The water solubility of transition metals constrain their use in the bioinorganic chemistry of organisms. Iron(III) is one of the most sought-after metals by organisms; however, it has poor solubility in water at physiological pH. Prior to the great oxidation event (GOE) 2.4 billion years ago, iron was more bioavailable in the 2+ oxidation state. This higher water solubility sustained a greater supply of iron to organisms that required it. Since the GOE and the oxidation of Fe(II) to Fe(III), organisms have been evolving methods to sequester iron from the environment. One method to obtain iron is through the use of low molecular weight molecules called siderophores which solubilize Fe(III) from their environment through chelation. The strongest siderophore to date is enterobactin (ent) which forms a Fe-ent complex with an association constant of 1049. While iron is utilized by most organisms, titanium is also a bioactive metal. Titanium is second to iron in abundance of transition metals in the Earth’s crust and is becoming increasingly relevant in medicine, technology, and commercial goods. Titanium(IV) (the most common oxidation state of titanium) also has a larger charge to size ratio than Fe(III) and may outcompete iron for binding. The already high abundance and increasing usage of titanium in the developed world leads to possible undocumented interactions between titanium and siderophores. In an attempt to address the natural selection of the chemical elements during bioinorganic evolution, the solution interactions of Ti(III), Ti(IV), Fe(II), and Zn(II) with sulfide in a solution mimicking the sulfidic early ocean were explored. Anaerobic metal solutions were added to solutions with ocean-mimicking salts, acetic acid buffer, and increasing concentrations of sodium sulfide. After equilibration, the soluble metal ion concentrations were measured using ICP-OES. Catechol ligands are important bioinorganic chelators. The synthesis and crystallization of the disodium, dilithium, and diammonium salts of the model catechol ligand 4,5-dihydroxy-1,3-benzenedisulfonate (tiron) are described. The dilithium and diammonium salts were synthesized from the disodium tiron salt by cation metathesis. The crystals were obtained by solvent evaporation in either alcohols or water. Crystal structures were acquired, solved, and analyzed revealing inter- and intramolecular interactions in the dication tiron crystal structures. The synthesis of the siderophore enterobactin (ent) was improved upon from published methods to increase purity. Following the synthesis of ent, spectrophotometric techniques were used to analyze Ti-ent complex stability. Spectrophotometric titrations were carried out from p[H] 2-12 revealed that the fully catecholate bound [Ti(ent)]2- complex was the only species in the p[H] range 2-9. Above a p[H] value of 9 the trilactone backbone apparently hydrolyzed. This result indicates that protons cannot outcompete Ti(IV) binding at low p[H] and hydroxide cannot outcompete ent for binding to Ti(IV) at high p[H]. Using various Ti(IV)-ligand complexes, the log b110 for Ti-ent was probed at pH 6. The complex [Ti(DFOB)]+ (log b110 = 38.1) with 1,000 fold excess desferrioxamine B (DFOB) was unable to compete with ent and every addition of ent lead to the formation of [Ti(ent)]2- yielding a lower limit log b110 of 52.9. The catecholate ligand tiron was used in a 4,000:1 ratio of tiron:Ti(IV) forming [Ti(tiron)3]8- (log b130 = 57.6) to attempt to compete with ent. This yielded a lower limit log b110 for [Ti(ent)]2- of 61.2. Kinetic studies of titanium sequestration from titanium containing species was also investigated. Using Ti(IV) complexes such as [Ti(DFOB)]+, [Ti(citrate)3]8-, and the titanium complex of human serum transferrin (Ti2HsTf) as the Ti(IV) source, the rate of titanium sequestration by ent was probed. Taken together, these findings indicate that ent is a strong binder of Ti(IV); however, it binds relatively slowly. Strong binding ligands are able to coordinate and dissolve metal ions from the surface of metal oxides. Titanium mostly exists in the solid oxide form in the Earth’s crust, thus solubilization of these oxides may be important for Ti(IV) incorporation into an organism. To determine the effects of ent in solution at a TiO2 interface, 50 mM ent was added to a series of suspensions of TiO2 at pH 5 ± 0.5 with different loadings for 176 h. The [Ti]max for all anatase trials averaged to be approximately 20 – 25 mM indicating 40 – 50% ent efficacy for TiO2 dissolution. This value was compared to previously reported TiO2 dissolution by 2 mM DFOB at pH 2 as well as dissolution of 50 mM and 150 mM DFOB and tiron, respectively, at pH 5 ± 0.5. Surface interactions between ent and anatase were probed using ATR-IR. One extension of this work related to the avid Fe(III) ligand deferasirox (DSX). DSX is an orally administered chelator for Fe(III) overload. A Ti(IV)-DSX complex may be useful as an anticancer drug. The solution chemistry of DSX with Ti(IV) was characterized utilizing spectrophotometric and mass spectrometric techniques. A series of titrations with Ti(IV) and DSX from pH 6.7-11.2 revealed two distinct species. Additions of [Ti(HDSX)(DSX)]- to bovine serum albumin (BSA) indicated that the [Ti(HDSX)(DSX)]- complex binds to BSA. This transfer is possibly an important step in the efficacy of Ti(IV) anticancer drugs and may relate to the possible mechanism for Ti(IV) delivery into cancerous cells. / Chemistry

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "May 1997." Typescript. Vita. Includes bibliographical references (leaves 212-236). Also available on the Internet.

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

Many gram-negative bacteria produce and secrete siderophores under iron-deficient conditions. Siderophores are low molecular weight compounds (600-1500 Daltons), which chelate ferric iron with an extremely high affinity, and the complex is actively transported across the outer and inner membranes of gram-negative bacteria. There are two main classes of siderophores: catechol and hydroxamate. Catechol-type siderophores chelate ferric iron via hydroxyl groups, and hydroxamate-type siderophores chelate ferric iron via a carbonyl group with an adjacent nitrogen. Rhizobia fix atmospheric nitrogen symbiotically in leguminous plants using the iron-containing enzyme nitrogenase. To satisfy their iron requirements, many rhizobia are known to produce siderophores. Rhizobium leguminosarum Strain IARI 312 is known to infect pigeon pea plants. R. leguminosarum Strain IARI 312 produces both a catechol-type and a hydroxamate-type siderophore when grown under iron deficient conditions. The catechol-type siderophore has been purified and chemically characterized, and is consistent with that of enterobactin.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2018-01-22T12:47:14Z No. of bitstreams: 1 PATRICIA VOLLÚ SILVA.PDF: 2575547 bytes, checksum: c2489f8abe58d46ea1c2ba8977ba02e1 (MD5) / Made available in DSpace on 2018-01-22T12:47:14Z (GMT). No. of bitstreams: 1 PATRICIA VOLLÚ SILVA.PDF: 2575547 bytes, checksum: c2489f8abe58d46ea1c2ba8977ba02e1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A ilha de patogenicidade pks de 54 kb é responsável pela produção de colibactin, uma molécula genotóxica, que causa rupturas de fita dupla de DNA (DSB) das células hospedeiras, e que parece estar relacionada ao aumento do risco de desenvolvimento de câncer colorretal. Colibactin foi inicialmente descrita em Escherichia coli, mas também pode ser encontrada em outras enterobactérias, como Klebsiella pneumoniae. A biossíntese de colibactin requer a atividade enzimática da fosfopantoteinil transferase (PPTase) ClbA, que também pode contribuir para a produção dos sideróforos enterobactin e yersiniabactin em E. coli. O objetivo deste trabalho foi avaliar a virulência em amostras de E. coli e de K. pneumoniae, presença da ilha de patogenicidade pks e determinar o papel de colibactin e do sideróforo enterobactin em infecções por K. pneumoniae. Para tal, uma coleção de 218 amostras de E. coli isoladas de pacientes com infecção do trato urinário atendidas no Instituto Nacional do Câncer, Brasil e 258 amostras de K. pneumoniae isoladas de sangue de pacientes internados no Centre Hospitalier Universitaire de Toulouse, França foram avaliadas. A tipificação filogenética e a presença do gene de virulência fyuA em E. coli e do gene clbN em ambas bactérias foram avaliadas por PCR. O fenótipo hipermucoviscoso (HMV) de K. pneumoniae foi determinado pelo teste string. Foi ainda investigado o papel de colibactin e enterobactin em um modelo de pneumonia em camundongos C57BL/6JRJ e em infecções de células HeLa, utilizando a cepa K. pneumoniae SB 4496 e mutantes isogênicos deficientes em clbA, clbN ou entD. Os resultados demonstraram que entre as amostras de E. coli pesquisadas, o grupo filogenético B2 foi o mais prevalente (44,9%), seguido pelos grupos filogenéticos A (13,8%), B1 (22,0%) e D (19,3%). O gene fyuA, relacionado a produção do sideróforo yersiniabactin, foi detectado em todos os grupos filogenéticos, no entanto, a detecção do referido gene foi mais frequente no grupo B2 (P = 0,0228), sendo detectado em 74,5% das amostras deste grupo. O gene clbN, relacionado a produção de colibactin, foi detectado em 14,7% das amostras de E. coli. Vale ressaltar que todas as amostras clbN positivas pertenciam ao grupo filogenético B2, as quais também portavam o gene fyuA. Adicionalmente, três amostras de E. coli apresentaram efeito citopático em células HeLa, independente da produção de colibactin e da presença dos genes hlyC/A, hlyF, cdt e cnf1. O gene clbN foi detectado em 5,8% das amostras de K. pneumoniae. Também foi observado que a detecção de clbN foi estatisticamente significantemente (P < 0,0001) entre as amostras caracterizadas como HMV, este gene foi observado em 35% das amostras HMV analisadas. Além disso, a síntese de colibactin não pôde ser mantida pela PPTase EntD e a produção de sideróforos pela K. pneumoniae SB 4496 foi continuada por outro sistema independente de PPTases EntD e ClbA. Os resultados obtidos neste trabalho parecem indicar que a produção de colibactin não afeta a sobrevivência de K. pneumoniae hipervirulenta (hvKP) nos tecidos pulmonares de camundongos C57BL/6JRJ, nas condições estudadas. No entanto, é importante salientar que a toxina colibactin produzida por hvKP é capaz de induzir genotoxicidade em células HeLa. Ambos os genes clbA e clbN foram necessários para a manutenção da megalocitose e DBS induzida por colibactin em K. pneumoniae / The 54-kb pks pathogenicity island produces a genotoxic molecule named colibactin, which causes double-strand DNA breaks (DSB) in the host cells and enhanced colorectal cancer development. Colibactin was initially described in Escherichia coli, but can be found in other enterobacteria, including Klebsiella pneumoniaE. colibactin biosynthesis requires the enzymatic activity of phosphopantetheinyl transferase (PPTase) ClbA, which may also support the enterobactin and yersiniabactin siderophores synthesis in E. coli. The goal of this work was to evaluate the virulence of E. coli and K. pneumoniae isolates, presence of pks pathogenicity island and to determine the role of colibactin and enterobactin siderophore in K. pneumoniae infections. For this purpose, it was evaluated a collection of 218 E. coli isolates obtained from patients with urinary tract infection assisted at Instituto Nacional do Câncer, Brazil, and 258 K. pneumoniae isolates collected from blood samples from patients admitted to the Centre Hospitalier Universitaire in Toulouse, France. Phylogenetic typing and the detection of fyuA virulence gene in E. coli and clbN gene in both bacteria were assessed by PCR. K. pneumoniae hypermucoviscous (HMV) phenotype was determined by the string test. The role of colibactin and enterobactin in a C57BL/6JRJ mice pneumonia model and in HeLa cells infection was investigated using K. pneumoniae SB 4496 strain and isogenic mutants deficient in clbA, clbN or entD. Among the E. coli isolates, the phylogenetic group B2 was more prevalent (44.9%) followed by phylogroups A (13.8%), B1 (22.0%) and D (19.3%). The fyuA gene, involved in the yersiniabactin production, was detected in all phylogenetic groups studied; however, its presence was more frequently detected among the phylogroup B2 (P = 0.0228), with a high percentage of 74.5%. The clbN gene, associated to colibactin production, was detected in 14.7% of E. coli isolates. It is noteworthy that all clbN positive isolates belong to the phylogroup B2 and carry the fyuA gene. In addition, three E. coli isolates studied showed cytopathic effect in HeLa cells independently of colibactin production and the presence of hlyC/A, hlyF, cdt and cnf1 genes. Regarding K. pneumoniae, clbN gene was detected in 5.8% of the isolates. It was also observed that this detection was statistically significant (P < 0.0001) among the isolates of the HMV phenotype group, being this gene observed in 35% of the HMV isolates analyzed. In addition, it was observed that colibactin synthesis could not be maintained by the PPTase EntD. Indeed, siderophores production by K. pneumoniae SB 4496 was successful continued by another system that does not require the activity of PPTases EntD and ClbA. Results obtained in this work seem to indicate that production of colibactin does not affect the survival of hypervirulent K. pneumoniae (hvKP) in C57BL/6JRJ lung mice. Despite that, it is interesting that colibactin produced by hvKP is capable of inducing genotoxicity in HeLa cells. Both clbA and clbN genes were required for the maintenance of megalocytosis and DBS induced by colibactin in K. pneumoniae

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the United States. Increasing resistance of Campylobacter to clinical antibiotics raises an urgent need for novel strategies to prevent and control infections in humans and animal reservoirs, which necessitates a better understanding of Campylobacter pathogenesis. We hypothesize that multidrug efflux pump CmeABC and ferric enterobactin (FeEnt) iron acquisition systems, which play a critical role in Campylobacter pathogenesis, are novel targets for developing effective measures against Campylobacter. To test this, the molecular, antigenic, functional, and protective characteristics of two outer membrane proteins, CmeC (an essential component of CmeABC drug efflux pump) and CfrA (a FeEnt receptor), were examined. Both CmeC and CfrA are highly conserved and widely produced in C. jejuni strains. Anti-CmeC and Anti-CfrA antibodies inhibited the function of CmeABC efflux pump and CfrA, resulting enhanced susceptibility to bile salts and reduced utilization of FeEnt of C. jejuni, respectively. Immunoblotting analysis also indicated that CfrA is expressed and immunogenic in vivo. Amino acid substitution mutagenesis demonstrated that a highly conserved basic amino acid R327 in CfrA plays a critical role in FeEnt acquisition. The purified recombinant CmeC and a Salmonella live vaccine expressing the protective epitope of CfrA were evaluated as subunit vaccines against Campylobacter infection in the chicken model. CmeC vaccination elicited immune response but failed to reduce C. jejuni colonization in the intestine. However, Salmonella-vectored vaccine conferred significant protection against C. jejuni challenge. To further elucidate the role of iron acquisition in the pathogenesis of Campylobacter, whole genome sequence of a unique C. jejuni strain was determined using a 454 GS FLX sequencer with Titanium series reagents. Comparative genomics analysis led to the identification of a novel Campylobacter Enterobactin Esterase (Cee) that is essential in the CfrB-dependent FeEnt utilization pathway. Extensive genetic manipulation revealed molecular pathways and mechanistic features of the two orchestrated FeEnt acquisition systems in Campylobacter. This project provides critical information about the feasibility of targeting CmeC and CfrA for immune protection against Campylobacter colonization in the intestine, and increases our understanding of the critical role of FeEnt acquisition in the pathophysiology of Campylobacter.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves : 212-236). Also available on the Internet.

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

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / "December 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

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.