Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Gram-positive bacteria are characterized by a single lipid bilayer with a thick peptidoglycan layer. This group of organisms contains bacteria commonly associated with human infection, including: Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis and Streptococcus pneumoniae among others. These bacteria have a common system for importing iron in the form of heme, which is acquired by proteins containing heme-binding NEAT (NEAr iron Transporter) domains. The heme acquisition system in S. aureus is termed the Iron Surface Determinant (Isd) system and in L. monocytogenes is termed Heme Binding Protein (Hbp) and Heme/Hemoglobin Uptake Protein (Hup). These proteins work together to obtain heme from hemoglobin and then transport the heme into the cytoplasm via well characterized ABC-transporters. Although there have been clinical trials with antibodies directed against Isd proteins, there are currently no antibiotics targeting iron uptake systems in bacteria in general. Building upon fluorescent approaches for detection of iron uptake in Gram negative organisms, this work develops fluorescent heme acquisition detection in Gram positive organisms. The spectrofluorimetric methodology facilitates the understanding of heme acquisition protein interactions and mechanisms in bacteria. This work could subsequently be used to identify inhibitors of Gram positive bacterial iron uptake systems, and develop a new target for antibiotic action.

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.

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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.

Les colicines sont des toxines protéiques sécrétées par Escherichia coli ou des espèces apparentées. Leur mécanisme d'action se décompose en plusieurs étapes impliquant des domaines structurellement distincts de la toxine : le domaine central interagit avec un récepteur spécifique de membrane externe, le domaine N-terminal est transloqué à travers la membrane externe via un translocateur, puis transite dans le périplasme et le domaine C-terminal porte l’activité létale. L’étape de transit des colicines dites du groupe A implique l'interaction du domaine N-terminal de la colicine avec les protéines du système Tol. Ce système est formé de cinq protéines : TolQ, TolR, TolA, TolB et Pal. Le système TonB, composé des protéines TonB, ExbB et ExbD, est quant à lui parasité par les colicines dites du groupe B. La combinaison de techniques in vivo et in vitro, nous a permis de mettre en évidence pour la première fois l’interaction d’une colicine avec la protéine TolQ. Nous avons également montré que le clivage protéolytique de la protéine TolA, une protéine clé du système Tol, contrôle les interactions séquentielles engagées entre les colicines du groupe A et les composants de leur machinerie d’import chez E. coli. La colicine interagit avec TolB, puis TolA et finalement avec TolR et/ou TolQ. Nous avons également pu attribuer un rôle à la protéase FtsH dans ce mécanisme de dégradation. Parallèlement, nous avons entrepris de caractériser la colicine TonB-dépendante appelée colicine M (ColM), la seule colicine connue à ce jour capable de perturber la synthèse de peptidoglycane et dont l’activité nécessite la présence de la protéine périplasmique FkpA, un chaperon possédant une activité peptidyl-prolyl isomérase. Nous avons proposé une nouvelle approche pour étudier la ColM et délimiter plus précisément ses domaines afin d’identifier la séquence minimale requise pour sa toxicité. Nous avons montré que dans E. coli, la production périplasmique de la ColM (sp-ColM) est toxique et que son activité dépend de FkpA. Le domaine minimal requis pour cette toxicité correspond aux 153 derniers acides aminés C-terminaux deColM. Contrairement à la ColM entière, la toxicité de ce domaine C-terminal dans le périplasme d’ E. coline requiert pas FkpA.L’ensemble des données montrent que les colicines sont capables de parasiter des systèmes bactériens pour pénétrer dans la cellule et aussi de détourner la fonction physiologique de certaines protéines pour atteindre leurs cibles. / Colicins are toxin proteins secreted by Escherichia coli or related bacteria species. The actionmechanism of the colicins can be divided into several steps that involve distinct structural domains: thebinding of its central domain to an outer membrane specific receptor, the translocation of its N-terminaldomain through the outer membrane, the transit of this same domain through the periplasm and the lethalactivity carried by the C-terminal domain. The transit step of the group A colicins requires the interactionof colicin N-terminal domain with the Tol system which is composed of five proteins: TolQ, TolR, TolA,TolB and Pal. The TonB system, composed of TonB, ExbB and ExbD, is parasitized by the group Bcolicins. Using a combination of in vitro and in vivo experiments, we identified for the first time aninteraction between a colicin and the TolQ protein. We have also shown that the proteolytic cleavage ofthe TolA protein, a key protein of the Tol system, controls the sequential interactions of the group Acolicins with the components of their import machinery in E. Coli and we assigned a role to FtsH proteasein this degradation mechanism. We defined that the colicin interacts first with the TolB protein, then withTolA, and finally with TolR and/or TolQ.In parallel, we undertook the characterization of the TonB-dependent colicin M (ColM), the only colicinknown to be able to disrupt the peptidoglycan synthesis and that requires for its toxic activity the presenceof FkpA, a chaperone and peptidyl propyl isomerase protein located in the periplasm. We proposed a newapproach to investigate the in vivo activity of ColM designed to identify the different domains of ColMand the minimal sequence that retains toxic activity. We have shown that in E. coli, the periplasmicproduction of ColM is toxic and that its activity is FkpA dependent. The minimal domain required fortoxicity corresponds to the C-terminal last 153 amino acids of ColM. Unlike the full-length protein, thisdomain produced in the E. coli periplasm does not require FkpA for toxic activity.All these data show that colicins are able to parasitize bacterial systems to enter the cell and also to divertthe physiological function of certain proteins to achieve their targets

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.

Porphorymonas gingivalis is one of the major bacterial pathogens responsible for the initiation and progression of periodontal disease. The bacterium requires hemin uptake for its growth and has developed sophisticated mechanisms to extract hemin from hemin containing proteins in the oral cavity. Hemin first binds to receptors on the surface of P. gingivalis and is then taken up in an energy dependent manner. TonB is an inner membrane bound protein that spans the periplasm and is believed to be involved in the passage of hemin through the double membrane of P. gingivalis. However, the TonB protein in P. gingivalis is yet to be identified. We identified PG0785 as a possible P. gingivalis TonB based on its bioinformatics data showing similarity to other known TonB proteins. We generated a P. gingivalis mutant lacking a functional PG0785 and then characterized the mutant to determine the role of PG0785. We performed metal content and protease assays, virulence studies and transcriptional and translational analysis of our mutant and wild type P. gingivalis strains. Phenotypic studies showed that the mutant cannot accumulate hemin on its surface. The mutant has significantly lower levels of iron compared to wild type based on metal content assays. The mutant also has significantly lower protease activity compared to the wild type. Virulence studies showed that the mutant interacted and invaded eukaryotic cells at much lower levels than the wild type. These results allowed us to speculate that PG0785 is very important in binding of hemin to surface of P. gingivalis. PG0785 also plays an important role in iron uptake, protease activity and virulence of P. gingivalis. Transcriptional and translational analyses have shown that numerous TonB related genes, metal uptake genes, hemin uptake genes and genes related to virulence have been differentially regulated in the mutant lacking a functional PG0785 gene compared to the wild type strain. In conclusion we believe that based on our results PG0785 is a putative P. gingivalis TonB protein that plays a significant role in the biology of P. gingivalis.

Rhizobium leguminosarum is a Gram negative nitrogen-fixing soil bacterium. Due to the limited bioavailability of iron, bacteria utilize siderophores that scavenge and bind available iron. The transport of iron-siderophore complexes is achieved by the TonB-ExbB-ExbD complex. We have previously shown that a functional TonB protein is necessary for iron transport by creating ΔtonB mutants and assessing their growth and 55Fe-siderophore transport ability. We attempted to identify and characterize the roles of putative exbB and exbD genes using a similar approach. Growth curves and sequence analyses suggest putative exbB and exbD may be the tolpal-associated genes tolQ and tolR. Phenotypic and sensitivity assays showed mutants do not exhibit the characteristic tol phenotype and are not sensitive to detergents or changes in ionic strength of the growth medium. We also expressed and purified the 120 amino acid fragment of the TonB C-terminus for further physical and chemical characterization.

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.