Thesis (honors)--Georgia State University, 2007. / Title from file title page. Under the direction of Dabney White Dixon. Electronic text (88 p. : col. ill.) : digital, PDF file. Description based on contents viewed Sept. 30, 2008. Includes bibliographical references (p. 46-47).

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on January 7, 2009). Research advisor: John D. Schuetz, Ph.D. Document formatted into pages (xi, 113 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 92-113).

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 29-34.

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ATP-binding cassette (ABC) transporters use the power of ATP binding and hydrolysis to deliver a wide variety of molecules across membrane bilayers. Crystal structures in the past two decades have provided snapshots of these transporters under various conditions, which revealed conformational changes of transporters upon substrate and ATP binding along the transport pathway. However, understanding of kinetics of substate translocation and the knowledge of transient intermediates along the transport pathway remain primitive, especially for Type II ABC importers. In this thesis work, I employed fluorescence resonance energy transfer (FRET) to study the transport mechanism of BtuCD, a model type II ABC importer that transports vitamin B12. I also exploited the photophysical property of the transport substrate, B12, that quenches the fluorescence of certain fluorophores nearby through FRET, which enables the monitoring of B12 binding and release. Our ensemble FRET data showed the binding of B12 and nucleotide both weaken the interaction between BtuCD and BtuF. Our single-molecule FRET (smFRET) experiments not only revealed stepwise movement of substrate molecule through the transporter in real-time and at the single-molecule level, but also yielded the rates of transition between individual conformational states, which had not been previously characterized in any other transporters. The results showed that ATP hydrolysis, instead of ATP binding, drives the power-stroke for the transport cycle. They also showed two sequential ATP hydrolysis events are required to complete a transport cycle, with the first ATP hydrolysis event delivering B12 into the cavity of BtuCD and the second resetting the BtuCD-F complex for a new cycle of transport.

ABC transporters consist of two transmembrane domains (TMDs) that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize transport via ATP binding and hydrolysis. Using site-directed mutagenesis, the role of highly conserved residues in the NBDs of Abcb1a was investigated. / In both NBDs of Abcb1a the A-loop aromatic residue is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine (618 in NBD1 and 1263 in NBD2) also appears to lie close to the ATP molecule. These four tyrosine residues were mutated to tryptophan and the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis was analyzed. Y618W and Y1263W enzymes had catalytic characteristics similar to wild-type (WT) Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including an ∼10-fold increase in KM(ATP). Thus, Y397 and Y1040 play an important role in Abcb1a catalysis. / Since it was speculated that ABC transporters utilize a catalytic base to hydrolyse the beta-gamma phosphodiester bond of ATP, a search for that residue was undertaken. Six pairs of highly conserved acidic residues in the NBDs of Abcb1a were investigated. Removal of the charge in D558N and D1203N as well as in E552Q and E1197Q produced enzymes with severely impaired transport. These mutants were purified and characterized with respect to ATPase activity. Mutants D558N and D1203N retained some drug-stimulated ATPase activity and vanadate (Vi) trapping of 8-azido-[alpha32P]nucleotide confirmed slower basal and drug-stimulated hydrolysis. The E552Q and E1197Q mutants showed absence of ATPase activity but Vi trapping of 8-azido-[alpha 32P]nucleotide was observed, at a level similar to that of WT Abcb1a. Photolabelling by 8-azido-[alpha32P]nucleotide, in the presence or absence of drug, was also detected in the absence of Vi. The ATPase activity, binding affinity, and trapping properties of these glutamate residues were further analyzed. In addition to the E→Q mutants, the glutamates were individually mutated to D, N, and A. The double mutants E552Q/E1197Q, E552Q/K1072R, and K429R/E552Q were also analyzed. The results obtained suggest that 1) the length of the side-chain is important for the catalytic activity, whereas the charge is critical for full turnover to occur, 2) formation of the catalytic transition state does occur in the mutant site in the single-site mutants, suggesting that E552 and E1197 are not classical catalytic carboxylates, 3) steps after formation of the transition state are severely impaired in these mutant enzymes, 4) NBD1 and NBD2 are functionally asymmetric, and 5) the glutamates are involved both in NBD-NBD communication and transition-state formation through orientation of the linchpin residue.

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ABCG2 est un transporteur de la famille ABC impliqué dans le phénotype de résistance aux drogues développé par certaines cellules, par exemple les cellules cancéreuses. Ce transporteur a aussi un rôle physiologique de détoxication de composés endogènes, notamment les porphyrines, molécules indispensables mais qui présentent une toxicité potentielle. Cette toxicité nécessite une prise en charge particulière, évitant à ces composés d’être libres en solution. Dans ce contexte, nous avons fait l’hypothèse qu’ABCG2 pourrait participer à cette détoxication en limitant l’accumulation des porphyrines dans les cellules en les présentant à un partenaire extracellulaire. Nous montrons qu’ABCG2 transporte de l’hème ainsi que certains de ses dérivés et précurseurs et que ces porphyrines, contrairement aux autres substrats d’ABCG2, se fixent sur un domaine extracellulaire spécifique d’ABCG2, ECL3, composé d’environ 70 acides aminés. L’affinité d’ECL3 pour les porphyrines est de 0,5 à 3,5 μM, suffisamment affine pour permettre leur fixation après transport.Nous montrons aussi que l’albumine sérique humaine, impliquée dans la détoxication de l’hème, récupère les porphyrines fixées sur ECL3 par une interaction directe avec ABCG2. L’ensemble de ce travail a donc permis d’une part de mieux comprendre le rôle d’ABCG2 dans la régulation de l’homéostasie des porphyrines, notamment l’hème, et d’autre part, de façon originale, d’identifier le mécanisme moléculaire par lequel cette détoxication s’effectue. / ABCG2 belongs to the ABC-transporter family, involved in drug resistance developed by cells, notably cancer cells. This transporter has also a physiological role of endobiotic detoxification, in particular porphyrins that are essential but potentially toxic molecules. This toxicity implies a specific handle, to avoid them to remain free in solution. In that context, we hypothesized that ABCG2 participate to this detoxification, limiting the intracellular porphyrin accumulation by presenting them to an extracellular partner. We show that ABCG2 transports heme and some of its derivatives and precursors. Interestingly, these porphyrins, unlike other ABCG2 (non-porphyric) substrates, can bind to an extracellular domain, specific of ABCG2, ECL3, 70 residues-long. ECL3 displays affinities for porphyrins in the range of 0.5 to 3.5 μM, high enough to allow their binding after transport. We also show that human serum albumin, implicated in heme detoxification, releases porphyrins bound to ECL3 by a direct interaction with ABCG2. This work established a better comprehension of ABCG2 role in porphyrin and in particular heme homeostasis regulation. In addition, our results contribute to elucidate part of the molecular mechanism by which such regulation is carried out.

La plasticité génomique de Staphylococcus aureus lui permet d’acquérir des gènes codant des facteurs de virulence mais aussi des gènes de résistance aux antibiotiques, notamment la cassette de résistance à la méticilline (SCCmec). La résistance à la méticilline est d’abord apparue dans des souches hospitalières à l’origine de grands clones pandémiques nosocomiaux, puis a ensuite émergé en milieu communautaire chez des souches virulentes possédant la leucocidine de Panton-Valentine. Nous avons observé en 2002, en milieu hospitalier et communautaire, l’émergence inquiétante de S. aureus résistant à la méticilline (SARM) portant le gène tst de la toxine du choc toxique staphylococcique (TSST-1). Notre travail a consisté à : (i) l’élaboration de nouveaux outils contribuant à la description de clones de SARM, notamment d’un outil de typage de la cassette SCCmec, (ii) la caractérisation phénotypique et moléculaire de ce nouveau clone, (iii) l’étude de l’épidémiologie de ce clone, (iv) l’exploration de la pathogénie de ce clone en recherchant les propriétés génétiques qui lui confèrent une telle virulence et épidémicité, et notamment en identifiant le rôle de la TSST-1. Le clone de SARM tst+ est un clone épidémique de fond génétique ST5 en Multi Locus Sequence yping, atteignant principalement les sujets jeunes, et responsable d’infections variées à la fois toxiniques et suppuratives. Il est doté d’une cassette SCCmec atypique de type I tronquée dont le rôle dans le potentiel épidémique et de virulence de ce clone reste à déterminer. Enfin, la TSST-1 ne semble pas jouer un rôle déterminant, direct ou indirect, dans la pathogénie pléiotropique de ce clone / The plasticity of the Staphylococcus aureus genome confers to this specie the ability to gain accessory genes encoding virulence factors as well as antibiotic resistance determinants such as Staphylococcal cassette chromosome mec SCCmec that encodes methicillin-resistance. Methicillin resistance first emerged in hospital-acquired strains originally of successful nosocomial pandemic clones, and is also risen in virulent community-acquired strains containing the Panton-Valentine leucocidin. In 2002, we observed the worrying emergence of a new methicillin-resistant S. aureus (MRSA) clone that contains the tst gene encoding toxic shock syndrome toxin (TSST-1) responsible for both hospital and community-acquired infections. Our study was focused on: (i) new tools for MRSA description, mainly on a SCCmec typing tool, (ii) the characterisation of this new clone by molecular and phenotypic methods, (iii) the epidemiology of this clone, (iv) the pathogenic potential of this clone and the investigation of the genetic features that enhance transmission and virulence, particularly the role of TSST-1. The tst+ MRSA clone is an epidemic clone of genetic background ST5 by multilocus sequence typing that occurs mainly in young people and is responsible for a wide diversity of clinical syndromes including toxin-mediated and suppurative diseases. This clone harbours a peculiar cassette “truncated SCCmec type I” which could play an important role in virulence and transmission but are still under investigation. TSST-1 does not play a determining role (either directly or indirectly) in the pleiotropic pathogenesis of this clone