<p>The research presented here aims to expand our understanding of the structural factors that contribute to selectivity for iron and to iron complex stability in siderophores, as well as iron transport processes in siderophore systems. This work will also investigate the factors that contribute to therapeutic applications of chelating agents, both for chelation therapy and for antimicrobial agents.</p> <p>The thermodynamics of iron(III) binding of a number of molecules, both natural and synthetic, are determined using pH-dependent spectrophotometric titrations and potentiometric titrations . Three of the synthetic siderophore analogs studied here are a tris-hydroxypyridinone and two bis-hydroxypyridinone ligands. A determination of the solution thermodynamics of the iron(III) complex of a water-soluble analog of Brasilibactin A, a membrane-bound mycobactin-type bacterial siderophore is also presented and related to the role of mycobactins in iron uptake of mycobacteria. The thermodynamics of chelation of iron(III) by a synthetic Trojan Horse antimicrobial agent featuring a 3-hydroxy-4-pyridinone moiety were also determined. In these studies, the thermodynamic stability constants of the iron-chelator complexes are determined through a series of spectrophotometric and potentiometric titrations. Also, the redox chemistry of the iron-chelator complexes are investigated using cyclic voltammetry. The structural features that contribute to complex stability in a series of tripodal tris-hydroxamate siderophores using computational techniques is presented, and it is shown that the position of the arm of an exocyclic siderophore system can contribute to differences in complex stability, as can the orientation of the donor group.</p> <p>Kinetics studies of the iron(III) exchange reactions of some polydentate chelators are presented. The study of the kinetics of some reactions of iron complexes featuring hydroxypyridinone donor-group chelators is performed by spectrophotometric kinetics experiments. A determination of the mechanism of proton-driven complex dissociation of a bishydroxypyridinone siderophore mimic is shown. Also, the mechanism of exchange between desferrioxamine B and an iron(III)-trishydroxypyridinone complex is determined through spectrophotometric monitoring of the reaction. The ability of a bidentate hydroxypyridinone chelator to catalyze the exchange of iron(III) from desferrioxamine B to EDTA is explored and the mechanism is determined.</p> <p>Finally, an investigation into the efficacy of chelation therapy treatments to protect from metal toxicity using the nematode C. elegans as a model organism is presented. The model developed therein can also be used as a model for soil remediation of toxic metals using chelating agents.</p> / Dissertation

Thesis (Ph. D.)--University of Canterbury, 2007. / Typescript (photocopy). Includes bibliographical references. Also available via the World Wide Web.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "December 1998." Typescript. Vita. Includes bibliographical references (leaves 157-166). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 113-117).

Despite recent advances, radiopharmaceuticals to detect and characterize bacterial infections have a number of limitations. Many of the clinically approved radiopharmaceutical agents are not specific to bacterial infections and accumulate at lesions of inflammation. Hence, new approaches are necessary to detect bacteria with high specificity and selectivity. A library of desferrioxamine B (DFO) derivatives were prepared to create radiolabeled siderophores to create a bacteria-specific imaging probe by exploiting the mechanism bacteria use to scavenge iron, which plays a key role in bacterial growth and biofilm formation Compounds were synthesized using two convenient carbamate forming strategies in 30% to 92% yield. The cold and radioactive gallium (67Ga) complexes were prepared and characterized and their uptake by S. aureus bacteria were assessed in vitro and in vivo. In vivo studies revealed that 67GaDFOethoxycarbamate had uptake comparable to GaDFO that was blockable, showing the compound was actively taken up via the siderophore pathway. In vivo studies in a mouse model resulted in a good infected to non-infected thigh ratio (11:1) and non-specific uptake by the GI tract. Bioorthogonal chemistry was also explored as an approach for imaging infection using trans-cyclooctene (TCO) functionalized vancomycin and a tetrazine functionalized 67GaDFO (67GaDFO-Tz) complex.2,3 In vitro results revealed that allowing vancomycin-TCO to bind S. aureus prior to the addition of 67GaDFO-Tz (pretargeting) showed higher (63%) uptake than with a conjugate formed prior to incubation with the bacteria (direct targeting, 28%). For the bioorthogonal approach, the distribution of the 67GaDFO-Tz was assessed in a S. aureus infection murine model, which showed significant uptake of 67GaDFO-Tz in the GI tract 1 h post intravenous injection. However, uptake in the infected joint was evident at 71 h post infection. The data suggests targeting bacteria using TCO-labeled antibiotics and radiolabeled tetrazines is a feasible strategy, but that further optimization of the vancomycin injection dose and injection time are necessary. / Thesis / Master of Science (MSc)

No description available.

Brucella causes undulant fever in humans and uterine and systemic infection leading to abortions in domestic animals and wild life. For the acquisition of iron in mammalian hosts, species of Brucella are known to produce two siderophores, 2, 3-dihydroxy benzoic acid (2, 3-DHBA) and brucebactin. Inability to synthesize of 2, 3-DHBA affects the ability of pathogen to metabolize erythritol, replicate in trophoblast cells and cause abortion in pregnant ruminant host. The entF gene has been implicated in the unresolved pathway allowing brucebactin biosynthesis in Brucella. The research effort presented in this thesis tries to relate the role of entF in iron acquisition and potential relation with erythritol metabolism by wild type B. abortus 2308. An entF deletion mutant (BAN1) of B. abortus 2308, generated using cre-lox methodology was found to be growth inhibited in iron minimal media compared to wild type strain. Growth inhibition was further enhanced with the addition of an iron chelator or 0.1% erythritol. Compared to wild type strain, no growth inhibition of BAN1 mutant was found in murine J774A.1 macrophages, which suggests that Brucella could acquire iron inside mammalian cells. The entF gene complemented mutant strains of BAN1 (BAN2A and BAN2B) were found to be intermediate in their ability to grow in iron minimal media supplemented with 0.0.05% erythritol, when compared to wild type and BAN1 strain. The results from the present thesis demonstrate that entF gene plays an important role in iron acquisition and erythritol metabolism by B. abortus 2308 under iron limiting conditions. / Master of Science

Le développement de l’industrie et de l’agriculture a entraîné l’accumulation d’éléments métalliques dans les sols. Dans ces écosystèmes, la fraction vivante intervient de façon importante dans la modification de la rétention des contaminants. Parmi les techniques de décontamination, la phytoremédiation apparaît comme un outil de choix. Si un des inconvénients majeurs de cette technique réside dans des durées de traitement pouvant atteindre plusieurs années, l’ajout de micro-organismes couplé aux plantes améliorerait les prélèvements et réduirait les temps de décontamination. Cette étude s’est intéressée aux possibilités de développer des procédés de phytoremédiation associés à des bactéries productrices de sidérophores, molécules ayant une forte affinité pour le fer mais également pour d’autres métaux. Dans ce contexte, un système de complexité croissante, jusqu’au modèle complet comprenant les constituants du sol, les bactéries, les métaux contaminants et les plantes, a été étudié avec pour objectif la compréhension des processus intervenant dans ces différentes interactions. Ces travaux ont conduit, dans un premier temps, à la compréhension, notamment par une approche de biologie moléculaire, des interactions intervenant entre des minéraux du sol, des argiles et des oxy-hydroxydes de fer, et une bactérie productrice de sidérophores, Pseudomonas aeruginosa. Les résultats ont mis en évidence le rôle majeur de l’un de ces sidérophores, la pyoverdine, ainsi que du biofilm, dans la dissolution des minéraux par cette bactérie. Une étude en fluorimétrie et dosages des métaux a, quant à elle, montré que la pyoverdine était capable de complexer des métaux contaminants adsorbés sur les argiles ou présents dans la structure cristalline des oxy-hydroxydes de fer. Enfin, ces travaux ont abouti à la mise en place du système d’étude dans sa globalité. Si la pyoverdine ou les bactéries productrices de sidérophores n’ont pas amélioré le prélèvement par les plantes du cadmium et du nickel, des résultats prometteurs ont cependant été obtenus pour le cuivre. De plus, les bactéries productrices de sidérophores s’avèrent être des cibles intéressantes en phytostabilisation. / The industrial and agricultural development caused a metallic element accumulation in the soils. In these ecosystems, the living fraction significantly operates in the changes of contaminants retention. Among the decontamination techniques, the phytoremediation appears as a tool of choice. If one of major disadvantages of this technique is the long-term remediation process, the addition of microorganisms with the plants could enhance the uptakes and decrease the time to remediate. This work focused on the possibility to develop processes for siderophore-producing bacteria-assisted phytoremediation, siderophores being molecules with a high affinity for iron and also able to chelate other metals. In this context, a system with an increasing complexity until a complete model including the soil constituents, the bacteria, the contaminant metals and the plants, has been studied to understand the processes involved in these different interactions. The understanding of the interactions between the soil minerals, clays and iron oxydydroxides, and a siderophore-producing bacteria, Pseudomonas aeruginosa, have been first performed with, in particular, a molecular biology approach. These results have highlighted the major role of the one of siderophores, the pyoverdine, and also the biofilm in the mineral dissolution by this bacteria. Fluorimetry and metal quantification assays showed that the pyoverdine was able to chelate the contaminant metals adsorbed on the clays or present in the crystal structure of the iron oxyhydroxides. Lastly, the global system has demonstrated that if the pyoverdine or the siderophore-producing bacteria do not enhance the cadmium and nickel uptake by plants, promising results were obtained for copper. Moreover, siderophore-producing bacteria turn out to be interesting asset for the phytostabilization.

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.

FbpA is the periplasmic binding protein of the transferrin and lactoferrin-iron transport systems. FbpA is conserved among neisserial species and is required for Neisseria gonorrhoeae to sustain growth on transferrin and lactoferrin. The identification of other putative TonB-dependent outer membrane transporters suggests that gonococci may employ other uncharacterized iron uptake systems that do not require FbpA. Previous work in our lab demonstrated that gonococcal strain FA19 utilizes iron from a number of xenosiderophores of the catecholate and hydroxamate classes. In this study we created conditional FbpA mutants to evaluate whether FbpA plays a role in the ability of gonococci to utilize iron from xenosiderophores. Strain FA19 was able to acquire iron from the xenosiderophores enterobactin and salmochelin in an FbpA-dependent and TonB-independent manner. We were also able to detect an extracellular population of FbpA indicating that FbpA may play a novel role in the internalization of iron in the absence of a dedicated transporter.