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.

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.

Iron is an essential element for most bacteria and is commonly acquired by siderophores, molecules secreted under iron restricted environment to bind ferric iron. Gram negative cells actively uptake these complexes via outer membrane-transport proteins such as FhuA in Escherichia coli. Structural analysis of receptors revealed a conserved β-barrel occluded by an N-terminal plug domain. The cell membrane TonB/ExbB/ExbD complex presumably supplies energy via interaction between the FhuA N-terminal TonB box and the C-terminal domain of TonB. In order to better understand the mechanism of action the FhuA mutant 104/149C, tethering the central β-strands 4 and 6 of the plug domain, was studied and showed severely reduced transport of radio-labeled ferrichrome. In the course of this study, this protein was HPLC purified for structural studies by crystallization and X-ray diffraction. In addition, protein interaction studies were performed with purified TonB-C terminal revealing no impact of the mutation on FhuA-TonB interactions.

The obligate human pathogen Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed “nutritional immunity” by expression of TonB-dependent transporters (TdTs): outer membrane receptors that facilitate nutrient transport in an energy-dependent manner. N. gonorrhoeae encodes eight TdTs, five of which facilitate utilization of iron or iron-chelates from host derived proteins including transferrin, lactoferrin and hemoglobin, in addition to siderophores from neighboring bacteria. The transferrin utilization system was previously shown to be critical for establishing infection in human males; demonstrating the possible contributions of TdTs to gonococcal pathogenesis. As such, studies describing the biological function and contribution to pathogenesis of the remaining three uncharacterized TdTs (TdfG, TdfH and TdfJ) are needed. In this study we report that neither TdfG, TdfH nor TdfJ are heme receptors as gonococcal heme utilization occurs passively, independent of energy derived from the TonB system. We also report that TdfH and TdfJ are zinc (Zn) regulated and identify virulence associated regulators that modulate expression of these TdTs, which is in some cases strain-specific. We report that both TdfH and TdfJ contribute to Zn acquisition in N. gonorrhoeae and we characterize TdfH as a calprotectin receptor. Calprotectin, an immune effector protein highly expressed in neutrophils, has antimicrobial activity due to its ability to sequester Zn and Mn. We present evidence that TdfH confers resistance to calprotectin and that TdfH facilitates gonococcal calprotectin binding and Zn accumulation in the presence or absence of calprotectin. Finally, we demonstrate that TdfH expression enhances N. gonorrhoeae NET survival. These studies identify for the first time a novel gonococcal defense strategy to host-mediated nutritional immunity, in which N. gonorrhoeae, via the TdT TdfH, utilizes calprotectin as a Zn source neutralizing its antimicrobial activity. These studies have yielded novel insights into the function and regulation of TdfG, TdfH and TdfJ in N. gonorrhoeae and have laid the framework for future investigation of TdT-mediated Zn acquisition and its role in bacterial pathogenesis.

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.

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.

The outer membrane (OM) of P. aeruginosa is a semi-permeable barrier that contributes to antibiotic resistance by reducing uptake. Finding strategies to circumvent this barrier is a major challenge. One approach involves screening in physiologically relevant conditions to identify novel activity in existing molecules. We discovered that thiostrepton (TS), a thiopeptide antibiotic with no reported activity against Gram-negative bacteria, hijacks the pyoverdine siderophore transporters FpvA and FpvB to cross the OM under iron limitation to inhibit translation. Using TS, we subsequently showed that FpvB is not primarily a pyoverdine transporter, but rather a promiscuous transporter for siderophores ferrichrome and ferrioxamine B. Our work with TS suggested that other thiopeptides may use siderophore transporters for entry into the cell. This hypothesis led to a screen to identify other thiopeptides with activity against P. aeruginosa, uncovering two other thiopeptides, thiocillin and micrococcin, that use the ferrioxamine transporter FoxA for uptake. We discovered another siderophore, bisucaberin, could also use FoxA for uptake and our collaborators solved the crystal structure of bisucaberin bound to FoxA. Through biochemical approaches, we characterized how FoxA accommodates structurally distinct ligands. Finally, we screened known large natural product antibiotics with no pseudomonal activity under nutrient limitation and discovered that the glycopeptide vancomycin inhibits growth by blocking peptidoglycan crosslinking. This pilot screen emphasizes the importance of screening for antibiotics under physiologically relevant conditions to avoid overlooking potential hits. Overall, the findings from these studies can be used to guide medicinal chemistry efforts to develop novel siderophore-antibiotic conjugates for the treatment of P. aeruginosa infections. These results also help us gain a deeper understanding of the mechanism of binding and uptake through siderophore transporters and the range of substrates that can be taken up. / Dissertation / Doctor of Philosophy (PhD) / Antibiotic resistance is a growing crisis that threatens modern medicine, and it is becoming more challenging to discover truly new antibiotics to combat this threat. Intrinsic resistance conferred by the outer membrane of Gram-negative bacteria restricts the entry of many antibiotics, especially larger antibiotics that would otherwise inhibit the growth of Gram-positive bacteria. Consequently, there are fewer treatment options for infections caused by Gram-negative bacteria and developing new antibiotics that can cross the outer membrane remains a significant challenge in drug discovery. My work describes the discovery of a class of antibiotics that can bypass the outer membrane using specific outer-membrane nutrient transporters. Using biochemical, structural biology, fluorescence microscopy, and molecular biology techniques, we uncover the molecular determinants of uptake of these antibiotics for their respective transporters. These results can inform the design of novel narrow-spectrum antibiotics that can overcome the outer membrane barrier to combat antimicrobial resistance.

Neisseria gonorrhoeae is an obligate human pathogen that causes the common STI gonorrhea, which presents a growing threat to global health. The WHO estimated 78 million new cases of gonorrhea worldwide in 2017, with estimates of 820,000 new cases in the United States alone according to the CDC. High-frequency phase and antigenic variation inherent in N. gonorrhoeae, coupled with its natural ability to rapidly acquire and stably integrate antimicrobial resistance factors into its genome, have culminated in an infection against which there is no effective vaccine, and for which the list of viable therapeutic options is quickly shrinking. Moreover, no protective immunity against subsequent infections is elicited upon exposure to N. gonorrhoeae, which highlights the need for research of novel antimicrobial and vaccination strategies. Within the human host, N. gonorrhoeae utilizes a unique strategy to overcome host sequestration of essential nutrients – termed nutritional immunity (NI) – such as ions of trace metals. The pathogen produces a family of outer membrane proteins called TonB-dependent transporters (TdTs) capable of binding to host NI factors and stripping them of their nutritional cargo for use by the pathogen. Importantly, these TdTs are very highly conserved and expressed among Neisseria species. TbpA is a well-characterized TdT that allows N. gonorrhoeae to acquire iron from human transferrin, and recent studies from our lab have shown that TdfH is capable of binding to a zinc-sequestering S100 protein called calprotectin and stripping it of its zinc ion. The S100 proteins are EF-hand calcium-binding proteins that naturally play an integral role in Ca2+ homeostasis, but due to their ability to bind transition metals, they have also demonstrated an innate immunity role by participating in nutrient sequestration. The S100 proteins are expressed in all human cells, and all are capable of binding transition metals including zinc, manganese, and cobalt. Calprotectin, S100A7, and S100A12 have demonstrated an ability to hinder the infection potential of pathogenic E. coli, S. aureus, C. albicans, and various other pathogens via zinc sequestration. Herein, we demonstrate that N. gonorrhoeae is able to overcome this phenomenon and actually utilize these proteins as a zinc source in vitro. Furthermore, we identify S100A7 as the specific ligand for TdfJ, which utilizes this ligand to internalize zinc during infection. S100A7 growth support in vitro is dependent upon a functional TonB, TdfJ, and the cognate ABC transport system ZnuABC, and isogenic mutants incapable of producing znuA or tdfJ recover S100A7 utilization by complementation. Whole-cell binding assays and affinity pulldowns show that S100A7 binds specifically to both gonococcal and recombinant TdfJ, and growth and binding experiments show that these described phenomena are specific to human and not mouse S100A7. Finally, we show that a His-Asn double mutant S100A7 that is incapable of binding zinc cannot be utilized for growth by gonococci. These data illustrate the unique nature of the gonococcus’ ability to co-opt host defense strategies for its own purposes, and further identify the TdTs as promising targets for strategies to combat and prevent gonococcal infection.

Pseudomonas is a diverse genus of Gram-negative bacteria that includes pathogens of plants, insects, and humans as well as environmental strains with no known pathogenicity. Pseudomonas fluorescens itself encompasses a heterologous group of bacteria that are prevalent in soil and on foliar and root surfaces of plants. Some strains of P. fluorescens suppress plant diseases and the genomic sequences of many biological control strains are now available. I used a combination of bioinformatic and phylogenetic analyses along with mutagenesis and biological assays to identify and compare the TonB-dependent outer-membrane proteins (TBDPs) of ten plant-associated strains of P. fluorescens and related species. TBDPs are common in Gram-negative bacteria, functioning in the uptake of ferric-siderophore complexes and other substrates into the cell. I identified 14 to 45 TBDRs in each strain of P. fluorescens or P. chlororaphis. Collectively, the ten strains have 317 TBDPs, which were grouped into 84 types based upon sequence similarity and phylogeny. As many as 13 TBDPs are unique to a single strain and some show evidence of horizontal gene transfer. Putative functions in the uptake of diverse groups of microbial siderophores, sulfur-esters, and other substrates were assigned to 28 of these TBDP types based on similarity to characterized orthologs from other Pseudomonas species. Redundancy of TBDP function was evident in certain strains of P. fluorescens, especially Pf-5, which has three TBDPs for ferrichrome/ferrioxamine uptake, two for ferric-citrate uptake and three for heme uptake. Five TBDP types are present in all ten strains, and putative functions in heme, ferrichrome, cobalamin, and copper/zinc uptake were assigned to four of the conserved TBDPs. The fluorescent pseudomonads are characterized by the production of pyoverdine siderophores, which are responsible for the diffusible UV fluorescence of these bacteria. Each of the ten plant-associated strains of P. fluorescens or P. chlororaphis has three to six TBDPs with putative roles in ferric-pyoverdine uptake (Fpv). To confirm the roles of the six Fpv outer membrane proteins in P. fluorescens Pf-5, I introduced deletions into each of the six fpv genes in this strain and evaluated the mutants and the parental strain for heterologous pyoverdine uptake. I identified at least one ferric-pyoverdine that was taken up by each of the six Fpv outer-membrane proteins of Pf-5. By comparing the ferric-pyoverdine uptake assay results to a phylogenetic analysis of the Fpv outer-membrane proteins, I observed that phylogenetically-related Fpv outer-membrane proteins take up structurally-related pyoverdines. I then expanded the phylogenetic analysis to include nine other strains within the P. fluorescens group, and identified five additional types of Fpv outer-membrane proteins. Using the characterized Fpv outer-membrane proteins of Pf-5 as a reference, pyoverdine substrates were predicted for many of the Fpv outer-membrane proteins in the nine other strains. Redundancy of Fpv function was evident in Pf-5, as some pyoverdines were recognized by more than one Fpv. It is apparent that heterologous pyoverdine recognition is a conserved feature, giving these ten strains flexibility in acquiring iron from the environment. Overall, the TBDPs of the P. fluorescens group are a functionally diverse set of structurally-related proteins present in high numbers in many strains. While putative functions have been assigned to a subset of the proteins, the functions of most TBDPs remain unknown, providing targets for further investigations into nutrient uptake by P. fluorescens spp.. The work presented here provides a template for future studies using a combination of bioinformatic, phylogenetic, and molecular genetic approaches to predict and analyze the function of these TBDPs. / Graduation date: 2012

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.