Comprehensive characterization of Mycobacterium tuberculosis strains after acquisition of isoniazid resistance

Includes bibliographical references.2016 Fall.Despite the global efforts to reduce tuberculosis (TB) rates, the emergence of drug resistant TB has not allowed effective control of this disease. In the last decade, there were roughly 10 million new TB cases per year and isoniazid resistant (INHr) TB accounted for 9.5% of these cases around the world. In 2012, United States had an interruption in the supply of isoniazid (INH), which increased the likelihood of INH resistance rates. Although INH resistance in Mycobacterium tuberculosis (Mtb) is multigenic, mutations in the catalase-peroxidase (katG) gene predominate amongst INHr Mtb strains. The characterization of the Mtb proteome before and after acquiring INH resistance remains understudied. Additionally, the effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. The purpose of this work is to describe a complete biochemical and immunological characterization of the INHr acquisition in Mtb. In this way, a global exploration of the protein and mycolic acids differences in Mtb cultures, as well as differences in the immune response and bacterial virulence in the mouse model comparing clonal susceptible and INHr pairs of Mtb were evaluated. After this, common trends were analyzed and the findings were interpreted in the context of bacterial metabolism and host-interaction. For this work, two clonal clinical Mtb strains and one laboratory clonal pair of the H37Rv strain with different susceptibility profiles to INH were studied. The H37Rv INHr strain was isolated from a mouse that was exposed to INH in the lab and developed the same katG mutation that one of the clinical INHr strain has (V1A). In all cases, the first strain was susceptible to all tested drugs (mostly known as the INHs strain in this dissertation) while the second strain was resistant only to INH (named INHr throughout this work). The clinical pairs were confirmed as clonal pairs of the Beijing and T genotype respectively by spoligotyping and restriction fragment polymorphism analysis that uses the patterns given by the distribution of the insertion sequence (IS)-6110. Previous whole genome sequencing analysis of the clinical clonal pairs showed a katG mutation and the presence of some additional non-synonymous polymorphisms in the INHr strains. After the proteomic analysis, a katG PCR sequencing confirmed two mutations in katG for the T INHr pair (V1A and E3V) while the L101R mutation previously identified for the Beijing INHr was not confirmed. This mutation was highly unstable and the Beijing INHr might have reversed its phenotype after the absence of INH during in vitro growth. Therefore, the analysis with the Beijing clonal pair is only presented in chapter II. Protein comparison of secreted and cellular fractions (membrane, cytosol and cell wall) between clinical and lab clonal pairs of Mtb before and after acquisition of INH resistance revealed at least 25 commonly altered proteins looking at the same cellular fractions. These proteins were involved in ATP synthase machinery, lipid metabolism, regulatory events, virulence, detoxification and adaptation processes. Western blot analysis supported some of our findings, particularly the lower level of bacterial enzyme KatG in the INHr strains. Mycolic acid (MA) analysis in these clonal pairs did not reveal a common trend in these molecules for INHr strains but generated supporting information about an alternative fatty acid biosynthetic pathway in the clinical INHr strain. These analyses are further described in chapter III. Additionally, differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of KatG were evaluated in the C57BL/6 mouse model. The results in the mouse study support the idea of the individual effect of specific located mutations in the katG gene together with the associated changes in the bacterial proteome induce differences in the Mtb virulence and pathogenicity. In addition, the in vivo results also suggest the contribution of innate immune response via TLR-2 in the clearance of the INHr-attenuated Mtb strains. Further details of this work are described in chapter IV. This work provides a better understanding of new compensatory mechanisms in Mtb after INH resistance acquisition providing novel information that could be used to address alternative combined therapies as well as the identification of new drug targets in INHr strains. The results presented here also contribute to the generation of new hypothesis regarding RNA decay in Mtb and the need to evaluate if the observed biochemical differences are also associated with the bacterial exposure to the first line drug therapy that occurred in the patient. After the results obtained in this study, a subsequent biochemical analysis of Mtb strains obtained from patients before and after drug treatment is proposed to improve the description of the evolution of the acquired drug resistant phenomena observed in TB cases that limit the global disease control and hence its eradication (chapter V)

Virulence factors of the mycobacterium tuberculosis complex

The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. Introduction.Fil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Gioffré, Andrea Karina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Sabio y García, Julia Verónica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Escuela de Ciencias Médicas. Cátedra de Microbiología, Parasitología y Virología; ArgentinaFil: Santangelo, María de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Design, synthesis and structural characterisation of inhibitors of 1-Deoxy-D-xylulose-5-phosphate Synthase

Due to the emergence of pathogenic organisms with resistance to classical antibiotics, the developmemt of new drugs is needed. The enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is a potential target for such a new antibiotic. DXPS is the first enzyme of the methylerythritol phosphate (MEP) pathway, one of two known pathways for the biosynthesis of essential terpene building-blocks. It is found in many bacteria and plants, whereas most other organisms, especially mammals, use the mevalonate pathway. Inhibition of the MEP pathway is therefore one way to impare the growth and survival of microorganisms. The focus of this thesis is the protein structure of DXPS and the identification and development of DXPS inhibitors. In Chapter 1.2 an overview of the enzyme and the metabolic pathway is given, Chapter 1.3 updates on developments since 2017. Chapter 1.4 introduces our general workflow for protein-templated dynamic combinatorial chemistry (ptDCC). The main part describes in Chapters 2.1 and 2.2 protein crystallographic work to improve the resolution of D. radiodurans DXPS and structural elucidation of DXPS homologous from pathogenic species. In parallel, the hit-identification strategies ligandbased virtual screening (Chapter 2.3) and ptDCC (Chapter 2.4) were applied to find DXPS inhibitors. Finally, Chapter 2.5 describes the development and crystallographic validation of bioisosters for acylhydrazone-based ptDCC hits.Aufgrund der Zunahme von antibiotika-resistenten Pathogenen ist die Entwicklung neuer Antibiotika erforderlich. Das Enzym 1-Desoxy-D-xylulose-5-phosphat-Synthase (DXPS) ist ein potenzielles Ziel für eine solche Neuentwicklung. DXPS ist das erste Enzym des Methylerythritolphosphat (MEP)-Weges, einer von zwei Stoffwechselwegen für die Biosynthese der essentiellen Terpen bausteine. Er kommt in vielen Bakterien und Pflanzen vor, wohingegen die meisten anderen Organismen, insbesondere Säugetiere, den Mevalonatweg nutzen. Die Hemmung des MEP-Weges ist daher eine Möglichkeit, das Wachstum und Überleben von Mikroorganismen gezielt zu beeinträchtigen. Der Schwerpunkt dieser Arbeit liegt auf der Proteinstruktur von DXPS sowie der Identifizierung und Entwicklung von DXPS-Inhibitoren. Zunächst wird ein Überblick über das Enzym, den MEP-Weg und den aktuellen Forschungsstand seit 2017 gegeben (Kapitel 1.2 und 1.3). Das Protokoll unserer Arbeitsgruppe für protein-templierte dynamische kombinatorische Chemie (ptDCC) wird anschließend in Kapitel 1.4 vorgestellt. Der Hauptteil beschriebt in den Kapiteln 2.1 und 2.2 proteinkristallographische Arbeiten zur Verbesserung der Auflösung von D. radiodurans DXPS sowie zur Strukturaufklärung von DXPS-homologen von Pathogenen. Parallel dazu wurden die Hit-identifikations- Strategien ligandenbasiertes virtuelles Screening (Kapitel 2.3) und ptDCC (Kapitel 2.4) angewandt, um DXPS-Inhibitoren zu finden. Abschließend wird in Kapitel 2.5 die Entwicklung und kristallographische Validierung von Bioisosteren für Acylhydrazon-basierte ptDCC-Hits beschrieben.LIFT gran

Redox-Sensing-Mechanismen unter Hypochlorit-Stress in Staphylococcus aureus

Glutathione (GSH) is the major low molecular weight (LMW) thiol of eukaryotic organisms and Gram-negative bacteria to maintain the redox balance (chapters 1-2). However, Gram-positive bacteria do not produce GSH. Bacillithiol (BSH) is utilized as alternative LMW thiol in Firmicutes, such as Bacillus subtilis and Staphyloccoccus aureus. Mycothiol functions instead as major LMW thiol in all Actinomycetes, such as Mycobacteria and Corynebacteria. Under oxidative stress, LMW thiols form mixed disulfides with proteins thiols, termed as S-thiolations which function as thiol-protection and redox-control mechanism. The main goal of this work was the identification of novel thiol-switches and S-thiolated proteins in the thiol-redox proteome of the human pathogens S. aureus and Corynebacterium diphtheriae under hypochlorous acid (HOCl) stress. HOCl is a highly reactive oxidant that is produced during neutrophil infections and is the major cause of bacterial killing. Using the thiol-redox proteomics approach OxICAT, 58 NaOCl-sensitive protein thiols with >10% increased oxidations could be identified in S. aureus USA300 (chapters 3 4). Among these are five S-bacillithiolated proteins, including the two aldehyde dehydrogenases GapDH and AldA which showed the highest oxidation increase of ~29 % in the OxICAT analysis. GapDH and AldA were S-bacillithiolated at their active site Cys residues, Cys151 in GapDH and Cys279 in AldA. GapDH represents the most abundant S-bacillithiolated protein contributing with 4% to the total Cys proteome of S. aureus. The catalytic active sites of GapDH and AldA are very sensitive to overoxidation and irreversible inactivation by ROS in vitro. In the presence of BSH, S-bacillithiolation protects the active sites against irreversible oxidation and functions in reversible inactivation. Using molecular docking it was further shown that BSH can undergo disulfide formation with the GapDH and AldA active site Cys residues without major conformational changes. In C. diphtheriae, the glycolytic GapDH was identified as main target for S-mycothiolation under HOCl stress (chapter 5). In addition, GapDH is also the most abundant protein in the Cys proteome of C. diphtheriae. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro, which was faster compared to the overoxidation pathway. Reactivation of S mycothiolated GapDH was catalyzed by the Trx and the Mrx1 pathways in vitro. De-mycothiolation by Mrx1 was faster compared to Trx. Thus, it is interesting to note that the glycolytic GapDH is a major target for S-thiolation by BSH and MSH across Gram-positive bacteria. To identify novel redox-sensing regulators in S. aureus USA300 that could provide protection under HOCl stress, we used an RNA- seq transcriptomic approach. We identified the novel Rrf2-family redox-sensing regulator HypR as most strongly induced under NaOCl stress in the transcriptome under NaOCl stress (chapter 6). HypR was characterized as redox- sensing repressor that negatively controls expression of the hypR-merA operon and directly senses and responds to NaOCl and diamide stress by a thiol-based redox switch. Mutational analysis identified Cys33 and the conserved Cys99 as essential for NaOCl-sensing while Cys99 is also important for repressor activity of HypR in vitro and in vivo. The redox-sensing mechanism of HypR involves Cys33-Cys99' intersubunit disulfide formation by NaOCl stress both in vitro and in vivo. Moreover, the HypR-controlled flavin disulfide reductase MerA was shown to protect S. aureus against NaOCl stress and increased survival in J774A.1 macrophage infection assays. We were further interested to investigate the changes in the BSH redox potential under NaOCl stress in S. aureus. Thus, we constructed a genetically encoded bacilliredoxin-fused Brx- roGFP2 redox biosensor for dynamic live-imaging of BSH redox potential changes in S. aureus during the growth, oxidative stress and under antibiotics treatment (chapter 7-8). The Brx-roGFP2 biosensor showed a specific and rapid response to low levels BSSB in vitro which required the active-site Cys of Brx. However, the biosensor was unresponsive to other LMW thiol disulfides in vitro. Dynamic live-imaging in two MRSA isolates USA300 and COL revealed fast and dynamic responses of the Brx-roGFP2 biosensor under NaOCl and H2O2 stress and constitutive oxidation of the probe in different BSH-deficient mutants. Using confocal laser scanning microscopy, the changes in the BSH redox potential in S. aureus were confirmed at the single cell level. In phagocytosis assays with THP-1 macrophages, the biosensor was 87 % oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes indicating that antibiotics do not cause oxidative stress in S. aureus. Our studies demonstrate that this novel Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be used for dynamic live imaging of the BSH redox potential inside S. aureus. Future studies are directed to apply this Brx-roGFP2 biosensor for screening of the BSH redox potential across S. aureus isolates of different clonal complexes to reveal the differences in pathogen fitness and in their ROS detoxification capacities as defense mechanisms against the host immune system. In addition, this biosensor can be applied in drug research to screen for new ROS-generating antibiotics that affect the BSH redox potential in S. aureus.Glutathion (GSH) ist die wichtigste niedermolekulare Thiolverbindung in eukaryontischen Organismen und Gram-negativen Bakterien, um die Redoxbalance aufrechtzuerhalten (Kapitel 1-2). Gram-positive Bakterien produzieren kein GSH, sondern dafür alternative Thiolverbindungen. Bacillithiol (BSH) fungiert als alternative Thiolverbindung in Firmicutes, wie z.B. in Bacillus subtilis und Staphyloccoccus aureus. Mycothiol kommt dagegen als wichtigste Thiolverbindung in allen Actinomycetes vor, wie z.B. in Mycobakterien und Corynebakterien. Niedermolekulare Thiolverbindungen spielen eine wichtige Rolle bei post-translationalen Modifikationen nach oxidativem Stress, wobei Cysteine zu S-Thiolierungen oxidiert werden können. S-Thiolierungen schützen die Thiolgruppe vor irreversibler Oxidation zur Cystein-Sulfonsäure und fungieren als Redox-Schalter. Das Hauptziel dieser Arbeit war es, neue Thiolschalter und S-Thiolierungen im Thiolredoxproteom in den pathogenen Bakterien S. aureus and Corynebacterium diphtheriae nach HOCl stress zu identifizieren. HOCl ist ein sehr reaktives Oxidant und wird von Neutrophilen während der Infektion produziert. HOCl ist deshalb für die Abwehr des angeborenen Immunsystems gegen Bakterien von großer Bedeutung. Im Thiolredoxproteom von S. aureus USA300 konnten mittels der OxICAT-Methode 58 NaOCl-sensitive Cysteine identifiziert werden, die >10% erhöhte Oxidation nach NaOCl-Stress aufwiesen (Kapitel 3 4). Dazu zählten fünf S-bacillithiolierte Proteine, wie z.B. die Aldehyd-Dehydrogenasen GapDH und AldA, die ca. 29 % stärker oxidiert waren in der OxICAT-Analyse. GapDH und AldA sind in ihrem katalytischen Zentrum S-bacillithioliert, am Cys151 von GapDH und am Cys279 von AldA. GapDH ist das am häufigsten vorkommende S-bacillithiolierte Protein, welches mit 4% zum Gesamt-Cystein-Proteom in S. aureus beiträgt. Die katalytischen aktiven Zentren von GapDH und AldA sind sehr sensitiv gegenüber Überoxidationen und irreversiblen Inaktivierungen durch ROS in vitro. In Gegenwart von BSH und ROS kommt es zur S-Bacillithiolierung der aktiven Zentren von GapDH und AldA. Die S-Bacillithiolierung dient als Schutz der Thiolgruppe vor Überoxidation und führt ebenfalls zur reversiblen Inaktivierung der Enzyme. Durch molekulares Docking konnte weiterhin gezeigt werden, dass die S-Bacillithiolierung der Cysteine in den aktiven Zentren von GapDH und AldA keine Konformationsänderungen erfordert. In C. diphtheriae wurde die glykolytische GapDH als S-mycothioliert nach HOCl-Stress identifiziert (Kapitel 5). GapDH ist ebenfalls das am häufigsten vorkommende Protein im Cystein-Proteom von C. diphtheriae. Nach Exposition von gereinigtem GapDH mit H2O2 und NaOCl kam es zur Überoxidation des aktiven Zentrums zur Sulfonsäure, was zur irreversiblen Inaktivierung führte. Die Oxidation von GapDH durch H2O2 und NaOCl in Gegenwart von MSH führte zur S-mycothiolierung und reversiblen GapDH Inaktivierung in vitro. Kinetische Messungen zeigten weiterhin, dass die S-Mycothiolierung schneller ablief als die Überoxidation zur Sulfonsäure. Die Reaktivierung von S mycothiolierten GapDH konnte sowohl durch den Trx-Pathway als auch durch Mrx1 katalysiert werden in vitro. Die Reduktion der Mycothiolierungen mittels Mrx1 verlief wesentlich schneller im Vergleich zur Reduktion durch Trx. Somit wurde hiermit die glykolytische Glyceraldehyd-3-Phosphat-Dehydrogenase GapDH als ein wichtiges S-thioliertes metabolisches Enzym in verschiedenen Gram-positiven Bakterien identifiziert und charakterisiert. Wir waren weiterhin interessiert, neue HOCl-spezifische redox-sensitive Regulatoren zu identifizieren. Dazu wurde eine RNA-seq Transkriptomanalyse nach NaOCl-Stress durchgeführt. Wir konnten einen neuen Regulator der Rrf2-Familie identifizieren, der sehr stark durch HOCl-Stress im Transkriptom induziert wurde (Kapitel 6). HypR wurde als neuer redox- sensitiver Repressor charakterisiert, der die Expression des hypR-merA-Operons negativ reguliert. HypR wird direkt nach NaOCl und Diamid-Stress über eine reversible Thioloxidation reguliert. Durch Mutagenese wurde gezeigt, dass Cys33 und das konservierte Cys99 essential für das Redox-sensing nach NaOCl- Stress sind. Cys99 ist ebenfalls wichtig für die Repressor-Aktivität von HypR in vitro und in vivo. HypR wird nach NaOCl-Stress durch eine intermolekulare Disufidbrückenbildung zwischen Cys33 und Cys99' in vitro und in vivo reguliert. HypR reguliert die Flavin-Disulfid-Oxidoreduktase MerA. Es konnte gezeigt werden, dass MerA am Schutz von S. aureus gegenüber NaOCl-Stress beteiligt ist und zum Überleben in Infektionsassays mit Makrophagen beiträgt. Unsere weiteren Untersuchungen zielten darauf ab, die Veränderungen im BSH- Redoxpotential in S. aureus nach oxidativen Stress zu messen. Dafür wurde ein genetisch-kodierter Bacilliredoxin-fusionierter Brx-roGFP2-Biosensor konstruiert für die Analyse des BSH-Redoxpotentials in S. aureus während des Wachstums, nach oxidativem Stress und nach Antibiotika-Behandlung (Kapitel 7-8). Der Brx-roGFP2-Biosensor zeigte eine spezifische und schnelle Oxidation nach Inkubation mit geringen Mengen BSSB in vitro, welche auf das aktive Zentrum von Brx zurückzuführen war. Keine Oxidation des Biosensors wurde nach Inkubation mit anderen niedermolekularen Thiolverbindungen gemessen. Biosensor-Messungen in zwei MRSA-Isolaten USA300 und COL zeigten eine schnelle und dynamische Oxidation des Brx-roGFP2 Biosensors nach NaOCl und H2O2-Stress. Der Biosensor war konstitutiv oxidiert in verschiedenen BSH-negativen S. aureus Mutanten. Durch konfokale Laser-Scanning-Mikroskopie konnten die Veränderungen im BSH-Redoxpotential in S. aureus auf Einzelzell-Ebene bestätigt werden. Nach Infektionsversuchen mit THP-1 Makrophagen wurde eine 87 %-ige Oxidation des Biosensors in S. aureus COL gemessen. Jedoch wurden keinen Veränderungen des BSH-Redoxpotentials nach Behandlung mit verschiedenen Antibiotika nachgewiesen. Dies weist darauf hin, dass Antibiotika in S. aureus keinen oxidativen Stress verursachen. Unsere Untersuchungen zeigten, dass der neue Brx-roGFP2 Biosensor eine spezifische Äquilibrierung zwischen den BSH und roGFP2 Redoxpaaren katalysiert. Deshalb kann der Biosensor weiterhin in S. aureus angewandt werden für dynamische Messungen des BSH-Redoxpotentials. In zukünftigen Studien soll der Brx-roGFP2 Biosensor für das Screening des BSH- Redoxpotentials in S. aureus-Isolaten verschiedender klonaler Komplexe eingesetzt werden. Somit könnten Unterschiede in der Fitness und Entgiftung von ROS zwischen verschiedenen S. aureus-Isolaten untersucht werden als Abwehrmechanismen gegen das Immunsystem des Wirts. Der Biosensor kann ebenfalls in der Antibiotika-Forschung eingesetzt werden, um nach neuen ROS- produzierenden Antibiotika zu screenen, die einen Einfluss auf das BSH- Redoxpotential von S. aureus haben

The compatible solutes ectoine and 5-hydroxyectoine: Catabolism and regulatory mechanisms

To cope with osmotic stress many microorganisms make use of short, osmotically active, organic compounds, the so-called compatible solutes. Examples for especially effective members of this type of molecules are the tetrahydropyrimidines ectoine and 5-hydroxyectoine. Both molecules are produced by a large number of microorganisms, not only to fend-off osmotic stress, but also for example low and high temperature challenges. The biosynthetic pathway used by these organisms to synthesize ectoines has already been studied intensively and the enzymes used therein are characterized quite well, both biochemically as well as structurally. However, synthesis of ectoines is only half the story. Inevitably, ectoines are frequently released from the producer cells in different environmental settings. Especially in highly competitive habitats like the upper ocean layers some bacteria specialized on a niche like this. The model organism used in this work is such a species. It is the marine bacterium Ruegeria pomeroyi DSS-3 which belongs to the Roseobacter-clade. Roseobacter species are heterotrophic Proteobacteria which can live in symbiosis with phytoplankton as well as turning against them in a bacterial warfare fashion to scavenge valuable nutrients. Ectoines can be imported by R. pomeroyi DSS-3 in a high-affinity fashion and be used as energy as well as carbon- and nitrogen-sources. To achieve this, both ectoines rings are degraded by the hydrolase EutD and deacetylated by the deacetylase EutE. The first hydrolysis products α-ADABA (from ectoine) and hydroxy-α-ADABA (from hydroxyectoine) are deacetylated to DABA and hydroxy-DABA which are in additional biochemical reactions transformed to aspartate to fuel the cell’s central metabolism. The role and functioning of the EutDE enzymes which work in a concerted fashion are a central aspect of this work. Both enzymes could be biochemically and structurally characterized, and the architecture of the metabolic pathway could be illuminated. α-ADABA and hydroxy-α-ADABA are not only central to ectoine catabolism, but also to the regulatory mechanisms associated with it. Both molecules serve as inducers of the central regulatory protein of this pathway, the MocR-/GabR-type regulator protein EnuR. In the framework of this dissertation molecular details could be clarified which enable the EnuR repressor molecule to sense both molecules with high affinity to subsequently derepress the genes for the import and catabolism of ectoines

Structural and functional studies on the regulation of pyruvate carboxylase by the bacterial second messenger cyclic-di-AMP

The primary focus of this dissertation is the metabolic enzyme pyruvate carboxylase (PC). The structure and function of this fascinating enzyme has been studies and characterized by many laboratories over many decades. This extensive background is reviewed in Chapter 1, with an overview of the biotin-dependent carboxylase family and a particular focus on PC. In this dissertation, we primarily use X-ray crystallography to study PC at a structural level. This dissertation is divided into two overarching sections, with the first section (Chapters 2-5) focusing on the bacterial second messenger cyclic-di-AMP (c-di-AMP). This project was initiated by our collaborators in the laboratory of Josh Woodward at the University of Washington, who performed the first screen to identify c-di-AMP binding proteins in the bacterium Listeria monocytogenes. In Chapters 2 and 3, the regulation of PC by c-di-AMP in L. monocytogenes as well as the bacterium Lactococcus lactis is discussed. Crystal structures of the PC from each of these species in complex with cyclic-di-AMP reveal the binding site and give insights into the molecular mechanisms of this regulation. In Chapters 4 and 5, structural studies of other c-di-AMP binding proteins identified in the screen are discussed. The second section (Chapters 6) focuses on a second class of PC enzymes called the two-subunit PCs, which are found in a subset of Gram-negative bacteria. In Chapter 6, the first crystal structure of a two-subunit PC from the bacterium Methylobacillus flagellatus is determined. In collaboration with the Lars Dietrich laboratory at Columbia University, we investigate the physiological function of the two-subunit PC in the pathogen Pseudomonas aeruginosa. A theme which emerges from these studies is that PC is an incredibly diverse enzyme which has been adapted for the peculiar physiological needs of each organism it inhabits. Because PC is found throughout nature in every kingdom of life, further studies of its unique properties and role in each organism are sure to provide more surprising insights in the years to come

Functional analyses of thiol-switches and their impact on the mycothiol redox potential in actinomycetes

In their natural habitat or during infections, bacteria are frequently exposed to reactive oxygen species (ROS) and reactive chloride species (RCS), which cause an oxidative stress response and the induction of antioxidant defense mechanisms. ROS and HOCl can damage all macromolecules of the cell, including proteins, nucleic acids and lipids. To cope with ROS and to restore the reduced state of the cytoplasm, bacteria produce low molecular weight (LMW) thiols as important antioxidants and scavengers of ROS. Gram-negative bacteria and eukaryotes utilize glutathione as major LMW thiol. However, Gram-positive firmicutes and actinomycetes do not encode the enzymes for GSH biosynthesis and instead produce alternative LMW thiol, such as bacillithiol (BSH) and mycothiol (MSH), respectively. The various functions of the LMW thiol BSH in Bacillus subtilis and Staphylococcus aureus are summarized in chapter 1. Under oxidative stress, the LMW thiols GSH, BSH and MSH were shown to form post-translational modifications with protein thiols, termed as protein S-glutathionylations, S-bacillithiolations and S-mycothiolations, respectively. Protein S-thiolations protect protein thiols from irreversible overoxidation to Cys sulfonic acids and function in redox regulation of proteins. In S. aureus, about 57 proteins were previously found S-bacillithiolated under HOCl stress including GapDH as major target. The reduction of S-bacillithiolated proteins is catalyzed by bacilliredoxins (Brx) which are regenerated by BSH and the NADPH-dependent BSSB reductase (YpdA) in the Brx/BSH/YpdA redox pathway as described in chapter 2. Using NADPH-coupled electron transfer assays I showed that YpdA acts as BSSB reductase which depends on the redox-active Cys14. I further revealed that the Brx/BSH/YpdA pathway can catalyze de-bacillithiolation of S-bacillithiolated GapDH in vitro. Interestingly, YpdA was shown to be involved in detoxification of S-thioallylated BSH, termed as allylmercaptobacillithiol (BSSA), under allicin stress which is presented in chapter 3. BrxA catalyzed reduction of S-thioallylated GapDH to regenerate in part GapDH activity. Thus, YpdA and Brx function to restore the pool of reduced LMW thiols and protein thiols in S. aureus under allicin stress. In eukaryotes, glutaredoxins have been fused to redox-sensitive GFP2 (Grx-roGFP2) to measure dynamic changes in the GSH redox potential at high spatio-temporal resolution. In actinomycetes, related mycoredoxins have been used to construct Mrx1-roGFP2 biosensors for measurements of the MSH redox potential in Mycobacterium tuberculosis (Mtb), revealing heterogeneity of the MSH redox potential (EMSH) during macrophage infections and in antibiotics resistant Mtb isolates. An overview of redox biosensor applications in pathogenic bacteria under oxidative stress and infections is presented in chapter 4. Most of these redox biosensors are expressed ectopically on plasmids, resulting in different expression levels of roGFP2 fusions. The first main goal of this PhD thesis was to construct a stable integrated Mrx1-roGFP2 biosensor for quantification of EMSH changes in Corynebacterium glutamicum, which is described in chapter 5. The Mrx1-roGFP2 biosensor was integrated in the genomes of C. glutamicum wild type and mutants lacking redox regulators and antioxidant enzymes to measure EMSH changes during the growth and under oxidative stress. Biosensor measurements revealed that C. glutamicum wild type cells maintain a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of -296 mV. Due to its H2O2 resistant phenotype, Mrx1-roGFP2 responds weakly to 20-40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response of Mrx1-roGFP2 in mshA, mtr, sigH, oxyR, mpx, tpx and katA mutants which are compromised in redox-signaling and the antioxidant defense. While the probe was constitutively oxidized in the mshA and mtr mutants, a small oxidative shift in basal EMSH was observed in the ∆sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification system required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy documented the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stable integrated Mrx1-roGFP2 biosensor was successfully applied as novel redox tool to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutant backgrounds revealing major roles of MSH, SigH and KatA for intracellular EMSH. We were further interested to identify novel thiol-based redox regulators that sense HOCl via thiol-oxidation in actinomycetes and confer protection under oxidative stress. Previous redox proteomics studies identified the novel MarR-type regulator MSMEG_4471 (HypS) as highly oxidized under HOCl stress. As second main goal of this PhD thesis, I have characterized the function and redox-regulatory mechanism of HypS in Mycobacterium smegmatis which is described in chapter 6. RNA-seq transcriptomics and qRT-PCR analyses of the hypS mutant revealed that hypS is autoregulated and represses transcription of the co-transcribed hypO gene which encodes a multidrug efflux pump. DNA binding activity of HypS to the 8-5-8 bp inverted repeat sequence upstream of the hypSO operon was inhibited under NaOCl stress. However, the HypSC58S mutant protein was not impaired in DNA-binding under NaOCl stress in vitro, indicating an important role of Cys58 in redox sensing of NaOCl stress. HypS was shown to be inactivated by Cys58-Cys58’ intersubunit disulfide formation under HOCl stress, resulting in derepression of hypO transcription. Phenotype results revealed that the HypR regulon confers resistance towards HOCl, rifamipicin and erythromycin stress. Thus, HypS was identified as a novel redox-sensitive repressor that contributes to mycobacterial resistance towards HOCl stress and antibiotics. In summary, the results of my PhD thesis contributed to a deeper understanding of the impact of redox regulators and antioxidant enzymes towards MSH homeostasis under basal growth conditions and oxidative stress in actinomycetes. I further characterized a novel thiol-based redox regulator that confers resistance to HOCl and antibiotics and could be a future drug target to fight life-threatening tuberculosis infections

Metabolome-based studies of virulence factors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen and an important causative agent of potentially life-threatening nosocomial infections in predisposed patients. The Gram-negative bacterium produces a large and diverse repertoire of small-molecule secondary metabolites that serve as regulators and effectors of its virulence. In this study, a range of mass spectrometry-based bacterial metabolomics approaches was used to investigate these small-molecule virulence factors and their interplay with pseudomonal metabolism as well as with phenotypic traits related to virulence. The groundwork was laid by exploring the metabolite inventory of P. aeruginosa and improving the coverage of its metabolome by the application of a custom software named CluMSID, that clusters analytes based on similarities of their MS² spectra. CluMSID led to the annotation of, i.a., 27 novel members of the class of alkylquinolone quorum sensing signalling molecules, which represent crucial players in the highly complex network that regulates pseudomonal virulence. The tool was developed towards a versatile and user-friendly R package hosted on Bioconductor, whose functionalities and benefits are described in detail. The new findings on the alkylquinolone chemodiversity led to further studies with a mechanistic focus that probed the substrate specificity of the enzyme complex PqsBC. It was demonstrated that PqsBC accepts different medium-chain acyl-coenzyme A substrates for the condensation with 2-aminobenzoylacetate and thereby produces alkylquinolones with various side chain lengths, whose distribution is a function of substrate specificity and substrate availability. Moreover, it was shown that PqsBC also synthesises alkylquinolones with unsaturated side chains. The focus was further broadened from metabolite and pathway-centred questions to a more global perspective on pseudomonal virulence and metabolism, which directed attention at PrmC, an enzyme with a partially unknown function indispensable for in vivo virulence. An untargeted metabolomics experiment yielded insights into the role of PrmC and its influence on the pseudomonal endo- and exometabolome. Finally, clinical P. aeruginosa strains with different virulence phenotypes were examined by untargeted metabolomics in order to disclose metabolic variation and interconnections between virulence and metabolism. The analysis resulted in the discovery of a putative virulence biomarker and enabled the construction of a random forest classification model for certain virulence phenotypes based only on metabolomics data. In summary, this study demonstrated the potential of metabolomics for the investigation of P. aeruginosa virulence factors and thereby contributed towards the comprehension of the complex interplay of metabolism and virulence in this important pathogen.Pseudomonas aeruginosa ist ein wichtiger opportunistischer Erreger potenziell lebensbedrohlicher nosokomialer Infektionen bei prädisponierten Patienten. Das Gram-negative Bakterium produziert ein vielfältiges Repertoire an niedermolekularen Sekundärmetaboliten, die als Regulatoren und Effektoren seiner Virulenz dienen. In dieser Studie wurde eine Reihe von Massenspektrometrie-basierten Ansätzen der bakteriellen Metabolomik verwendet, um diese niedermolekularen Virulenzfaktoren und ihre Wechselwirkungen mit dem pseudomonalen Metabolismus sowie mit virulenzassoziierten phänotypischen Merkmalen zu untersuchen. Die Grundlage bilden die Untersuchung des Metaboliteninventars von P. aeruginosa und die Verbesserung der analytischen Abdeckung des Metaboloms durch die Anwendung einer selbstentwickelten Software namens CluMSID, die MS²-Spektren nach Ähnlichkeit clustert. CluMSID führte zur Annotation von u.a. 27 neuen Mitgliedern der Klasse der Alkylchinolone, die als Quorum-Sensing-Signalmoleküle entscheidende Akteure im hochkomplexen Netzwerk der Virulenzregulation darstellen. Das Tool wurde zu einem R-Paket entwickelt, das auf Bioconductor verfügbar ist und dessen Funktionalitäten und Vorteile ausführlich beschrieben werden. Die neuen Erkenntnisse über die Chemodiversität der Alkylchinolone führten zu weiteren Studien mit mechanistischem Schwerpunkt, die die Substratspezifität des Enzymkomplexes PqsBC untersuchten. Es wurde nachgewiesen, dass PqsBC verschiedene mittelkettige Acyl-Coenzym-A-Substrate für die Kondensation mit 2-Aminobenzoylacetat akzeptiert und dadurch Alkylchinolone mit verschiedenen Seitenkettenlängen produziert, deren Verteilung eine Funktion der Substratspezifität und der Substratverfügbarkeit ist. Zudem konnte gezeigt werden, dass PqsBC auch Alkylchinolone mit ungesättigten Seitenketten synthetisiert. Im Weiteren wurde der Fokus von Metaboliten- und Stoffwechselweg-zentrierten Fragen hin zu einer globaleren Perspektive der pseudomonalen Virulenz und des Metabolismus erweitert, was die Aufmerksamkeit auf PrmC lenkte, ein Enzym mit teilweise unbekannter, für die in vivo-Virulenz unverzichtbarer Funktion. Ein globales Metabolomik-Experiment lieferte Einblicke in die Rolle von PrmC und seinen Einfluss auf das pseudomonale Endo- und Exometabolom. Schließlich wurden klinische P. aeruginosa-Stämme mit unterschiedlichen Virulenzphänotypen mittels ungerichteter Metabolomik untersucht, um metabolische Variationen und Zusammenhänge zwischen Virulenz und Metabolismus aufzudecken. Die Analyse resultierte in der Entdeckung eines putativen Virulenzbiomarkers und ermöglichte die Konstruktion eines Random-Forest-Klassifikationsmodells für bestimmte Virulenzphänotypen, das nur auf Metabolomik-Daten basiert. Zusammenfassend hat diese Studie das Potenzial der Metabolomik für die Untersuchung der Virulenzfaktoren von P. aeruginosa aufgezeigt und damit zum Verständnis des komplexen Zusammenspiels von Metabolismus und Virulenz bei diesem wichtigen Pathogen beigetragen

Studies on natural products: resistance modifying agents, antibacterials and structure elucidation

This thesis describes research starting in 1999 on three areas of natural product science, namely bacterial resistance modifying agents, antibacterials and structure elucidation of natural products. Plants produce an array of structurally-complex and diverse chemical scaffolds and whilst there is an expanding volume of published literature on structure elucidation, there remains a need to understand why these compounds are produced and how they function in terms of biological activity. That can only be properly realised by a full and determined attempt at structure elucidation. This is an important concept as molecular structure describes and precedes function. The chirality and functional group chemistry of natural products defines the way in which a compound specifically binds to a receptor, protein or drug target. My independent research career started with studies on the ability of plant extracts and phytochemicals to modulate the activity of antibiotics that are substrates for bacterial multidrug efflux. These investigations are described in the first section, “Natural Product Resistance Modifying Agents”. Studies were, in the first instance, simple assays to look at potentiation and synergy of extracts and pure phytochemicals to potentiate the activity of antibiotics against resistant bacteria. This research evolved to study efflux inhibition, where we learnt much from the collaborations with Professors Piddock (Birmingham), Kaatz (Wayne State) and Bhakta (Birkbeck). Latterly, we were inspired by the highly imaginative and creative work of Dr Paul Stapleton (UCL), to study the plasmid transfer inhibitory effects of natural products; the rationale being that plasmids carry antibiotic-resistance genes and virulence factors. Inhibition of transfer could result in a reduction in the spread of antibiotic resistance and a reduction in pathogenicity. The second section of this thesis describes antibacterial natural products that were evaluated against clinically-relevant species of bacteria, in the main Gram-positive organisms such as Staphylococcus aureus and its methicillin- (MRSA) and multidrug-resistant variants and Mycobacterium tuberculosis, the causative agent of tuberculosis, which still continues to affect millions of people globally and for which antibiotic resistance is considerable. The papers described in this section detail the extraction of the plant and the bioassay-guided isolation of the active compounds, which were then subjected to structure elucidation, using in the majority of cases, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Mass Spectrometry, and Infrared and Ultraviolet-Visible Spectroscopy. Natural products from the acylphloroglucinol, terpenoid, polyacetylene, alkaloid and sulphide classes are well represented in these publications with some of these antibacterial natural products displaying minimum inhibitory concentrations (MIC) values of less than 1 mg/L against MRSA and Mycobacterium tuberculosis strains. These activity levels approach those of existing clinically used antibiotics and this highlights the value of plant natural products as a resource for antibacterial templates. Mechanistic studies have also been conducted on selected compounds, for example the natural products from Hypericum acmosepalum were found to inhibit ATP- dependent MurE ligase, a key enzyme involved in bacterial cell wall biosynthesis. Other examples included the main component of cinnamon (Cinnamomum zeylanicum), an ancient medicinal material cited in the Bible in Exodus, which has been used in antiquity as an anti-infective substance. The main compound from this medicinal material is trans-cinnamaldehyde, a simple phenylpropanoid which has been shown to inhibit Acetyl-CoA Carboxylase, a pivotal enzyme that catalyses the first committed step in fatty acid biosynthesis in all animals, plants and bacteria. In collaboration with the marine natural product chemist Professor Vassilios Roussis, we have also been able to characterise the antibacterial activities of marine plants, particularly compounds of the diterpene class that display promising levels of antibacterial activity against MRSA and S. aureus strains. Work on the antibacterial properties of Cannabis sativa showed that some of the main cannabinoids display excellent potency towards drug-resistant variants of S. aureus and support the ancient medicinal usage of Cannabis as an anti-infective and wound healing preparation. The acylphloroglucinol class of plant natural products are also noteworthy, particularly from Hypericum and Mediterranean medicinal plant species such as Myrtle (Myrtus communis), again with MIC values reaching 1 mg/L against pathogenic bacteria. We synthesised some of these acylphloroglucinols and made analogues and not surprisingly, were unable to improve the activity as nature really is the best chemist of all. The final section describes early and continuing research into the isolation and structure elucidation of natural products from plants and microbes. The rationale for this research is manifold: training for isolation to understand the medicinal use of a plant or microbe, chemotaxonomic investigations, the ecological relevance of phytochemicals in plants that are halophytic and xerophytic and in some cases just plain academic curiosity. These studies use classical phytochemical techniques to isolate and determine the structures of the species of investigation and where possible, absolute stereochemistry is undertaken. It should be noted however that isolation can be exceptionally challenging and frustrating. This can be due to the paucity of biomass, low concentrations of compounds, complexity of the resulting natural product mixtures and finally a lack of chemical stability of the products. All of these issues need to be faced before structure determination can even be attempted. A word of caution is therefore needed to the young natural product chemist embarking on their first isolation project. However, words of encouragement are also needed: the isolation of new, chemically complex and exquisitely biologically active molecules is a beautiful endeavour and exceptionally rewarding on many levels.This thesis describes research starting in 1999 on three areas of natural product science, namely bacterial resistance modifying agents, antibacterials and structure elucidation of natural products. Plants produce an array of structurally-complex and diverse chemical scaffolds and whilst there is an expanding volume of published literature on structure elucidation, there remains a need to understand why these compounds are produced and how they function in terms of biological activity. That can only be properly realised by a full and determined attempt at structure elucidation. This is an important concept as molecular structure describes and precedes function. The chirality and functional group chemistry of natural products defines the way in which a compound specifically binds to a receptor, protein or drug target. My independent research career started with studies on the ability of plant extracts and phytochemicals to modulate the activity of antibiotics that are substrates for bacterial multidrug efflux. These investigations are described in the first section, “Natural Product Resistance Modifying Agents”. Studies were, in the first instance, simple assays to look at potentiation and synergy of extracts and pure phytochemicals to potentiate the activity of antibiotics against resistant bacteria. This research evolved to study efflux inhibition, where we learnt much from the collaborations with Professors Piddock (Birmingham), Kaatz (Wayne State) and Bhakta (Birkbeck). Latterly, we were inspired by the highly imaginative and creative work of Dr Paul Stapleton (UCL), to study the plasmid transfer inhibitory effects of natural products; the rationale being that plasmids carry antibiotic-resistance genes and virulence factors. Inhibition of transfer could result in a reduction in the spread of antibiotic resistance and a reduction in pathogenicity. The second section of this thesis describes antibacterial natural products that were evaluated against clinically-relevant species of bacteria, in the main Gram-positive organisms such as Staphylococcus aureus and its methicillin- (MRSA) and multidrug-resistant variants and Mycobacterium tuberculosis, the causative agent of tuberculosis, which still continues to affect millions of people globally and for which antibiotic resistance is considerable. The papers described in this section detail the extraction of the plant and the bioassay-guided isolation of the active compounds, which were then subjected to structure elucidation, using in the majority of cases, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Mass Spectrometry, and Infrared and Ultraviolet-Visible Spectroscopy. Natural products from the acylphloroglucinol, terpenoid, polyacetylene, alkaloid and sulphide classes are well represented in these publications with some of these antibacterial natural products displaying minimum inhibitory concentrations (MIC) values of less than 1 mg/L against MRSA and Mycobacterium tuberculosis strains. These activity levels approach those of existing clinically used antibiotics and this highlights the value of plant natural products as a resource for antibacterial templates. Mechanistic studies have also been conducted on selected compounds, for example the natural products from Hypericum acmosepalum were found to inhibit ATP- dependent MurE ligase, a key enzyme involved in bacterial cell wall biosynthesis. Other examples included the main component of cinnamon (Cinnamomum zeylanicum), an ancient medicinal material cited in the Bible in Exodus, which has been used in antiquity as an anti-infective substance. The main compound from this medicinal material is trans-cinnamaldehyde, a simple phenylpropanoid which has been shown to inhibit Acetyl-CoA Carboxylase, a pivotal enzyme that catalyses the first committed step in fatty acid biosynthesis in all animals, plants and bacteria. In collaboration with the marine natural product chemist Professor Vassilios Roussis, we have also been able to characterise the antibacterial activities of marine plants, particularly compounds of the diterpene class that display promising levels of antibacterial activity against MRSA and S. aureus strains. Work on the antibacterial properties of Cannabis sativa showed that some of the main cannabinoids display excellent potency towards drug-resistant variants of S. aureus and support the ancient medicinal usage of Cannabis as an anti-infective and wound healing preparation. The acylphloroglucinol class of plant natural products are also noteworthy, particularly from Hypericum and Mediterranean medicinal plant species such as Myrtle (Myrtus communis), again with MIC values reaching 1 mg/L against pathogenic bacteria. We synthesised some of these acylphloroglucinols and made analogues and not surprisingly, were unable to improve the activity as nature really is the best chemist of all. The final section describes early and continuing research into the isolation and structure elucidation of natural products from plants and microbes. The rationale for this research is manifold: training for isolation to understand the medicinal use of a plant or microbe, chemotaxonomic investigations, the ecological relevance of phytochemicals in plants that are halophytic and xerophytic and in some cases just plain academic curiosity. These studies use classical phytochemical techniques to isolate and determine the structures of the species of investigation and where possible, absolute stereochemistry is undertaken. It should be noted however that isolation can be exceptionally challenging and frustrating. This can be due to the paucity of biomass, low concentrations of compounds, complexity of the resulting natural product mixtures and finally a lack of chemical stability of the products. All of these issues need to be faced before structure determination can even be attempted. A word of caution is therefore needed to the young natural product chemist embarking on their first isolation project. However, words of encouragement are also needed: the isolation of new, chemically complex and exquisitely biologically active molecules is a beautiful endeavour and exceptionally rewarding on many levels