<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

Iron is essential for life and growth in most organisms. Although it is abundant, iron exists mostly as insoluble iron-oxyhydroxide. Bacteria secrete siderophores to chelate iron and transport it into the cell via specific outer membrane receptors. The FhuA receptor protein transports ferrichrome, a siderophore produced by Ustilago sphaerogena. We determined the binding affinity of variants from the conserved 'lock region' of FhuA and also created and characterized variants of the highly conserved R452 to determine its role in ferrichrome transport. We hypothesize that during transport the plug domain of FhuA does not leave the barrel; rather it undergoes a conformational change to form a channel. We mutated selected amino acids to cysteine to form disulfide bonds to tether the plug, preventing its displacement or unfolding during transport. The tetra-cysteine mutant 72/615/109/356C was able to bind and transport radiolabeled ferrichrome. One double-cysteine mutant, 104/149C, was purified for crystallization.

The cyclic trihydroxamic acid, N, N', N' '-triacetylfusarinine C, produced by Mycelia sterilia EP-76, is shown to be a ferric ionophore for this organism. The association constant for ferric-N, N', N' '-triacetylfusarinine C complex was determined to be log K=32.5. Other iron chelating agents, such as rhodotorulic acid, citric acid, or the monomeric subunit of triacetylfusarinine C, N-acetyl- fusarinine, delivered iron to the cells by an indirect mechanism involving iron exchange into triacetylfusarinine C. In vitro ferric ion exchange was found to be rapid with triacetylfusarinine C. Gallium uptake rates comparable to those of iron were observed with the chelating agents that transport iron into the cell. Ferrichrome, but not ferrichrome A, was also capable of delivering iron and gallium to this organism, but not by an exchange mechanism. Unlike triacetylfusarinine C, the 14C-ligand of ferrichrome was retained by the cell. A mid-point potential of -690 mV versus the saturated silver chloride electrode was obtained for the ferric-N, N', N' '-triacetylfusarinine C complex, indicating that an unfavorable reduction potential was not the reason for utilizing a hydrolytic mechanism of intracellular iron release from the ferric triacetylfusarinine C chelate. The iron transport system recognizes only the  -cis coordination isomer of ferric-N, N’, N' '-triacetylfusarinine C metal ligand complex even though the -cis configuration predominates in solution. Ferrichrome and ferric-N, N', N' '-triacetylfusarinine C are both transported by the same receptor.

Pseudomonas syringae pv. syringae B728a, an aggressive bacterial pathogen of bean, utilizes large surface populations and extracellular signaling to initiate a fundamental change from an epiphytic to a pathogenic lifestyle. Extracytoplasmic function (ECF) sigma (σ) factors serve as important regulatory factors in responding to various environmental signals. Bioinformatic analysis of the B728a genome has revealed 10 ECF sigma factors, five of which have high levels of sequence similarity to the FecI-type of ECF sigma factors and play a known role in the regulation of various iron transport systems. Because iron is essential for the induction of major virulence factors in B728a, I hypothesized that these FecI-type sigma factors may play a critical role in the bacterium’s transition between lifestyles. Deletion mutants of two FecI-type sigma factors, Psyr_1040 and Psyr_1107, in B728a have been created using homologous recombination based on the phage λ Red recombinase method. This study shows that the B728a FecI-type sigma factors, Psyr_1040 and Psyr_1107 are affected by conditions of iron stress, and influence the expression of putative outer membrane receptors and transmembrane sensors associated with these genes. Moreover, Psyr_1107 contributes to the expression of a cluster of predicted pili assembly genes downstream of it. Mutations in Psyr_1040 and Psyr_1107 affect the population levels of B728a in bean plants, since in planta growth of deletion mutants of B728a lacking Psyr_1040 and Psyr_1107 appears to be slower than wild-type B728a. In this thesis, the possible roles of Psyr_1040 and Psyr_1107 in the adaptation of B728a to a pathogenic lifestyle are addressed using a combination of phenotypic characterization and quantitative real-time PCR (qRT-PCR) analyses.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Phillip E. Klebba / The goal of the research included in this dissertation is to provide a more complete model of the role of TonB, an energy transducing protein that resides in the inner membrane and is an essential component of the iron transport of Escherichia coli under iron-starved conditions. Using fluorescent hybrid proteins, the anisotropy of TonB in the cytoplasmic membrane (CM) of Escherichia coli was determined. With the aim of understanding the bioenergetics of outer membrane (OM) iron transport, the dependence of TonB motion on the electrochemical gradient and the effect of CM proteins ExbB and ExbD on this phenomenon was monitored and analyzed. The native E. coli siderophore, enterobactin chelates Fe⁺³ in the environment and ferric enterobactin (FeEnt) enters the cell by energy- and TonB-dependent uptake through FepA, its OM transporter. The TonB-ExbBD complex in the CM is hypothesized to transfer energy to OM transporters such as FepA. We observed the polarization of GFPTonB hybrid proteins and used metabolic inhibitors (CCCP, azide and dinitrophenol) and chromosomal deletions of exbBD to study these questions. The results showed higher anisotropy (R) values for GFP-TonB in energy-depleted cells, and lower R-values in bacteria lacking ExbBD. Metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings suggest that TonB undergoes constant, energized motion in the bacterial CM, and that ExbBD mediates its coupling to the electrochemical gradient. By spectroscopic analyses of extrinsic fluorophore labeled site-directed Cys residues in 7 surface loops of Escherichia coli FepA, binding and transport of ferric enterobactin (FeEnt) was characterized. Changes in fluorescence emissions reflected conformational motion of loops that altered the environment of the fluorophore, and we observed these dynamics as quenching phenomena during FeEnt binding and transport in living cells or outer membrane vesicles. Cys residues in each of the 7 surface loops (L2, L3, L4, L5, L7 L8, and L11) behaved individually and characteristically with regard to both fluorophore maleimide reactivity and conformational motion. Fluorescence measurements of FeEnt transport, by either microscopic or spectroscopic methodologies, demonstrated that ligand uptake occurs uniformly throughout the cell envelope, and susceptibility of FeEnt uptake to the proton ionophore m-chlorophenyl hydrazone (CCCP) at concentrations as low as 5 uM. The latter result recapitulates the sensitivity of inner membrane major facilitator transporters to CCCP (Kaback, 1974), providing further evidence of the electrochemical gradient as a driving force for TonB-dependent metal transport.

No description available.

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.

A limited understanding of iron uptake mechanisms is available for Streptococcus pyogenes, a hemolytic human pathogen capable of using a variety of hemoproteins in addition to ferric and ferrous iron. This study characterizes the transporters of iron-complexes siuADBG (for streptococcal iron uptake) and siaABC (for streptococcal iron acquisition). These ABC-type transporters are encoded by iron regulated operons and their protein products are homologous to components of heme and siderophore transporters found in both Gram-positive and Gram-negative bacteria. Mutants of the membrane permeases siuG and siaB were constructed and characterized. Mutations in both transporters demonstrated growth reduction in comparison to the parent strain when grown in complex medium containing iron in the form of hemoglobin. The addition of heme to the growth medium inhibited ferric uptake by the wild-type while the addition of protoporphyrin IX did not, suggesting that heme utilization as an iron source is responsible for the inhibition of ferric accumulation. Inactivation of siuG reduced the ability of heme to inhibit ferric incorporation by the cells. Inactivation of siaB in addition to siuG had a cumulative effect, indicating that both siu and sia transporters are involved in heme utilization. We also demonstrated that purified rSiaA, the surface receptor of SiaABC, binds heme and hemoglobin in vitro, and we propose a mechanism of heme binding by SiaA. Studies in a zebrafish infection model revealed that the siuG mutant was attenuated in producing disease. While the siaB mutant also presented virulence attenuation, infection by this mutant was characterized by an increase in the host inflammatory response. These observations show that iron acquisition is important for S. pyogenes virulence. We propose that the SiaABC and SiuADBG, together with the multi-metal transporter MtsABC, are involved in iron acquisition from different iron sources present in the human body, thus contributing to the survival and pathogenesis of S. pyogenes.

Le fer est un élément essentiel à la vie car il possède un rôle clé dans de nombreux processus biologiques.Malgré son abondance au niveau de la croûte terrestre, le fer est très faiblement biodisponible. Pour contourner ce problème, la majorité des micro-organismes a développé différents systèmes particulièrement efficaces pour l’acquisition de cet élément. Le mécanisme le plus répandu implique la production et la sécrétion de petites molécules chélatrices ayant une forte affinité pour le fer. Après sécrétion dans le milieu extracellulaire, ces composés chélatent le Fe3+ et le transportent ensuite au travers de la membrane externe via des transporteurs TonB-dépendants (TBDT). Durant cette thèse, nous avons mis en place un protocoleefficace permettant d’aller rapidement du clonage à la cristallisation de ces cibles afin d’étudier la structure tridimensionnelle de cette famille de protéines. Ainsi, nous avons pu résoudre et étudier la structure de plusieurs TBDT, de bactéries Gram-négatives. Ainsi nous avons mis en évidence un mouvement du domaine de signalisation en présence du ligand, proposé un mécanisme de transporteur de la molécule d’hème par le système shu chez Shigella dysenteriae. Chez les bactéries du genre Pseudomonas, nous avons élucidé et caractérisé au niveau structural les mystères de l’énantiosélectivité des pyochélines. En parallèle, nous nous sommes intéressé au devenir du ferri-sidérophore au niveau du périplasme, chez P. aeruginosa, ainsi qu’au transport du fer au travers de la membrane interne grâce à un transporteur ABC FpvCDEF ayant laparticularité de posséder deux protéines périplasmiques associées capables d’interagir avec le sidérophore. / Iron is essential for life because it has a key role in many biological processes. Despite its abundance in the earth's crust, iron is poorly bioavailable. To circumvent this problem, most micro-organisms have developed different systems particularly effective for the acquisition of this element. The most common mechanism involves the production and secretion of small chelating molecules having high affinity for iron. After secretion into the extracellular medium, these compounds chelate and transport ferric iron through the outer membrane via TonB-dependent transporters (TBDTs). In this thesis, we have developed an efficient protocol to easily go from cloning to crystallization of these targets and then studied the three-dimensional structure of this protein family. Thus, we were able to solve and study the structure of several TBDT of Gram-negative bacteria. We have identified a movement of the signaling domain in the presence of ligand. We proposed a mechanism for heme translocation through the shu system, in Shigella dysenteriae. In Pseudomonas species, we elucidated and characterized at the structural level the mysteries of the pyochelin enantioselectivity. In Pseudomonas aeruginosa, we studied the ferri-siderophore become in the periplasmic space, as well as iron transport across the inner membrane by an ABC transporter, named FpvCDEF, with the particularity of having two periplasmic proteins associated able to interact with the siderophore.

<p>Two species of <i>Neisseria</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>, are obligate human pathogens that cause meningitis and gonorrhea, respectively. Although generally asymptomatic, <i>N. meningitidis</i> can cause invasive meningococcal disease with high mortality rate. Due to emerging antibiotic resistance strains of <i>N. gonorrhoeae</i>, the Centers for Disease Control and Prevention (CDC) have designated it as an urgent threat to public health. Therefore, immediate interventions are required for fight against these Neisserial pathogens. Iron is an essential nutrient for all bacteria, including <i>Neisseria</i>. However, free iron is scarce in human, therefore, <i>Neisseria</i> have evolved to acquire iron from host proteins. These iron acquisition systems are immunogenic and important for infection and are promising therapeutic targets.</p> <p> In the host, lactoferrin sequesters free iron and limits iron availability to pathogens. However, <i>Neisseria</i> have evolved machinery to hijack iron directly from lactoferrin itself. Lactoferrin binding proteins, LbpA and LbpB, are outer membrane proteins that together orchestrate the acquisition of iron from lactoferrin. Additionally, LbpB serves an additional role in providing protection against host cationic antimicrobial peptides and innate immune response. Despite studies aimed at deciphering the roles of LbpA and LbpB, the molecular mechanisms underpinning iron acquisition and immune protection remain unknown. Here, we investigated the role of the lactoferrin binding proteins in iron acquisition and protection against cationic antimicrobial peptides. We obtained three-dimensional structures of <i>Neisseria</i> LbpA and LbpB in complex with lactoferrin using cryo-electron microscopy and X-ray crystallography. These structures show that both LbpA and LbpB bind to C-lobe of lactoferrin, albeit at distinct sites. Structural analyses show that while lactoferrin maintains its iron-bound closed conformation in the LbpB-lactoferrin complex, it undergoes a large conformational change from an iron-bound closed to an iron-free open conformation upon binding to LbpA. This observation suggest that LbpA alone can trigger the extraction of iron from lactoferrin. Our studies also provide an explanation for LbpB’s preference towards holo-lactoferrin over apo-lactoferrin and LbpA’s inability to distinguish between holo- and apo-lactoferrin. Furthermore, using mutagenesis and binding studies, we show that anionic loops in the C-lobe of LbpB contribute to binding the cationic antimicrobial peptide lactoferricin. Solution scattering studies of the LbpB-lactoferricin complex showed that LbpB undergoes a small conformational change upon peptide binding.</p> Together, our studies provide structural insights into the role of the lactoferrin binding proteins in iron acquisition and evasion of the host immune defenses. Moreover, this work lays the foundation for structure-based design of therapeutics against <i>Neisseria</i> targeting the lactoferrin binding proteins.