Previous reports have shown that killed cells of S. aureus potentiate the lethal effect of crude staphylococcal alpha toxin for mice. In the present study this synergistic effect has been demonstrated with highly purified preparations of alpha toxin. Time studies indicate that the active principle does not appear to be released until approximately two hours following administration of the whole cells.

Skin and nasal bacterial dysbiosis is common in children with atopic dermatitis (AD). However, there is limited data of these bacterial changes in sub-Saharan children with AD. Therefore, this study investigated the bacterial alterations in skin and nasal bacterial communities in AD compared to healthy children in rural and urban South African settings. Staphylococcus aureus was more common in children with AD (cases) than healthy children (controls). S. aureus carriage was also associated with increased disease severity. Using spa typing, we also showed that cases and controls were colonised by distinct spa types. This led us to comprehensively explore genomic differences of S. aureus in cases and controls using whole-genome sequencing. Here, we showed that S. aureus strains from cases and controls had distinct genomic features, with cases harbouring genes associated with antibiotic resistance, DNA damage repair and virulence while controls had genes associated with adhesion. Recent reports indicate the potential role of coagulase-negative Staphylococcus (CoNS) in AD pathology. This study found that CoNS and S. aureus were commonly co-carried on nonlesional skin among cases (regardless of location) and anterior nares among urban cases than the control group. The carriage of S. capitis on nonlesional skin and anterior nares was positively associated with more severe disease in both rural and urban cases. 16S rDNA amplicon sequencing analysis revealed that bacterial diversity was higher on the nonlesional skin and anterior nares of controls. Bacterial community structure differed on lesional skin, nonlesional skin and anterior nares based on AD disease status. The relative abundance of Streptococcus, Granulicatela, Veillonella and Prevotella was high in lesional skin specimens, Anoxybacillus and Cutibacterium on nonlesional skin, and Staphylococcus, Veillonella and Sphingomonas in the anterior nares among cases Overall, the findings presented in this thesis indicate that S. aureus and other CoNS, particularly S. capitis, may predominate among cases and are associated with increased disease severity. However, the increased relative abundance of genera such as Streptococcus, especially among skin samples, indicates that other bacterial genera may be contributing to disease activity on lesional skin in AD than the traditionally reported Staphylococcus.

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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. / Biologische Systeme unterliegen ständig dynamischen Interaktionen. Diese werden geprägt von Konkurrenz und Kooperation. Im Falle einer Infektion wird die Konkurrenz zwischen einem bakteriellen Organismus und dem infizierten Wirt von der Einwirkung vieler Stressoren in allen biologischen Nischen geprägt. Eine fundamentale Rolle spielen dabei reaktive chemische Verbindungen die als universale Stressoren alle biologischen Systeme mit ihren fundamentalen Makromolekülen (Nukleinsäuren, Proteine, Kohlenhydrate und Lipide) potenziell schädigen. Reaktive chemische Verbindungen, entweder endogen oder exogen gebildet, beeinträchtigen das Überleben aller Pathogene, auch das Überleben des in dieser Arbeit behandelten gram-positiven Bakteriums Staphylococcus aureus (S. aureus). Um die lebensbedrohende Toxizität der reaktiven Verbindungen zu umgehen, haben Bakterien eine Vielzahl hoch spezialisierter Überlebensstrategien entwickelt. S. aureus ist ein weit verbreiteter opportunistischer Krankheitserreger. Er unterliegt dem permanenten Kontakt mit dem umgebenden Mikrobiom und den verschiedenartigen Wirtsfaktoren. Das Wissen um die Mechanismen der bakteriellen Stressabwehr während einer Pathogen-Wirts-Beziehung könnte als Grundlage für die Identifizierung neuer antibakterieller Zielstrukturen dienen. Eine spezifische Inaktivierung solcher Strukturen könnte dann den pathogenen Organismus schädigen ohne die normale Flora zu schwächen. Ferner können Untersuchungen an der Stressantwort von S. aureus genutzt werden, um die bakterielle Resistenz gegen wirtseigene Faktoren zu schwächen. Im Mittelpung dieser Arbeit steht die Charakterisierungeines neuartigen Faktors in der Stressantwort von S. aureus, der sowohl gegen elektrophilen Stress als auch gegen reaktive Sauerstoff- und Hypochlorit-Verbindungen aktiv ist. Die Ergebnisse der Arbeiten tragen zu einem besseren Verständnis der Stressantwort von dem wichtigen pathogenen Bakterium S. aureus bei. Trotz der Tatsache, dass die Untersuchung bakterieller Stressantworten Gegenstand der aktuellenForschung ist, sind viele Prozesse und die daran beteiligten Faktoren nur unzureichend charakterisiert. Daher war die Zielsetzung dieser Thesisdie Funktion eines Vertreters einer neuen Proteinfamilie, die mglw. in vielen Abwehrsystemen gegen chemische Stressoren eine wichtige Rolle spielt, zu untersuchen. Die von Otto Warburg erstmalig als “old yellow enzymes” (OYEs) bezeichnete Proteinfamilie ist im Bakterien-, Pilz- und Pflanzenreich weit verbreitet. Nach der erstmaligen Isolation des gelben Flavoproteins, werden OYEs seit vielen Jahrzehnten als Biokatalysatoren verwendet, um aktivierte C=C-Doppelbindungen in α,β-ungesättigte Carbonylverbindungen zu reduzieren. Die Promiskuität der enzymatischen Katalyse ist für industrielle Anwendungen sehr vorteilhaft. Nichtsdestotrotz konnte die physiologische Funktion von OYEs besonders in Bakterien bislang nur ansatzweise aufgeklärt werden und die Beteiligung der OYEs unter Infektionsbedingungen ist weiterhin unbekannt. In dieser Arbeit wurden zwei Vertreterder OYEs (OYE flavin oxidoreductase OfrA und OfrB) im Genom von Staphylokokken und Firmicuten identifiziert. OfrA (SAUSA300_0859) ist in Staphylokokken stärker konserviert als OfrB (SAUSA300_0322) und ist Teil des Kerngenoms. Es wurde ein Reportersystem etabliert, um die Expression von ofrA in S. aureus-Stämmen zu untersuchen. Die Daten dieser Arbeit zeigen, dass ofrA unter elektrophilen, oxidativen und hypochloriten Stressbedingungen induziert wird. OfrA schützt S. aureus vor Stress durch Quinone, Methylglyoxal, Wasserstoffperoxid und Hypochlorit. Weiterhin liefern die Ergebnisse Evidenz, dass OfrA die Thiol-abhängige Redox-Homöostase unterstützt. Weiterhin ist OfrA an der Fitness und dem Überleben von S. aureus nach Phagozytose in murinen Makrophagen beteiligt. Das Überleben von S. aureus in humanem Vollblut war ebenfalls sehr stark von der OfrA Expression abhängig. Somit kann auf eine wichtige Rolle von OfrA während des Infektionsgeschehens z.B. bei Bakteriämie geschlossen werden. Weiterhin zeigt sich, dass Mutationen in ofrA, die Produktion des Virulenzfaktors Staphyloxanthin über den oberen Mevalonatweg beeinflussen. Insgesamt liefert die vorliegende Arbeit neue Einblicke in die Funktion und Verbeitung von OfrA, einem neuen Vertreter aus der Klasse der OYEs. Die vorliegenden Ergebnisse ermöglichen somit auch ein besseres Verständnis konservierter Strategien der Stressantwort bei Bakterien und deren Bedeutung während des Infektionsgeschehens.

O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.

O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.