No description available.

No description available.

Neisseria gonorrhoeae, the etiologic agent of gonorrhea, utilizes TonB-dependent transporters to import essential nutrients such as iron. Study of TonB-dependent transporters is extremely important due to the fact that they make excellent vaccine targets. In order to learn more about the structure, function, expression, and regulation of selected TonB-dependent transporters, three goals were established for this study. The first goal was to examine the role of two highly conserved regions of TbpB in lipidation. One of the conserved regions of TbpB, the LSAC motif, was shown to be critical for lipidation. The second goal was to determine whether MisR/MisS regulates expression of TbpA and TbpB. MisR/MisS was shown to regulate the expression of TbpA and TbpB. The third goal was to assess the ability of recombinant TdfJ to bind hemin when expressed in E. coli. Recombinant TdfJ was shown to specifically bind hemin when expressed in E. coli.

Rhizobium leguminosarum is a gram-negative soil bacterium that requires iron for survival. However, iron becomes insoluble in the presence of oxygen at physiological pH. In response, Rhizobia species have used siderophore mediated iron transport systems to meet their iron requirements. R. leguminosarum ATCC 14479 produces the trihydroxymate siderophore vicibactin and we hypothesize that the import of the ferric iron-vicibactin complex is energized by the TonB-ExbB-ExbD system. Here, we have identified a putative tonB gene. A tonB mutant was created and compared with wild type in its ability to transport 55Fe-vicibactin. Also, the putative TonB of R. leguminosarum ATCC 14479 is interesting due to its estimated size compared to the TonB of E. coli.. Many groups have attempted structural analysis of the C-terminus of TonB in E. col with inconsistent results. We were successful in expressing 2 different sized TonB C-terminals (120 and 200 amino acids) using pET17b in E. coli.

No description available.

Neisseria gonowhoeae has evolved a repertoire of high-affinity iron acquisition systems to facilitate essential iron uptake in the human host. Acquisition of iron requires both the energy-harnessing cytoplasmic membrane protein, TonB, as well as specific outer membrane TonB-dependent transporters (TdTs). The overall goal of this study was to investigate the extra- and intracellular iron acquisition mechanisms of N. gonorrhoeae and determine the role of the TonB and TdTs in this process.The ability of gonococci to acquire potential exogenous iron sources was determined by plate bioassay. Gonococcal growth was promoted by various catecholate and hydromate siderophores; however, growth was not dependent upon TonB expression.As all previously characterized siderophore-iron uptake is dependent upon this protein, apotential TonB-bypass mechanism is suggested.The role of the Ton system and TdTs in gonococcal survival within human cervicalepithelial cells was also determined for two gonococcal strains, FA1090 and MS 1 1. Wedemonstrate that intracellular survival of both strains was dependent upon host cell ironacquisition, yet the expression of the Ton system was only critical to the survival ofFA1090. One characterized difference between these two strains is possession of thegonococcal genetic island (GGI) which is present in approximately 80% of gonococcalstrains. This study demonstrates that the GGI provides a mechanism to bypassintracellular TonB-dependent iron acquisition.In the strain lacking the genetic island, none of the characterized TdTs provided abenefit to the gonococcus when grown intracellularly. However, expression of oneuncharacterized TdT, TdfF, was necessary for successful intracellular survival. To ourknowledge, this is the first demonstration of a specific requirement for a single irontransporter in the survival of a bacterial pathogen within host epithelial cells.In the GGI-containing strain, TonB function was not critical to survival withincervical epithelial calls. The presence of the GGI was associated with the ability to bypass TonB-dependant uptake. Specifically, this bypass mechanism was mediated bycomponents of the T4S machinery encoded by the GGI, and replication was directlyrelated to iron acquisition. To our knowledge, this study provides the first direct linkbetween bacterial iron acquisition and a type IV secretion system.

Master of Science / Department of Biochemistry and Molecular Biophysics / Phillip Klebba / Iron acquisition is a component of Gram-negative bacteria pathogenesis, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. Energy poisons azide, arsenate, and 2,4-dinitrophenol were evaluated to determine sensitivity to known transport inhibitors. We developed and optimized methods to screen for iron transport inhibitors using a cell-based high-throughput screening platform. These inhibitors may have broad spectrum bacteriostatic antibiotic properties.

The bacterium Burkholderia pseudomallei causes the disease melioidosis, a significant public health threat in endemic regions and is a potential biowarfare agent. Treatment of melioidosis is intensive and prolonged and there is no licensed vaccine to protect against it. The aim of this study was to characterise novel targets for antimicrobials to improve treatment of melioidosis. A holistic down selection process was undertaken in order to identify a range of possible novel and exploitable antimicrobial targets in Burkholderia pseudomallei. Four targets: FtsA, FtsZ, MraW and TonB were selected for characterisation by mutagenesis study. FtsA and FtsZ are early effectors of cell division and are considered potential antimicrobial drug targets in other pathogenic bacteria. Genes for both were shown likely to be essential for viability in Burkholderia pseudomallei, following attempted deletion of the genes, thus confirming their potential for drug targeting for treatment of melioidosis. MraW, a highly conserved methyltransferase, and TonB, the energiser for high affinity iron uptake in Gram negative bacteria, were also selected for characterisation as antimicrobial targets. In-frame deletions of the genes encoding these targets were constructed in B. pseudomallei K96243. In order to determine the roles played by MraW and TonB during infection, these mutants were characterised in several models of Burkholderia pseudomallei infection. Deletion of mraW rendered the bacteria non-motile and led to attenuation during infection of Balb/C mice. A small growth defect was seen early during infection of macrophages by this mutant, whilst no attenuation was seen on deletion of mraW in Galleria mellonella. Burkholderia pseudomallei ΔtonB required free iron supplementation for growth. This mutant had an improved ability to invade murine macrophages, though the mutant was attenuated in both Galleria mellonella and Balb/C mice. Attenuation of both mutants in a mammalian model of infection, support the strategy to target either of these proteins as novel targets for inhibition with small molecules during Burkholderia pseudomallei infection. However, an improved ability to infect macrophages by Burkholderia pseudomallei ΔtonB and non-complementation of this mutant by iron supplementation to Galleria mellonella suggests additional roles to iron uptake alone for TonB in Burkholderia pseudomallei, such as bacterial iron sensing and signalling.

Neisseria gonorrhoeae is an obligate human pathogen that causes the common sexually- transmitted infection, gonorrhea. Gonococcal infections cause significant morbidity, particularly among women, as the organism ascends to the upper reproductive tract, resulting in pelvic inflammatory disease, ectopic pregnancy and infertility. Antibiotic resistance rates have risen dramatically, leading to severe restriction of treatment options for gonococcal disease. Gonococcal infections do not elicit protective immunity nor is there an effective vaccine to prevent the disease. Thus, further characterization of expression, function and regulation of surface antigens could lead to better treatment and prevention modalities in the future. N. gonorrhoeae express a repertoire of TonB-dependent transporters for the acquisition of iron. All of these transporters are under the transcriptional regulation of Fur. We investigated putative intracellular iron sources utilized by gonococci and the role that the TonB-dependent transporter, TdfF, played in this acquisition. We determined that ascorbate which could prevent ferritin degradation or withhold iron from gonococci, inhibited intracellular survival. The utilization of iron from the iron binding moiety 2, 5-DHBA of the putative mammalian siderophore was also examined. In this study we continued to investigate the regulation of TdfF and further investigate potential host-specific inducing molecules for TdfF expression. We investigated the regulation of tdfF expression and the role of MpeR, an AraC-like regulator, in tdfF expression. We determined that MpeR, interacted specifically with the DNA sequence upstream of fetA and activated FetA expression. We confirmed that the outer membrane transporter, FetA, allows gonococcal strain FA1090 to utilize the xenosiderophore, ferric-enterobactin, as an iron source. However, we further demonstrated that FetA has an extended range of substrates that encompasses other catecholate xenosiderophores, including ferric-salmochelin and the dimers and trimers of dihydroxybenzoylserine. We demonstrated that fetA is encoded as part of an iron-repressed, MpeR-activated operon, which putatively encodes other iron transport proteins. These iron transport proteins also play a role in xenosiderophore acquisition. We also identified genetic differences that may explain why some gonococcal strains are capable of xenosiderophore internalization in a TonB-dependent pathway and other strains are restricted to TonB-independent pathways. Interestingly, the chromosomal locus that codes for mpeR and tdfF is pathogen specific. Thus understanding more about the TonB-dependent transporter and AraC-like regulator may further elucidate the pathogenicity of N gonorrhoeae.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Siderophore uptake systems are one the most prominent methods of Fe³+-iron acquisition in Gram negative bacteria. The catecholate siderophore enterobactin is synthesized and utilized by many members of Enterobacteriaceae as well as several of the ESKAPE pathogens. The outer membrane (OM) transporter of ferric enterobactin (FeEnt), FepA is a ligand-gated porin (LGP) that requires interaction with the inner membrane (IM) protein TonB in order to accomplish active transport. TonB is thought to transduce the electrochemical energy created by the proton gradient across the IM to LGPs like FepA in the OM, to promote siderophore transport through their occluded channels. However, we do not yet have a clear picture of either how TonB transfers energy to FepA, or what kind of conformational changes occur in the occluding domain of FepA to allow ligand passage. The experiments described herein investigate these two questions, building on previously outlined models and observations. Using fluorescence labeling of strategically substituted cysteines in the surface loops of FepA, we unraveled a hierarchy of loop motion during binding of FeEnt to FepA. Additionally, by rendering parts of the FepA protein immobile as a result of engineered disulfide bonds, I identified residues or regions within its occluding domain that may normally unfold to open a size-specific channel for FeEnt. I also elucidated the role of the peptidoglycan polymer beneath the OM a framework for protein-protein interactions between IM and OM proteins. This includes the proposed interaction between a rotating TonB and FepA, or other LGPs, that may transfer kinetic energy to the OM transporter. The role of iron in microbial survival and pathogenesis makes iron-uptake pathways an attractive target for therapeutic intervention. Using the FeEnt-FepA uptake system as a model, we used a fluorescence based high-throughput screening method to identify novel small molecule inhibitors of TonB action in E. coli. The approach used can be potentially adopted to screen bigger chemical libraries as well as used to find inhibitors of ESKAPE pathogens that use FeEnt such as, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Finally, we discoverd a TonB-dependent OM transporter of heme/hemoglobin called HutA in the oligotrophic bacterium Caulobacter crescentus.