Etwa ein Drittel der Weltbevölkerung ist mit Mycobacterium tuberculosis (Mtb), der Erreger der Tuberkulose (TB), infiziert. Daher ist es unbedingt notwendig vorhandenen Behandlungsstrategien weiter zu verbessern. Diese Study beschäftigt sich mit dem Mtb Protein Ag85C, einer Mycolyltransferase, als ein neues Ziel für die medikamentöse Behandlung der TB. Ag85C ist eines von drei verwandten Proteinen, Ag85A, B und C, welche zusammen an der Biogenese der Zellwand von Mtb beteiligt sind. Eine Gruppe von chemischen Molekülen mit den Namen Ag85C-1 bis -4 wurde als Inhibitoren von Ag85C getestet. Alle Verbindungen waren in der Lage das Wachstum von Mtb in Flüssigkulturen zu inhibieren, aber nur Ag85C-3 hatte ebenfalls einen Effekt auf intrazelluläre Bakterien, welches in einem Makrophagen-Infektions-System getestet wurde. Hervorzuheben ist, dass Ag85C-3 darüber hinaus auch das in vitro Überleben eines MDR Stammes inhibierte. Dies macht dieses Molekül zu einem interessanten Kandidaten für neue anti-mycobakterielle Therapieansätze. Desweiteren wurden detaillierte, funktionelle Charakterisierungen der Effekte von Ag85C-3 auf Mtb durchgeführt. Die Verbindung modifiziert die Lipide der mykolischen Säuren in der Zellwand durch die Blockierung der Ag85 Funktionen. Dieser Effekt führt dann zu einer Veränderung in der Durchlässigkeit der Außenhülle von Mtb. Mit Hilfe der microarray Analyse wurden die Regulierungen der Signalwege durch Ag85C-3 umfassend untersucht. Es konnte gezeigt werden, dass lebensnotwendige Siderophore durch das Molekül modifiziert werden, was auf mehrere Wirkungsmechanismen schließen lässt. Diese Erkenntnisse machen Ag85C, als Ziel, und Ag85C-3, als Inhibitor, zu vielversprechende Kandidaten für zukünftige Medikamentenforschung auf dem Gebiet der TB-Therapien. Diese Studie hebt zudem die zielbasierte Identifizierung von chemischen Inhibitoren als wichtigen und wertvollen Ansatz für die Medikamentenentwicklung hervor. / Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) infects about one-third of the world’s population. Therefore there is an urgent need to improve existing intervention strategies. This study evaluated the Mtb Ag85C protein, a mycolyl transferase, as a novel target for drug mediated intervention. Ag85C belongs to a family of three cognate proteins, Ag85A, B and C. They are involved in the final steps of Mtb cell envelope biogenesis. A panel of chemical molecules, Ag85C-1-4, which bind to Ag85C were utilized as inhibitors of Ag85C. All compounds inhibited growth of Mtb in vitro in liquid medium cultures but only Ag85C-3 had an effect on intracellular bacteria in macrophage infection system. Importantly, Ag85C-3 can inhibit in vitro survival of a MDR strain of Mtb making it a relevant molecule in the search for novel classes of anti-mycobacterial compounds. Furthermore a detailed functional characterization of Ag85C-3 effect on Mtb was performed. It modified the cell wall mycolic acid containing lipid amounts by blocking Ag85 function that led to changes in permeability of Mtb envelope. A comprehensive analysis of Mtb signalling pathways regulated by Ag85C-3 was investigated through microarray analysis. It showed modification of vital siderophore biosynthesis indicating multiple mechanisms of action. Thus the target, Ag85C and the inhibitor, Ag85C-3 are promising candidates for future TB drug research aimed at combating broad spectrum resistance development. This study also reinforces target based identification of chemical inhibitors as a valid and valuable approach in drug development.

Activation of otherwise unreactive substrates is a common strategy in chemistry and in nature. Adenylate-forming enzymes use adenosine monophosphate to activate the hydroxyl of their carboxylic substrate, creating a better leaving group. In a second step this reactive group is replaced in a nucleophilic elimination reaction to form esters, amides or thioesters. Recent studies have revealed that NRPS- independent siderophore (NIS) synthetases are also adenylate-forming enzymes, but are not included in the current superfamily description. NIS enzymes are involved in biosynthesis of high-affinity iron chelators which are used for iron acquisition by many pathogenic microorganisms. This is an important area of study, not only for potential therapeutic intervention, but also to illuminate new enzyme chemistries. Here the structural and biochemical studies of AcsD from Pectobacterium chrysanthemi are reported. AcsD is a NIS synthetase involved in achromobactin biosynthesis. The co-complex structures of ATP and citrate provide a mechanism for the stereospecific formation of an enzyme-bound citryl-adenylate. This intermediate reacts with L-serine to form a likely achromobactin precursor. A detailed characterization of AcsD nucleophile profile showed that it can not only catalyze ester formation, but also amide and possibly thioester formation, creating new stereospecific citric acid derivatives. The structure of a N-citryl-ethylenediamine product co-complex identifies the residues that are important for both recognition of L-serine and for catalyzing ester formation. The structural studies on the processive enzyme AlcC, which is involved in the final step of alcaligin biosynthesis of Bordetella pertussis, show that it has a similar topology to AcsD. It also shows that ATP is coordinated in a manner similar to that seen in AcsD. Biochemical studies of a substrate analogue establish that AlcC is not only capable of synthesizing substrate dimers and trimers, but also able to assemble the respective dimer and trimer macrocycles. A series of docked binding models have been developed to illustrate the likely substrate coordination and the steps along dimerization and macrocyclization formation. Structural and mechanistic comparison of NIS enzymes with other adenylate-forming enzymes highlights the diversity of the fold, active site architecture, and metal coordination that has evolved. Hence, a new classification scheme for adenylate forming enzymes is proposed.

Le fer est un élément essentiel pour les bactéries. Puisqu’elles ne peuvent le synthétiser elles-mêmes, elles utilisent un ou plusieurs systèmes d’acquisition de fer afin de se le procurer dans l’environnement, ou chez l’hôte pour leurs propres métabolismes. Différentes stratégies coexistent chez les bactéries pathogènes dues à la faible concentration de cet élément, autant chez l’hôte que dans l’environnement. Salmonella enterica sérovar Typhi (S. Typhi) est une entérobactérie Gram négative causant une maladie systémique, soit la fièvre typhoïde, qui est spécifique à l’homme. Les mécanismes de pathogènese de ce sérovar sont peu connus jusqu’à ce jour, puisque son tropisme pour l’humain empêche l’utilisation d’un modèle animal adéquat. L’objectif de cette recherche est de caractériser le système d’acquisition de fer chez S. Typhi encodé par le locus iro. Les gènes du locus, iroBCDEN ont fait l’objet de plusieurs recherches chez différents pathogènes, notamment E. coli et Salmonella Typhimurium. Bien qu’un rôle dans la virulence ait été établi pour ce locus chez ces bactéries, très peu d’informations sont disponibles quant au rôle chez S. Typhi, qui emprunte plutôt la voie systémique d’infection. Nous avons évalué le rôle de la synthèse, de l’exportation et de l’importation du sidérophore salmochéline, codé par les gènes iroBCDEN. En inactivant le locus puis par la suite les gènes de façon indépendante par échange allélique, il a été possible d’observer leurs implications in vitro lors d’infections de cellules humaines. Le rôle dans l’adhésion et l’invasion des cellules épithéliales ainsi que le rôle dans la phagocytose et la survie dans les macrophages ont donc été déterminés. De plus, le mécanisme de sécrétion par lequel la salmochéline peut traverser la membrane externe est inconnu à ce jour. La pompe à efflux TolC est responsable de la sécrétion de plusieurs molécules, y compris l’entérobactine, un sidérophore analogue à la salmochéline. Par mutagénèse, nous avons effectué un mutant de délétion tolC afin de vérifier son implication dans l’interaction avec les cellules épithéliales et les macrophages. Afin de caractériser in vitro les mutants, nous avons effectué des courbes de croissance dans différents milieux. La sensibilité au peroxyde d’hydrogène a été vérifiée par la suite, puis dû aux résultats d’infections, la mobilité de la souche ΔtolC a été évaluée. Ces différents tests nous ont permis de mieux comprendre l’implication du locus iro, de ses composantes et de la pompe à efflux TolC lors de l’interaction avec les cellules cibles d’une infection systémique causée par Salmonella Typhi. / Iron is an essential element for most bacteria. Since they are unable to synthetize it themselves, bacteria have developed systems to acquire iron from the environment or from host, for their own metabolic needs. Different strategies coexist in the same pathogen as a result of the low iron concentration available in the environment and in their host. Salmonella enterica serovar Typhi (S. Typhi) is a Gram negative entericbacteria that causes a systemic disease called typhoid fever, which is specific to humans. The pathogenic mechanisms of this serovar remain poorly characterized due to its tropism for humans and the lack of an adequate animal model. The objective of this project is to characterize the iron uptake system encoded by the iro locus of S. Typhi. The iroBDCEN genes of this locus are well known in other pathogens, especially in E. coli and Salmonella Typhimurium. Even if a role in virulence has been established for this locus in several bacteria, it has not been verified in S. Typhi, which importantly uses the systemic route of infection. We evaluated the role of synthesis, export and import of the salmochelin siderophores coded by the iroBCDEN genes. Inactivating the whole locus and each individual gene by allelic exchange, provided a means to observe their effects in vitro by infecting human cells. The role in adhesion and invasion of epithelial cells and their role in phagocytosis and survival in macrophages were determined. Moreover, the secretion mechanism by which salmochelin siderophores are able to pass through the external membrane is unknown to date. The TolC efflux pump is responsible for secretion of several molecules, including enterobactin, a siderophore related to salmochelins. By mutagenesis, we created a tolC deletion mutant to verify its implication in interactions with epithelial cells and macrophages. To characterize the mutants in vitro, we tested their ability to grow in different media. Sensitivity to hydrogen peroxide was verified for all mutants and due to the infection results for ΔtolC, we tested the motility of this mutant. These experiments allowed a better understanding of the implication of the iro locus, its components and the efflux pump TolC with human host cells that are targeted during systemic infection caused by Salmonella Typhi.

A sobrecarga de ferro é uma condição prejudicial para os pacientes, que apresentam uma diminuição significativa na qualidade de vida. Os fármacos quelantes são moléculas que têm capacidade de uso clínico para atuar como atenuadores da sobrecarga de metais. Neste trabalho apresentamos uma análise de sideróforos do tipo hidroxamato e quinona, com o objetivo de ampliar a gama de terapia de sobrecarga de ferro. Para cada composto foi realizado um ensaio competitivo com a sonda calce- ína para verificar a capacidade de ligação do ferro, e um ensaio antioxidante baseado na supressão da oxidação dependente de ferro da dihidrorrodamina (DHR) sob ascorbato. Foi observado que o hidroxamato cíclico piridoxatina apresentou capacidade de sequestrar ferro de substratos de alta afinidade, tanto em meio tamponado quanto em meio intracelular. Em ambas as situações também se mostrou um antioxidante eficiente. Entretanto, parece ser o mais tóxico do grupo dos hidroxamatos (que ainda continha o hidroxamato linear desferricoprogênio e o aromático desferriastercromo). Outros compostos naturais também foram estudados como possíveis candidatos a fármacos para sobrecarga de ferro. Complexos de ferro foram caracterizados por espectrofotometria para avaliar a estequiometria possível, considerando os sítios de ligação para cada composto. Ensaios de fluorescência revelaram que entre os quatro compostos em estudo (ácido clorogênico, lapachol, hemateína e hematoxilina), o complexo entre ferro e hemateína apresenta maior estabilidade relativa do que outros. / Iron overload is a harmful condition for patients, who have a significant decrease in life quality. Chelating drugs are molecules that have the capacity for clinical use to act as attenuators of metal overload. In this work we present an analysis of hydroxamate and quinone-type siderophores, intending to broaden the range of iron overload therapy. For each compound it was conducted a competitive assay with the fluorescent probe calcein to verify the iron binding ability, and an antioxidant assay based on suppression of the iron-dependent oxidation of dihydrorhodamine (DHR) under ascorbate. It was observed that cyclic hydroxamate pyridoxatin displayed good ability to scavenge iron from high affinity substrates both in buffer and in intracellular medium. It was also an efficient antioxidant in both setups. However, pyridoxatin seems to be the most toxic from the hydroxamate group (composed also by the linear desferricoprogen and the aromatic desferriasterchrome). Other natural compounds have also been studied as possible candidates for iron-overload drug therapy. Iron complexes were characterized by spectrophotometry to assess the possible stoichiometry considering the binding sites for each compound. Fluorescence assays revealed that among the four compounds in study (chlorogenic acid, lapachol, hematein and hematoxylin), the complex between iron and hematein has higher relative stability than others.

<p>This work examines the characterization of supramolecular assemblies and, more specifically, host-guest complexes involved in molecular recognition events. The supramolecular assemblies studied take root from metal ion delivery in biological uptake pathways, specifically the delivery of iron to microbial cells. These assemblies are studied in an effort to further understand the nature of molecular recognition events, specifically the nature and strength of interactions between a host and a guest, and possible applications of these systems. </p> <p>The development of a mass spectral method by which to characterize supramolecular assemblies involving the cation binding hosts 18-crown-6, benzo-18-crown-6, dicyclohexano-18-crown-6, and dibenzo-18-crown-6 macrocycles, and the linear ionophore lasalocid with cationic guests, including substituted protonated amines and the iron siderophore ferrioxamine B is presented. Methodology was developed using ESI-MS to successfully quantitate host-guest interactions in binary and complex mixtures. Binding constants were obtained in the range of log Ka = 3 - 5 and correspond to similar systems previously studied in the literature. The studies presented here further our understanding of the molecular recognition events that must occur between a siderophore and a receptor and provide an improved method by which to measure the strength of their interaction. </p> <p>The effects of redox hosts on host-guest complex formation with ferrioxamine B and the characterization of the host-guest complexes formed and the strength of the interactions between them were studied using cyclic voltammetry, ESI-MS, FAB-MS and ITC. A shift in redox potential towards more positive values is observed upon addition of a cationic siderophore guest to a solution of a redox-active para-Wurster's aza crown or mono-substituted Wurster's aza crown macrocycle. Mass spectral evidence indicates the formation of a host-guest complex between the cationic siderophore and the redox host. A redox switch mechanism is proposed, whereby the redox state of the host influences the binding affinity between the host and guest and, consequently, host-guest complex formation. These systems offer a unique means by which to modulate the uptake or release of ionic guests from a cavity by using externally controlled methods and can be applied to selective metal ion compartmentalization. </p> <p>Finally, the application of supramolecular assemblies as a tool in the field of drug delivery is presented. The covalent attachment of an antimalarial drug, artemisinin, by our collaborators to a siderophore produced by M. Tuberculosis, mycobactin, facilitates the subsequent delivery of the drug into the microbial cell by taking advantage of the natural biological iron uptake pathway. Here, the molecular recognition event and supramolecular assembly of interest is that occurring between the siderophore-drug assembly and the microbial receptor. Characterization of the siderophore-drug assembly using cyclic voltammetry shows that there is an interaction between the Fe-mycobactin and artemisinin when these are covalently attached in the form of a conjugate. Increased current output is observed due to an intramolecular electron transfer between the two components. Based on these in vitro data, we propose a redox mechanism by which the drug-siderophore conjugate exhibits a therapeutic effect in vivo.</p> / Dissertation

Vicibactin is a small, high-affinity iron chelator produced by Rhizobium leguminosarum ATCC 14479. Previous work has shown that vicibactin is produced and secreted from the cell to sequester ferric iron from the environment during iron-deplete conditions. This ferric iron is then transported into the cell to be converted into ferrous iron. This study uses UV-Vis spectroscopy as well as ion trap-time of flight mass spectroscopy to determine that vicibactin does form a complex with copper(II) ions, however, at a much lower affinity than for iron(III). Stability tests have shown that the copper(II)-vicibactin complex is stable over time. The results of this study show that vicibactin could be used in order to remove copper(II) ions from the soil or other media if they are present in toxic amounts. It also suggests that vicibactin’s purpose for the rhizobia could be expanded to include both copper sequestering and to reduce extracellular copper concentrations to prevent toxicity.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21050号 / 薬科博第93号 / 新制||薬科||10(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 竹本 佳司, 教授 二木 史朗 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Iron is essential to the majority of microorganisms; it is an important cofactor in many cellular processes and enzymes. However in an aerobic environment and at biological pH, iron is primarily found as insoluble oxyhydroxides and is unavailable to microorganisms. Many bacteria have the ability to produce siderophores, low molecular weight compounds that have a high affinity for Fe3+. Siderophores are part of a multi-component system that actively transports the iron-siderophore complex into the cytoplasm. Rhizobia are characterized by their ability to form symbiotic relationships with leguminous plants, where they can fix nitrogen for the host plant and the plant provides the bacteria with nutrients. Under iron-limiting conditions, Rhizobia are known to produce siderophores. Rhizobium leguminosarum IARI 917 produces one dihydroxamate-type siderophore. This siderophore has been purified and chemically characterized. Results indicate that this strain is producing schizokinen, which has not been described in a member of the Rhizobia family.

Iron is the 4th most abundant metal on the earth's crust and is required by most organisms as a cofactor for many enzymes; however, at physiological pH and aerobic conditions iron forms insoluble ferric oxyhydroxide polymers. Siderophores are low molecular weight compounds that scavenge ferric ions, bind with high affinity, and transport it into the cell via multicomponent transport systems. Rhizobia are soil dwelling organisms that form symbiotic relationships with host plants and fix atmospheric nitrogen, while the bacteria receive nutrients. R. leguminosarum IARI 917 produces a siderophore characterized as 'schizokinen'. In the present study, we have characterized the binding and transport kinetics of 'schizokinen' and have also attempted to identify the genes involved in its biosynthesis using mini Tn5 random mutagenesis. DNA sequence analysis of a non siderophore producing transconjugant revealed a gene involved in PAC/PAS signal transduction systems that respond to many extracellular cues.

Nature uses inorganic elements for biological processes based on the useful chemistry, abundance, and availability of each metal. Transition metals are critical in the biogeochemical cycling of essential elements and the bioinorganic chemistry of organisms. Hydrolysis-prone metals such as iron and titanium are abundant on Earth but are mostly insoluble in oxic aqueous environments. Nearly every organism requires iron for survival, therefore Nature evolved to stabilize iron from hydrolysis and hydrolytic precipitation through protein and small molecule mechanisms. Like iron, titanium primarily exists as insoluble mineral oxides and is second only to iron as the most abundant transition metal in the Earth’s crust. Despite the reputation as an inert and insoluble metal, titanium can be solubilized and made bioavailable through by chemical and biological weathering. Currently there is no known native role for titanium, however it is quite bioactive. As a stronger Lewis acid, titanium can compete with iron in binding to biomolecules and proteins. It is of interest to investigate the interactions between hydrolysis-prone metals and biological systems, from whole cell organisms to proteins and small molecules. The non-pathogenic bacterium Rhodococcus ruber GIN-1 was isolated for its ability to strongly adhere to titanium dioxide (TiO2) over other metal oxides, providing an opportunity to study the interactions between whole bacterial cells and metal oxides. The GIN-1 strain incorporates Ti(IV) ions into its biomass after adherence to anatase, rutile, and a mixture of the two morphologies. Six metals were quantitated in TiO2-exposed and control (unexposed) cells by inductively coupled plasma optical emission spectroscopy. The exposure to TiO2 caused a significant uptake of titanium with concomitant loss of iron, zinc, and possibly manganese. A collaborative project with the Strongin laboratory at Temple University works to develop stable, biomaterial photocatalysts for environment remediation of toxic inorganic contaminants. Ferritins are a class of proteins that mineralizes and stores iron as a non- toxic ferrihydrite nanoparticle. These proteins can be photoactivated with ultraviolet light to release iron from its core to remediate environmental contaminants. Ferritin can be sensitized with plasmonic gold nanoparticles to extend the photoactivity of the catalyst to the visible spectrum. Work in this thesis highlights the contribution to this collaboration from the Valentine laboratory, included the expression and purification of proteins in E. coli (human H-chain ferritin, human L-chain ferritin, and bacterial DNA protection from starved cells protein), mutation of proteins to improve sensitization of catalyst, and biomineralization with iron and titanium. The trafficking of hydrolysis prone metals is vital for the survival of nearly every organism. Iron transport proteins such as transferrins are studied to understand how nature utilizes a difficult essential metal across the domains of life. Most transferrins have two homologous lobes and are believed to have evolved from a gene duplication of a monolobal transferrin. The ascidian Ciona intestinalis has genes for both a bilobal and monolobal transferrin. Nicatransferrin (nicaTf), the monolobal transferrin from C. intestinalis, is a primitive protein that may provide insight on the evolution of transferrins in higher organisms. It is advantageous to use E. coli expression systems to produce recombinant proteins, however protein misfolding and aggregation can be a concern. To improve expression of nicaTf in E. coli, codon optimization and disulfide bonded protein expression were used. Finally, siderophores are small, high affinity iron-chelating molecules secreted from lower organisms that scavenge iron in iron-limiting conditions. R. ruber GIN-1 and R. ruber DSM 43338 strains both secrete siderophores in artificial seawater media. There are several siderophores identified from Rhodococcus species, however none have been reported from any R. ruber strain. A new siderophore was isolated and preliminary work has been done to purify and characterize the molecule. Understanding the siderophore- metal ion interactions may help elucidate the mechanism of how R. ruber cells obtain titanium from the metal-oxide particles. / Chemistry