Les méthionine sulfoxyde réductases (Msr) sont des enzymes ubiquitaires impliquées dans la résistance au stress oxydant, les processus de vieillissement mais également dans la virulence de certaines bactéries pathogènes. Deux classes de Msr : MsrA et MsrB, structuralement distinctes, catalysent respectivement la réduction des stéréoisomères S et R de la fonction sulfoxyde de la méthionine sulfoxyde selon un même mécanisme catalytique en trois étapes impliquant la formation d’un intermédiaire acide sulfénique suivie de celle d’un pont disulfure intramoléculaire ensuite réduit par la thiorédoxine (Trx). Des études de relation structure-fonction, ont permis 1) de caractériser les résidus du site actif de la MsrB de Neisseria meningitidis (N. meningitidis) impliqués dans la reconnaissance du substrat, et dans la catalyse de l’étape réductase conduisant à la formation de l’intermédiaire acide sulfénique, et de proposer un scénario pour la catalyse de l’étape réductase dans lequel le résidu His 103 joue un rôle majeur de catalyseur acide / base; 2) de caractériser le mécanisme des autres sous-classes de MsrB, qui diffèrent de la sous-classe représentée par la MsrB de N. meningitidis par l’absence de la Cys de régénération en position 63, notamment celui de la MsrB de Xanthomonas campestris, qui possède une Cys de régénération en position 31 située dans une boucle flexible ; et 3) de caractériser la protéine PilB de N. meningitidis, protéine à trois domaines, localisée dans le périplasme portant non seulement les activités MsrA et MsrB mais aussi une activité disulfure oxydoréductase au niveau de son domaine N-terminal dont le rôle est de régénérer les activités Msr. / Ubiquitous enzyme methionine sulfoxide reductases (Msrs) are involved in oxidative stress resistance, aging process but also in bacteria pathogenicity like for Neisseria genius. The two Msrs classes: MsrA and MsrB structural-unrelated catalyze the reduction of the two stereoisomeric forms R and S of the sulfoxide function from the methionine sulfoxide. They share a similar three-step chemical mechanism including formation of a sulfenic acid intermediate following by intramolecular disulfide bond formation, reduced in the last step by the thioredoxin (Trx). The structure function studies are conduced to 1) characterization of active site amino acids involved in substrate recognition and reductase step catalysis leading to sulfenic acid formation in Neisseria meningitidis (N. meningitidis) MsrB, we have proposed a scenario for the reductase step with a major role of the acid / base catalyst His 103 2) characterization of different MsrB sub-classes mechanisms, Xanthomonas campestris MsrB present a Cys 31 located in a flexible loop compare to the Cys 63 from N. meningitidis MsrB also located in a flexible loop, the Mycoplasma pulmonis MsrB don’t posses recycling Cys; and 3) characterization of the N. meningitidis PilB, a three domains protein located in the periplasm, PilB possess MsrA and MsrB activities, and a oxydoreductase activity carried by the N-terminal domain, moreover this domain can reduced the oxidized MsrA and MsrB domains.

Les résidus méthionine sont facilement oxydables en sulfoxydes in vivo. Cette oxydation est réversible via la méthionine sulfoxyde réductase (Msr), un enzyme ubiquitaire. Dû aux deux configurations possibles du sulfoxyde, deux classes d'enzyme structuralement différents existent : les MsrA sont spécifiques de l'isomère S, et les MsrB de l'isomère R. Les deux possèdent un même mécanisme catalytique en deux étapes. La première étape est dédiée à la réduction du substrat ; elle aboutit à l’oxydation d’une cystéine en acide sulfénique. Puis le recyclage de l’enzyme s’opère avec la formation d’un pont disulfure intramoléculaire qui est finalement réduit par une thiorédoxine (Trx). Chez Neisseria meningitidis la protéine PilB porte MsrA et MsrB sous forme de deux domaines. Un troisième domaine à activité du type Trx existe en N-terminal. Les trois domaines isolés ont été étudiés par cristallographie. (i) Le domaine N-terminal : la structure confirme son homologie avec la Trx, mais son analyse révèle des éléments probablement à l’origine d’un fonctionnement particulier. (ii) La MsrA : la structure de deux mutants a permis d’observer un complexe avec un substrat et l’acide sulfénique. Leurs études s’additionnent à celles de l’enzyme sauvage sous formes réduite et oxydée. (iii) La MsrB : la structure d’un mutant a également permis d’obtenir un complexe et complète les structures des formes réduite et oxydée du sauvage. En addition, la structure de la MsrB de Xanthomonas campestris met à jour des différences conformationnelles entre MsrB d’organismes différents. Enfin, l’étude structurale de PilB entier a été entamée en solution par la méthode de diffusion aux petits angles. / Methionine residues are easily oxidized to sulfoxides, in vivo. This oxidation is reversed via a ubiquitous enzyme named methionine sulfoxide reductase (Msr). Due to the two possible configurations of the sulfoxide group, two structurally-different classes of enzymes exist: MsrAs are specific for the isomer S, and MsrBs for the isomer R. Both classes act through a two-step mechanism. The first step is dedicated to substrate reduction. It results in the sulfenic form of the catalytic cysteine. Then recycling of the enzyme starts with the formation of an intramolecular disulfide bridge, finally reduced by thioredoxin (Trx). In Neisseria meningitidis, the protein PilB bears MsrA and MsrB as two adjacent domains. A third domain with a Trx-like activity exists at the N-terminal end. The three isolated domains have been studied by X-Ray crystallography. (i) The N-terminal domain: its structure confirmed its homology to Trx and DsbEs, but its analysis revealed elements that probably explain its peculiar properties. (ii) MsrA: the structures of two mutants allowed the observation of a complex with the substrate and of the sulfenic acid. These results add to the structure of the reduced and oxidized forms of the wild type domain so that the catalytic mechanism can be analyzed. (iii) MsrB: the structures of the reduced and oxidized forms were completed by that of a complex with the substrate obtained from a mutant. In addition, comparison with the Xanthomonas campestris MsrB structure enlightened conformational differences between MsrBs from distinct organisms. Finally, structural studies of the whole PilB protein have been initiated in solution using small angle X-Ray scattering.

Nous montrons que la structure RMN, en solution, du domaine N-terminal de DsbD de Neisseria meningitidis (nDsbD) présente, à l’état réduit, un repliement de type immunoglobuline avec un site actif adoptant une conformation fermée. Toutefois, l’analyse des mouvements internes du squelette peptique de nDsbD montre que la région dite « couvercle » de la protéine et qui protège les résidus actifs dans les formes réduite et oxydée, est dotée de mouvements internes. Cela démontre les capacités intrinsèques d’ajustement structural de nDsbD. Nous montrons aussi que les structures RMN, en solution, du domaine N-terminal de PilB de N. meningitidis (NterPilB) sous ses deux formes, réduite et oxydée, présentent un repliement de type thiorédoxine. Ces deux formes, très proches d’un point de vue structural, apparaissent comme étant globalement rigides. Par conséquent, la boucle FLHE, caractéristique de NterPilB et bordant le site actif de la protéine, ne dévoile pas de nouveaux indices structuraux et/ou dynamiques traduisant son implication dans la spécificité de substrat. Finalement, nous montrons, grâce à l’étude structurale et dynamique, en solution, d’un complexe entre nDsbD et NterPilB de N. meningitidis, que nDsbD fait preuve d’une grande adaptabilité à l’état complexé. La région « couvercle » s’ouvre pour venir se positionner au dessus de l’hélice a qui contient les cystéines actives de NterPilB. Par contre, la boucle FLHE de NterPilB ne semble pas intervenir dans la stabilisation du complexe. Nous proposons que des phénomènes dynamiques puissent faciliter d’une part, l’adaptabilité relative des deux partenaires dans le complexe, et d’autre part, la dissociation finale de ces derniers / We show, on one hand, that the NMR solution structure of DsbD N-terminal domain from Neisseria meningitidis (nDsbD) displays, in its reduced state, an immunoglobulin fold with a closed conformation of its active site. Nonetheless, our backbone dynamics study shows that the cap-loop region of the protein, which covers active residues in both oxidized and reduced forms, displays internal motions. This illustrates the inner structural adjustment capacities of nDsbD. On the other hand, we show that NMR solution structures of the oxidized and reduced forms of N. meningitidis NterPilB display a thioredoxin-like fold. These two structures appear to be very similar and globally rigid. Consequently, the NterPilB characteristic FLHE loop, which covers one edge of the active site, does not reveal new structural and/or dynamics properties for its involvement in the substrate specificity. Finally, we point out, from the structural and dynamics study of a complex between nDsbD and NterPilB from N. meningitidis, that nDsbD exhibits a powerful adaptability in its complex state. Its cap-loop region opens and comes over the a helix containing the NterPilB active cysteines. In contrast, the NterPilB FLHE loop does not seem to play a role in the complex stabilization. We propose that internal dynamics should facilitate, on one hand, the relative adaptability between the two partners of the complex and, on the other hand, their subsequent dissociation

Disease caused by antibiotic resistant (ABR) bacteria has become a widespread global public health issue as humanity's existing collection of effective antibiotics dwindles. ABR bacteria are responsible for approximately 5 million deaths worldwide annually, which is predicted to reach 10 million yearly by 2050. Antivirulence therapeutics have been explored in recent times as another approach to tackling the global ABR pandemic by disrupting the function of virulence factors that promote disease development. The bacterial type IV pilus (T4P) is a prevalent virulence factor in many ABR pathogens, contributing to bacterial pathogenesis by facilitating cell motility, surface adhesion, and biofilm formation. Critically, the T4P facilitates early stages of disease, providing a means to invade and colonize a host. T4P assembly is driven by the PilB/PilF motor ATPase that localizes to the cytoplasmic face of the inner membrane to drive pilus biogenesis by ATP hydrolysis. The thesis work here explores computational and experimental methods for the discovery of antivirulence therapeutics targeting the T4P assembly ATPase PilB. A computational model of Chloracidobacterium thermophilum PilB was generated by homology modeling and molecular docking was performed to analyze the binding characteristics of six anti-PilB inhibitory compounds identified in previous studies. Computational docking aligns with the existing body of work and reveals important protein-ligand interactions and characteristics, particularly involving the ATP binding domain of PilB. This work supports the use of PilB in structure-based virtual screening to identify novel compounds targeting PilB. Additionally, through heterologous expression and chromatography methods, the ATPase core of Neisseria gonorrhoeae PilF was successfully expressed and purified as an active ATPase. This work optimized conditions for its ATPase activity in vitro. Additionally, this thesis documents the experimental attempt to express and purify Clostridioides difficile PilB as an active ATPase. Two of the seven C. difficile PilB variant proteins expressed led to soluble protein while one construct remains to be explored. The results of these studies provide insight for future methodology design for antivirulence therapeutic research targeting the T4P assembly ATPase using both in silico and in vitro methods. / Master of Science / Antibiotic resistant bacterial infections are responsible for nearly 5 million deaths worldwide every year. These infections are becoming increasingly more difficult to treat as bacterial pathogens acquire greater means to overcome our dwindling antibiotic repertoire. This has prompted researchers to explore alternative therapeutic strategies, including the antivirulence approach that aims to disable the function or production of bacterial virulence factors. Virulence factors serve as arms and armor that help bacteria cause disease, but they may be disrupted in such a way that renders potentially pathogenic bacteria harmless to humans. One major virulence factor in many antibiotic resistant bacteria is the type IV pilus (T4P), which is important in the early stages of host invasion by mediating adhesion and biofilm formation. This work explores both computational and experimental strategies to antivirulence drug discovery targeting the T4P, specifically the primary motor protein PilB/PilF. Newly identified PilB inhibitors were evaluated by molecular docking and molecular dynamics simulation to assess the use of PilB for drug discovery via virtual screening in silico. This revealed key characteristics and protein-ligand interactions that contribute to successful PilB inhibition and supports the use of CtPilB for structure-based virtual screening. Additionally, the PilF motor protein from Neisseria gonorrhoeae was successfully purified and demonstrated to be active for inhibitor discovery in the future. This work also covers efforts to establish Clostridioides difficile PilB as potential model enzyme for inhibitor discovery in the future.

Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors. / Doctor of Philosophy / Bacteria can be motile or sessile. Motile bacteria can use hair like structures on their surface, called pili, to move in their natural environment, whereas sessile bacteria produce intricate structures attached to solid surfaces known as biofilms. Bacteria are able to switch between being motile and sessile states depending on their environment conditions. However, it isn't clear how this switch is controlled in bacteria that use pili to move. To answer this question, we studied how PilB the protein that assembles pili, might control this switching process. We specifically investigated PilB because it has two known roles. The first is that it can assemble pili, to enable pili-mediated motility. The second is that it can stimulate or promote biofilm formation. This places PilB at the intersection of these two lifestyles, suggesting that this protein may play a key role in deciding whether a bacterium is to be motile or sessile. Another important reason to understand how PilB functions is because pili are used by some antibiotic resistant pathogenic bacteria. Since PilB is essential for the formation of pili, if the actions of PilB could be blocked, bacteria would be unable to make pili. This could stop bacteria from causing disease. By searching for new chemicals which stop PilB from creating pili, we can potentially identify new drugs to treat bacterial infections.

The type IV pilus (T4P) is a dynamic long thin fiber found on the surface of many bacterial groups. T4P is a versatile nanomachine; it plays many important roles such as for surface attachment, virulence factor, and surface motility apparatus. This research focuses on understanding the kinetics of PilB, the T4P assembly ATPase. PilB crystal structure exhibits an elongated hexamer with 2-fold symmetry indicating a symmetric rotary mechanism model. Except for its structure, the symmetric rotary mechanism of PilB has not been demonstrated experimentally. Its conformation and relatively low activity constrained previous in vitro studies of PilB. This study identified PilB from thermophilic organism Chloracidobacterium thermophilum (Ct) to be a model for in vitro studies. An active CtPilB was successfully expressed and purified as a hexamer. Malachite green phosphate assay was used to examine CtPilB ATPase activity. The examination indicated that CtPilB is a robust ATPase with a complex kinetics profile. The profile has a stepwise incline in ATPase activity as a function of [ATP] that led to a decline in higher [ATP]. The decline was confirmed to be a substrate inhibition by the enzyme-coupled assay. As for the incline, the detailed mechanism is still less clear to explain the multiphasic profile. The overall incline did not conform with classical Michaelis-Menten kinetic but the first part of the incline was shown to conform with Michaelis-Menten kinetics. The complex kinetics profile of PilB is consistent with the symmetric rotary mechanism of catalysis. / Master of Science

O pilus tipo IV (T4P) são finos e flexíveis filamentos encontrados na superfície de uma ampla gama de bactérias Gram-negativas, Gram-positivas e archaea. O T4P desempenha um rol crucial no estilo de vida bacteriano ao estar envolvido em uma variedade de funções incluindo motilidade, aderência, formação de biofilme, patogenicidade, transformação natural e na infecção por fagos. Várias das proteínas requeridas para a biossíntese e regulação do T4P se estendem através do periplasma conectado a membrana interna e externa. O T4P são estruturas dinâmicas que sofrem ciclos de extensão e retração energizados por duas ATPases associadas com a membrana interna bacteriana. Durante a extensão, PilB, a ATPase de biossíntese do T4P, estimula a polimerização do pilus a partir de monômeros de pilinas localizados na membrana interna, através de um mecanismo ainda desconhecido. Duas proteínas, FimX e PilZ estão envolvidas na regulação da biossíntese do T4P via interações com PilB e nocautes de esses genes acabam com a biogênese e função do T4P. Neste trabalho, nós determinamos a estrutura cristalográfica do complexo binário formado pelo domínio N-terminal de PilB (PilBNt, resíduos 12-163) e a PilZ com uma resolução de 1.7 Å. As interações entre PilB e PilZ envolve uma superfície hidrofóbica formada por aminoácidos altamente conservados na família não canônica de domínios PilZ. Mutações ou deleções de alguns destes resíduos em PilZ enfraquecem a interação PilB-PilZ e afeta a função do T4P. Nós também observamos que esta interação induz mudanças conformacionais no domínio PilBNt, revelando a possibilidade de um rearranjo estrutural funcionalmente relevante da região Nterminal de PilB permitindo a sua interação com PilM, conectando a ATPase PilB como a maquinaria do T4P. Nós mostramos que PilB, PilZ e FimX podem formar um complexo ternário estável com uma massa molar aparente de ~600 kDa, sugerindo uma estequiometria de 6PilB:6PilZ:2FimX. Também observamos que FimX incrementa a atividade ATPase do complexo PilB-PilZ. O c-di-GMP e o ATPγS (um análogo não hidrolisável do ATP) induz mudanças conformacionais em FimX e no complexo PilB-PilZ, respectivamente, e estabiliza o complexo ternário PilB-PilZ-FimX. Além disso, PilB, PilZ e FimX localizam em um dos polos da célula (polo líder) em células de X. citri e a localização polar dirige a orientação da motilidade twitching. Finalmente, o T4P é necessário para a exitosa infecção de X. citri pelo fago ΦXacm4-11. Nossos resultados sugerem que asinterações entre PilB-PilZ-FimX estariam envolvidas na regulação da função de PilB, onde sinais especificas sentidas pelos domínios de FimX seriam transmitidas por PilZ até PilB. / Bacterial type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria and play a crucial role in their lifestyles due to their involvement in a variety of functions including motility, adherence, biofilm formation, pathogenicity, natural transformation and phage infection. Several proteins required for the biogenesis and regulation of T4P span the periplasm connecting both the inner and outer membranes. T4P are dynamic structures that undergo cycles of extension and retraction powered by two hexameric ATPases associated with the bacterial inner membrane. During extensions, the T4P assembly ATPase PilB stimulates the polymerization of pilin monomers from the inner membrane, though the precise mechanism is unknown. Two proteins, FimX and PilZ are involved in the regulation of T4P biogenesis via interactions with the PilB and knockouts of these proteins abolish T4P biogenesis. Here, we determined the crystal structure of the binary complex made up of the PilB N-terminal domain (PilBNt, residues 12- 163) bound to PilZ at 1.7Å resolution. PilZ interactions with PilB involve a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains. Mutations or deletion of some these amino acids in PilZ weaken the PilZ-PilB interaction and affect T4P function. This interaction induces significant conformational changes in the PilBNt domain, suggesting that structural rearrangements of the PilB N-terminal domains could be important for its interaction with PilM, connecting the ATPase PilB with T4P machinery. We show also that full-length PilB, PilZ and FimX can form a stable ternary complex with apparent molecular weight of ~600 kDa, suggestive of a 6PilB:6PilZ:2FimX stoichiometry and that FimX increases the ATPase activity of the PilB PilZ complex. C-diGMP and ATPγS (non-hydrolysable analog of ATP) induce conformational changes in FimX and in PilB-PilZ, respectively, and stabilize the ternary PilB-PilZ-FimX complex. In addition, we show that PilB, PilZ and FimX localize at one cell pole (leading pole) that drives the movement in X. citri. Finally, the T4P is necessary for successful infection of X. citri cells by phage ΦXacm4-11. Our results suggest how FimXPilZPilB interactions could be involved in the regulation of PilB function, where specific environmental signals sensed by FimX domains could be transmitted via PilZ to PilB.

Les méthionine sulfoxyde réductases (Msr) permettent de restaurer la fonction des protéines oxydées sur leur résidus Methionine. Dans un premier temps, le mécanisme catalytique de la MsrA et de la MsrB de la protéine PilB de la bactérie pathogène Neisseria meningitidis a été étudié. Les deux classes de Msr, qui sont structuralement différentes, partagent un même mécanisme catalytique en trois étapes avec formation d'un intermédiaire acide sulfénique suivie de la formation d'un pont disulfure intra moléculaire qui est réduit par la thiorédoxine (Trx). Elles présentent en revanche une stéréospécificité de substrat inverse vis-à-vis de la fonction sulfoxyde. Dans un deuxième temps, les trois étapes du mécanisme catalytique de la MsrB ont été caractérisées au niveau cinétique. L'étude du rôle des acides aminés du site actif dans la catalyse, la caractérisation biochimique de l'interaction MsrB-Trx et, enfin, l'étude du rôle du métal coordiné ont également été abordées.