beiden Proteine Stk und Stp haben einen großen Einfluss auf die Signalweiterleitung, den zentralen Metabolismus, die Stressantwort, die Antibiotikaresistenz und die Virulenz von S. aureus. Im ersten Teil dieser Arbeit wird dargelegt, dass Stk und Stp in der bakteriellen Membran lokalisiert sind, dort miteinander interagieren und antagonistisch Zielproteine phosphorylieren bzw. dephospho-rylieren. Die Deletion der Phosphatase Stp bewirkt, dass zahlreiche Proteine in der Zelle permanent phosphoryliert und daher vermutlich nur noch eingeschr{\"a}nkt funktionst{\"u}chtig sind. Die ausbleibende Dephosphorylierung der Proteine in der stp-Mutante hat einen dramatischen Effekt auf die Zellwand-synthese und die Virulenz von S. aureus. So hat die stp-Mutante eine verdickte Zellwand und ist weniger virulent als die stk-Mutante und der Wildtypstamm. Im Rahmen dieser Arbeit wird erstmals eine Erkl{\"a}rung pr{\"a}sentiert, die die strukturellen Besonderheiten von Stk und deren Auswirkung auf die Zellwandsynthese zusammenf{\"u}hrt: In der stp-Mutante akkumulieren Zellwandvorl{\"a}ufer in der Zelle, da vermutlich die entsprechenden Zellwandsyntheseproteine durch Stk-vermittelte Phosphorylierung gehemmt werden. Die Proteine FemXAB nehmen eine zentrale Rolle in der Zellwandsynthese ein, indem sie die Pentaglycin-Interpeptidbr{\"u}cke des Zellwandvorl{\"a}ufers Pentaglycin-Lipid II syntheti-sieren. Stk wird durch die Bindung seiner extrazellul{\"a}ren Dom{\"a}nen an Pentaglycin-Lipid II aktiviert. In der vorliegenden Arbeit konnte FemX als in vitro Substrat von Stk und Stp identifiziert werden. Die permanente Phosphorylierung von FemX in der stp-Mutante f{\"u}hrt zur verminderten Synthese der Pentaglycin-Br{\"u}cken am Lipid II und infolgedessen zum Einbau von unvollst{\"a}ndigen Muropeptiden in den neuen Peptidoglycanstrang. Diese strukturelle Ver{\"a}nderung f{\"u}hrt zur Verdickung der Zellwand und folglich zur verminderten Empfindlichkeit gegen{\"u}ber der Glycyl-Glycinpeptidase Lysostaphin. Neben FemX interagiert Stk mit weiteren Zellwandsyntheseproteinen wie FemAB und einigen Zellteilungsproteinen. Diese Ergebnisse verdeutlichen, dass Stk das Vorkommen seines extrazellul{\"a}ren Liganden Lipid II detektiert und dementsprechend die Zellwandsynthese {\"u}ber FemX reguliert. Im zweiten Teil der Arbeit wurde anhand verschiedener Omics-Techniken die stk-, stp- und stk/stp-Mutante im Vergleich zum S. aureus NewmanHG Wildtyp charakterisiert. Dabei zeigten sich teilweise große Unterschiede zwischen der stp-Mutante und den anderen St{\"a}mmen. Mit diesen Unter-suchungen konnten Ergebnisse aus anderen Studien best{\"a}tigt und mit weiteren Daten untermauert werden. So l{\"a}sst sich die verminderte Virulenz der stp-Mutante mit der reduzierten Expression und Sekretion von Toxinen wie H{\"a}molysinen und Leukozidinen erkl{\"a}ren. Dies f{\"u}hrt zu einer verminderten H{\"a}molyse von Erythrozyten und einer verminderten Immunantwort gegen diese Toxine im Infektions-versuch. Stk und Stp phosphorylieren bzw. dephosphorylieren Transkriptionsfaktoren und Antwort-regulatoren von Zweikomponentensystemen, was zu der ver{\"a}nderten Expression und Sekretion der Virulenzfaktoren f{\"u}hrt. Die Analyse der Mutanten offenbart, dass Stk ein negativer und Stp ein positiver Regulator der Virulenz in S. aureus ist. Außerdem regulieren Stk und Stp zentrale Aspekte des Metabolismus in S. aureus. So ist die Konzentration an Nukleotidtriphosphaten in der stp-Mutante reduziert, was auf eine verminderte Expression der Gene der Pyrimidinsynthese zur{\"u}ckzuf{\"u}hren ist. Anhand dieser Ergebnisse wird deutlich, dass Stk und Stp wesentliche Aspekte der Zellphysiologie wie die Zellwandsynthese, den zentralen Metabolismus und die Virulenz von S. aureus regulieren.},
  subject      = {Kinase},
  language  = {de}
}
@phdthesis{HagmanngebKischkies2016,
  author    = {Hagmann [geb. Kischkies], Laura Violetta},
  title     = {Stringent response regulation and its impact on ex vivo survival in the commensal pathogen \(Neisseria\) \(meningitidis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144352},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments.},
  subject      = {Neisseria meningitidis},
  language  = {en}
}
@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}
}
@phdthesis{Oesterreich2017,
  author    = {Oesterreich, Babett},
  title     = {Preclinical development of an immunotherapy against antibiotic-resistant Staphylococcus aureus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123237},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The Gram-positive bacterium Staphylococcus aureus is the leading cause of nosocomial infections. In particular, diseases caused by methicillin-resistant S. aureus (MRSA) are associated with higher morbidity, mortality and medical costs due to showing resistance to several classes of established antibiotics and their ability to develop resistance mechanisms against new antibiotics rapidly. Therefore, strategies based on immunotherapy approaches have the potential to close the gap for an efficient treatment of MRSA. In this thesis, a humanized antibody specific for the immunodominant staphylococcal antigen A (IsaA) was generated and thoroughly characterized as potential candidate for an antibody based therapy. A murine monoclonal antibody was selected for humanization based on its binding characteristics and the ability of efficient staphylococcal killing in mouse infection models. The murine antibody was humanized by CDR grafting and mouse and humanized scFv as well as scFv-Fc fragments were constructed for comparative binding studies to analyse the successful humanization. After these studies, the full antibody with the complete Fc region was constructed as isotype IgG1, IgG2 and IgG4, respectively to assess effector functions, including antibody-dependent killing of S. aureus. The biological activity of the humanized antibody designated hUK-66 was analysed in vitro with purified human PMNs and whole blood samples taken from healthy donors and patients at high risk of S. aureus infections, such as those with diabetes, end-stage renal disease, or artery occlusive disease (AOD). Results of the in vitro studies show, that hUK-66 was effective in antibody-dependent killing of S. aureus in blood from both healthy controls and patients vulnerable to S. aureus infections. Moreover, the biological activity of hUK-66 and hUK-66 combined with a humanized anti-alpha-toxin antibody (hUK-tox) was investigated in vivo using a mouse pneumonia model. The in vivo results revealed the therapeutic efficacy of hUK-66 and the antibody combination of hUK-66 and hUK-tox to prevent staphylococcal induced pneumonia in a prophylactic set up. Based on the experimental data, hUK-66 represents a promising candidate for an antibody-based therapy against antibiotic resistant MRSA.},
  language  = {en}
}
@phdthesis{Selle2018,
  author    = {Selle, Martina},
  title     = {Interaktionen zwischen sekretierten Proteinen von Staphylococcus aureus und der Immunantwort des Wirtes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128031},
  school      = {Universit{\"a}t W{\"u}rzburg},
  pages     = {XVII, 216},
  year      = {2018},
  abstract  = {Staphylococcus aureus ist ein grampositives Bakterium, welches h{\"a}ufig als kommensaler Besiedler auf der Nasen- und Rachenschleimhaut von S{\"a}ugetieren vorkommt. Dar{\"u}ber hinaus besitzt dieser fakultativ pathogene Mikroorganismus die F{\"a}higkeit schwer zu behandelnde Krankenhausinfektionen auszul{\"o}sen. Aufgrund der weiten Verbreitung von Antibiotikaresistenzen und dem Mangel an effektiven Therapien, verursachen S. aureus Infektionen j{\"a}hrlich enorme Kosten f{\"u}r das Gesundheitssystem. S. aureus wird meist von der Nase zum prim{\"a}ren Infektionsort {\"u}bertragen, wodurch zun{\"a}chst sehr h{\"a}ufig Wund- und Weichteilinfektionen hervor gerufen werden. Von diesem prim{\"a}ren Infektionsort ausgehend, kann der Erreger tiefer liegende Gewebsschichten infizieren oder sich {\"u}ber den Blutstrom im gesamten Organismus ausbreiten. Das Spektrum an Krankheitsbildern reicht von leichten Abszessen der Haut bis zu schweren, lebensbedrohlichen Erkrankungen wie Pneumonien und akuter Sepsis. F{\"u}r die erfolgreiche Kolonisierung und Infektion des Wirtes exprimiert S. aureus eine Vielzahl unterschiedlicher Virulenzfaktoren. Die wohl gr{\"o}ßte Gruppe an Virulenzfaktoren umfasst die Proteine, die an der Immunevasion und der Umgehung von verschiedenen Abwehrstrategien des Immunsystems beteiligt sind. Das bisherige Wissen {\"u}ber die Interaktion von S. aureus mit dem Immunsystem des Wirtes und die zugrunde liegenden Pathogenit{\"a}tsmechanismen ist bisher limitiert. Um neue Erkenntnisse {\"u}ber die Interaktion von Wirt und Pathogen zu erlangen, wurden im Rahmen dieser Arbeit bislang unbekannte sekretierte und Oberfl{\"a}chen-assoziierte Proteine von S. aureus funktionell charakterisiert. Die Funktion der ausgew{\"a}hlten Proteine wurde in vitro hinsichtlich Einfluss auf Komponenten des Immunsystems, Adh{\"a}sion an Wirtsfaktoren und Invasion in eukaryotische Zellen untersucht. Mit Hilfe der vorangegangenen in-vitro-Charakterisierung der putativen Virulenzfaktoren, konnte f{\"u}r die cytoplasmatische Adenylosuccinat-Synthase PurA eine neuartige Funktion identifiziert werden. PurA ist bekannt als essentielles Enzym der de novo Purin-Synthese. In dieser Arbeit wurde nun gezeigt, dass PurA zudem an der Immunevasion beteiligt ist. Durch die Bindung des humanen Faktor H des Komplementsystems sch{\"u}tzt PurA S. aureus vor der lytischen Aktivit{\"a}t des Komplementsystems und verhindert die Opsonisierung des Pathogens. Basierend auf diesen Ergebnissen wurde PurA detailliert charakterisiert. In Bindungsstudien mit rekombinantem Faktor H und PurA wurde eine direkte Interaktion beider Proteine nachgewiesen, wobei Faktor H mit dem N-terminalen Bereich von PurA interagiert. Weiterhin konnte PurA durch Immunfluoreszenz und FACS-Analysen auf der Zelloberfl{\"a}che nachgewiesen werden, wo es wahrscheinlich mit der Zellwand assoziiert vorliegt. Dort rekrutiert es Faktor H an die bakterielle Oberfl{\"a}che und verhindert das Fortschreiten der Komplement-Kaskade und damit die Lyse des Pathogens. Aufgrund der Multifunktionalit{\"a}t z{\"a}hlt PurA somit zur Gruppe der Moonlighting Proteine. Des Weiteren wurde die Rolle von PurA im Infektionsgeschehen in zwei unabh{\"a}ngigen Tiermodellen untersucht. In beiden Modellen wurde ein signifikant reduziertes Virulenzpotential der ΔpurA-Mutante beobachtet. Zuk{\"u}nftig soll gekl{\"a}rt werden, ob die verminderte Virulenz in der fehlenden Komplementevasion oder im Defekt in der Purin-Synthese begr{\"u}ndet ist. Aufgrund der sehr starken Attenuation in allen untersuchten Infektionsmodellen sollte PurA als potentielles Target f{\"u}r eine Therapie von S. aureus Infektionen weiter charakterisiert werden. Im Ergebnis dieser Arbeit wurde demnach mit PurA ein neues Moonlighting Protein identifiziert, das als Inhibitor des Komplementsystems wesentlich zur Immunevasion von S. aureus beitr{\"a}gt. F{\"u}r das bessere Verst{\"a}ndnis der humoralen S. aureus-spezifischen Immunantwort, Unterschieden in der Antik{\"o}rperantwort und der gebildeten Antik{\"o}rperspezifit{\"a}ten wurde weiterhin das w{\"a}hrend der Kolonisierung und Infektion gebildete S. aureus-spezifische Antik{\"o}rperprofil untersucht. Dazu wurden Plasmen von humanen nasalen Tr{\"a}gern und Nicht-Tr{\"a}gern sowie murine Seren von infizierten Tieren untersucht. Insbesondere wurde das Pathogen-spezifische Antik{\"o}rperprofil in unterschiedlichen Infektionsmodellen mit Hilfe eines Proteinarrays analysiert, der im Rahmen dieser Arbeit in einer Kooperation mit der Firma Alere Technologies (Jena, Deutschland) und universit{\"a}ren Forschergruppen der Universit{\"a}ten Greifswald, M{\"u}nster und Jena mitentwickelt wurde. Die Antik{\"o}rperprofile von intramuskul{\"a}r und intraven{\"o}s infizierten Tieren resultierten in jeweils spezifischen Antik{\"o}rperprofilen. Diese Ergebnisse deuten auf einen Zusammenhang zwischen der Art der Infektion und der gebildeten Antik{\"o}rperspezifit{\"a}ten hin. Wahrscheinlich beruht dies auf einer gewebespezifischen Genexpression als Anpassung an die individuellen Bed{\"u}rfnisse im Wirtsorganismus. Das ausgebildete Antik{\"o}rperprofil gibt somit einen Einblick in das Expressionsmuster von Virulenzfaktoren von S. aureus unter in vivo Bedingungen und tr{\"a}gt damit zum Verst{\"a}ndnis der komplexen Interaktion von Pathogen und Wirt bei. Diese Untersuchungen erg{\"a}nzen zudem die bisherigen Kenntnisse {\"u}ber die Anpassung der humoralen Immunantwort an eine asymptomatische Kolonisierung im Gegensatz zu einer akuten Infektion durch S. aureus. Dar{\"u}ber hinaus k{\"o}nnen die gewonnenen Ergebnisse f{\"u}r diagnostische Zwecke und zur Identifikation von neuen Zielstrukturen f{\"u}r eine Vakzin-Entwicklung genutzt werden.},
  subject      = {Staphylococcus aureus},
  language  = {de}
}