Type IV pili (T4P) are long, thin, flexible surface appendages used by various bacteria for surface adhesion, cell-cell aggregation, DNA uptake, biofilm formation and motility. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, and uses T4P as a key virulence factor to infect immunocompromised individuals. Four subcomplexes make up a functional T4P system in P. aeruginosa and the role of the alignment subcomplex is to physically connect the outer membrane pore with the inner membrane motor, allowing for efficient extrusion of the pilus fibre from the cell. Two alignment subcomplex proteins, PilN and PilO, form heterodimers and are required for proper function of the system. These proteins may be able to transduce signals between various T4P components to indicate extension and/or retraction of the pilus fibre. This thesis focused on characterization of the interaction interfaces between PilN and PilO, and on understanding the dynamics required for proper function of the system. We show that although PilN and PilO make extensive interaction contacts throughout their lengths, single point substitutions at key residues can successfully disrupt the function of the T4P system. Crosslinking PilN and PilO as homo- or heterodimers can disrupt motility and surface piliation, indicating that interfaces between these proteins must be dynamic to allow proper T4P function. A high resolution X-ray crystal structure of PilO was solved and exhibits new structural features previously unidentified. This work furthers our understanding of the structures and regions of interaction between PilN and PilO, as well as defining a role for these proteins in extension and retraction. / Dissertation / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is an opportunistic bacterium, able to infect individuals with weakened immune systems. It attaches to and moves along surfaces using long, thin, sticky, retractable fibres known as type IV pili. Similar to a grappling gun, a functional type IV pilus system requires four subcomplexes working in unison to allow for the extension, adherence, and retraction of pilus fibres, which pulls the cell forward towards the point of attachment. Two key proteins, PilN and PilO, are bound to each other and allow for efficient extension and retraction of the pilus fibre. This study focused on characterization of the interactions of PilN and PilO, and on understanding whether dynamic rearrangements of the interfaces between these proteins is required for proper function of the system. We show that although these proteins have extensive interaction interfaces, single residue substitutions in either of them can disrupt the ability of the bacteria to properly extend and/or retract their pili. This work furthers our understanding of the structures and regions of interaction between PilN and PilO, providing information that might allow disruption of these interfaces to block bacterial attachment or motility, both of which are important for infection.

Type IVa pili (T4aP) are long, retractile, filamentous, surface appendages involved in cellular surface adhesion, biofilm formation, DNA uptake, and a unique form of motility called ‘twitching’. They are a critical virulence factor in a number of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, a major cause of hospital-acquired infections. T4aP function is controlled by a number of different regulatory proteins and systems. A putative chemosensory system termed ‘Chp’, controls levels of the second messenger molecule cyclic adenosine monophosphate (cAMP). cAMP works with a cAMP receptor protein called Vfr to control expression of ~200 virulence genes, including those that are required to make T4aP. cAMP levels are regulated by proteins outside the Chp system, including the bitopic inner membrane protein, FimV. This study examines the role of the Chp system and FimV in T4aP regulation. Both proteins are required for regulation of cAMP levels, while the Chp system also has a cAMP-independent role in regulating twitching. FimV has been shown to regulate cAMP levels, possibly connecting to the Chp system through a scaffold protein, FimL. We present the structure of a conserved cytoplasmic region of FimV, and show that this region is required for connecting FimV to the Chp system. We also characterize the cAMP-independent role of FimV, confirming that it is distinct from that of the Chp system, and is involved in localizing T4P regulatory proteins. We also provide evidence that the cAMP-independent role of the Chp system is to mediate the balance between T4P extension and retraction, possibly through denoting the ‘front’ of a motile cell. Together, these data help to resolve the cAMP-independent and –dependent pathways controlling twitching motility. / Thesis / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is a bacterium that causes infection in people with weakened immune systems. One key factor it uses to cause infection is the type IVa pilus (T4aP), a filamentous appendage displayed on the cell surface. T4aP can repeatedly extend and retract, and are involved in attachment to host cells, and movement along surfaces. When T4aP cannot extend or retract, the bacteria cannot cause infection. Many proteins work together to control T4aP function – this study focuses on two of them. They have one overlapping function, controlling levels of a signalling molecule needed to make T4aP. We also show that they have a second, non-overlapping function. One is involved in controlling the extension/retraction balance, possibly by marking the front of a cell, while the other may localize pilus-related proteins within a cell. This work helps us understand how P. aeruginosa makes T4aP, and provides information helpful to understanding control of virulence.

Xylella fastidiosa é o agente etiológico de diversas doenças em plantas, incluindo a Clorose Variegada dos Citros (CVC), uma séria ameaça à indústria citrícola. Os níveis de transcritos sob diferentes disponibilidades de ferro foram medidos com microarranjos de DNA representando 2608 (91,6%) sequências codificadoras (CDS) da cepa 9a5c de X. fastidiosa. Na presença de 100 uM pirofosfato férrico, 218 e 256 CDS foram consideradas como reguladas positiva e negativamente, respectivamente. Quando tratada com o quelante de ferro 2,2\'-dipiridil, 193 CDS foram consideradas como reguladas positivamente e 216 negativamente. A expressão diferencial de um subconjunto de 44 CDS foi também avaliada por RT-qPCR, que mostrou uma correlação de Pearson de 0,77 com os resultados dos microarranjos. As CDS diferencialmente expressas nas variações de concentração de ferro participam em diversas funções celulares. Muitas CDS envolvidas com funções regulatórias, patogenicidade e estrutura celular foram moduladas em ambas as condições testadas, sugerindo que grandes mudanças na arquitetura celular e metabolismo ocorrem quando células de X. fastidiosa são expostas a variações extremas na concentração de ferro. Interessantemente, as CDS moduladas incluem as de síntese e secreção de bacteriocinas similares à colicina tipo V e funções ligadas a formação de pilus e fímbria. Nós também investigamos a contribuição do regulador transcricional Fur no stimulon do ferro em X. fastidiosa. Para tal, as regiões promotoras do genoma da cepa 9a5c foram varridas em busca de Fur boxes putativas. Nossas análises identificaram que regiões promotoras de 49 CDS contem elementos com características de Fur boxes. A funcionalidade de pelo menos uma das Fur boxes identificadas foi confirmada através de ensaios de alteração de mobilidade eletroforética que demonstraram sua interação específica com a proteína recombinante Fur de X. fastidiosa. Entretanto, nem todos os genes cuja expressão é modulada por alterações na concentração de ferro, são diretamente regulados por Fur, apoiando a hipótese de que Fur não é o único responsável pela modulação do stimulon de ferro em X. fastidiosa. Em conjunto, nossos dados apresentam novas evidências da relação entre a disponibilidade de ferro e a regulação de determinantes de patogenicidade e virulência em X. fastidiosa. / Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to citrus industry. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. In the presence of 100 uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. When treated with the iron chelator 2,2\'-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. Differential expression for a subset of 44 CDS was further evaluated by RT-qPCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. Thus, the promoter regions of strain 9a5c genome were screened for putative Fur boxes. Our analyses identified Fur boxes-like elements in promoter regions of 49 CDS. The functionality of at least one Fur box was confirmed by electrophoretic mobility shift assays which demonstrated its interaction with X. fastidiosa recombinant Fur. However, not all genes that appeared to be modulated by iron concentration shift are directly regulated by Fur. This supports the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa. Taken together, our data present novel evidence for iron regulation of pathogenicity and virulence determinants in X. fastidiosa.

O cancro cítrico é considerado na atualidade uma das doenças mais perigosas e prejudiciais à citricultura brasileira e mundial, devido aos danos causados na produção e qualidade dos frutos, sendo a Xanthomonas axonopodis pv. citri (Xac) a bactéria fitopatogênica responsável por tais prejuízos. Nosso laboratório iniciou estudos de identificação e análise funcional das interações proteína-proteína de Xac envolvendo sistemas importantes para sua patogenicidade (Alegria et. al., 2004). Nosso objetivo principal foi o estudo funcional e fisiológico de interações já identificadas entre proteínas do sistema de secreção do tipo III (T3SS) da Xac. O foco de nossa pesquisa foi tentar desvendar a importância biológica, na patogenicidade de Xac, das interações proteína-proteína: HrpB2-HrcU; HpaA-HpaB-HrcV; HrpD6-HrpB1- HrpW. Com este intuito clonamos, expressamos e purificamos as proteínas recombinantes. Produzimos soros policlonais específicos contra cada uma das proteínas citadas acima. Estudamos a interação entre as proteínas in vitro por meio de técnicas como Far-Western Blot, Pull Down, fluorescência e dicroísmo circular. Outro enfoque do nosso trabalho foi monitorar a contribuição individual destas proteínas no desenvolvimento da doença in planta. Para isso produzimos cepas de Xac mutantes para os genes hrpB2, hrcU, hpaA, hpaB, hrpB1 e hrpG. Os nocautes não polares foram infiltrados em plantas de laranja pêra, assim como também as cepas de complementação correspondentes, e assim foi testada a habilidade de desenvolver o cancro cítrico e/ou reverter os sintomas da doença. Também foi monitorada a capacidade de multiplicação e sobrevida in planta das cepas Xac ΔhrpB2, ΔhrcU e ΔhpaB, assim como a secreção das proteínas HrpB2 e HpaA pelo T3SS de Xac. Estudamos com mais detalhe a possível função de HrpB2 no T3SS de Xac, desenvolvendo experimentos para determinar a região da proteína imprescindível para sua função permanecer inalterada. Realizamos mutações sítio dirigidas, a fim de introduzir códons de terminação em diferentes regiões da proteína e testar a habilidade desses fragmentos de reverter os sintomas da doença na planta. Monitoramos a capacidade de proteínas mutantes de reverter fenótipos de patogenicidade em citrus, ausentes na cepa Xac ΔhrpB2 e revertidos na cepa de complementação Xac ΔhrpB2+pUFR047_hrpB2. Desta maneira, determinamos que os últimos seis aminoácidos de HrpB2 estão envolvidos no desenvolvimento da/s função/ões em Xac. / Citrus canker, caused by the bacterial pathogen Xanthomonas axonopodis pv citri (Xac), is a disease with significant economic consequences for the Brazilian and global citrus industry due to reductions in production and fruit quality. Our laboratory has initiated studies for the identification and functional analysis of protein-protein interactions involving Xac systems involved in pathogenicity (Alegria et. al., 2004). One objective has been to study functional and physiological interactions between proteins that make up the Xac Type III secretion system (T3SS). The focus of the present study is to unravel the biological significance in Xac pathogenicity of the following previously identified protein-protein interactions: HrpB2-HrcU; HpaA-HpaBHrcV; HrpD6-HrpB1-HrpW. With therefore cloned, expressed and purified the above-mentioned recombinant proteins. Specific polyclonal serum were produced and interactions between the proteins were studied in vitro using a variety of methods, including Far-Western Blot, Pull Down, fluorescence and circular dichroism. To monitor the individual contribution of these proteins in disease development in planta, we produced mutant Xac strains in which the hrpB2, hrcU, hpaA, hpaB, hrpB1 and hrpG genes were disrupted. The nonpolar knockouts as well as the corresponding complementation strains were infiltrated into Citrus sensensis plants and the development of citrus canker symtoms and bacterial proliferation in planta was evaluated. We also evaluated the T3SS-dependent secretion of proteins HpaA and HrpB2 by these Xac mutant strains. Structure-function relationships of the HrpB2 protein were studied in more detail. We developed experiments to determine the region of the protein essential for its function. We produced a series of hrpB2 mutants which were used to complement the hrpB2 knockout strain and evaluated their abilities to reverse the symptoms of the disease in the plant. The results demonstrate that the last six amino acids HrpB2 are important for its function in the development of disease symptoms by Xac.

Xylella fastidiosa é o agente etiológico de diversas doenças em plantas, incluindo a Clorose Variegada dos Citros (CVC), uma séria ameaça à indústria citrícola. Os níveis de transcritos sob diferentes disponibilidades de ferro foram medidos com microarranjos de DNA representando 2608 (91,6%) sequências codificadoras (CDS) da cepa 9a5c de X. fastidiosa. Na presença de 100 uM pirofosfato férrico, 218 e 256 CDS foram consideradas como reguladas positiva e negativamente, respectivamente. Quando tratada com o quelante de ferro 2,2\'-dipiridil, 193 CDS foram consideradas como reguladas positivamente e 216 negativamente. A expressão diferencial de um subconjunto de 44 CDS foi também avaliada por RT-qPCR, que mostrou uma correlação de Pearson de 0,77 com os resultados dos microarranjos. As CDS diferencialmente expressas nas variações de concentração de ferro participam em diversas funções celulares. Muitas CDS envolvidas com funções regulatórias, patogenicidade e estrutura celular foram moduladas em ambas as condições testadas, sugerindo que grandes mudanças na arquitetura celular e metabolismo ocorrem quando células de X. fastidiosa são expostas a variações extremas na concentração de ferro. Interessantemente, as CDS moduladas incluem as de síntese e secreção de bacteriocinas similares à colicina tipo V e funções ligadas a formação de pilus e fímbria. Nós também investigamos a contribuição do regulador transcricional Fur no stimulon do ferro em X. fastidiosa. Para tal, as regiões promotoras do genoma da cepa 9a5c foram varridas em busca de Fur boxes putativas. Nossas análises identificaram que regiões promotoras de 49 CDS contem elementos com características de Fur boxes. A funcionalidade de pelo menos uma das Fur boxes identificadas foi confirmada através de ensaios de alteração de mobilidade eletroforética que demonstraram sua interação específica com a proteína recombinante Fur de X. fastidiosa. Entretanto, nem todos os genes cuja expressão é modulada por alterações na concentração de ferro, são diretamente regulados por Fur, apoiando a hipótese de que Fur não é o único responsável pela modulação do stimulon de ferro em X. fastidiosa. Em conjunto, nossos dados apresentam novas evidências da relação entre a disponibilidade de ferro e a regulação de determinantes de patogenicidade e virulência em X. fastidiosa. / Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to citrus industry. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. In the presence of 100 uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. When treated with the iron chelator 2,2\'-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. Differential expression for a subset of 44 CDS was further evaluated by RT-qPCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. Thus, the promoter regions of strain 9a5c genome were screened for putative Fur boxes. Our analyses identified Fur boxes-like elements in promoter regions of 49 CDS. The functionality of at least one Fur box was confirmed by electrophoretic mobility shift assays which demonstrated its interaction with X. fastidiosa recombinant Fur. However, not all genes that appeared to be modulated by iron concentration shift are directly regulated by Fur. This supports the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa. Taken together, our data present novel evidence for iron regulation of pathogenicity and virulence determinants in X. fastidiosa.

O cancro cítrico é considerado na atualidade uma das doenças mais perigosas e prejudiciais à citricultura brasileira e mundial, devido aos danos causados na produção e qualidade dos frutos, sendo a Xanthomonas axonopodis pv. citri (Xac) a bactéria fitopatogênica responsável por tais prejuízos. Nosso laboratório iniciou estudos de identificação e análise funcional das interações proteína-proteína de Xac envolvendo sistemas importantes para sua patogenicidade (Alegria et. al., 2004). Nosso objetivo principal foi o estudo funcional e fisiológico de interações já identificadas entre proteínas do sistema de secreção do tipo III (T3SS) da Xac. O foco de nossa pesquisa foi tentar desvendar a importância biológica, na patogenicidade de Xac, das interações proteína-proteína: HrpB2-HrcU; HpaA-HpaB-HrcV; HrpD6-HrpB1- HrpW. Com este intuito clonamos, expressamos e purificamos as proteínas recombinantes. Produzimos soros policlonais específicos contra cada uma das proteínas citadas acima. Estudamos a interação entre as proteínas in vitro por meio de técnicas como Far-Western Blot, Pull Down, fluorescência e dicroísmo circular. Outro enfoque do nosso trabalho foi monitorar a contribuição individual destas proteínas no desenvolvimento da doença in planta. Para isso produzimos cepas de Xac mutantes para os genes hrpB2, hrcU, hpaA, hpaB, hrpB1 e hrpG. Os nocautes não polares foram infiltrados em plantas de laranja pêra, assim como também as cepas de complementação correspondentes, e assim foi testada a habilidade de desenvolver o cancro cítrico e/ou reverter os sintomas da doença. Também foi monitorada a capacidade de multiplicação e sobrevida in planta das cepas Xac ΔhrpB2, ΔhrcU e ΔhpaB, assim como a secreção das proteínas HrpB2 e HpaA pelo T3SS de Xac. Estudamos com mais detalhe a possível função de HrpB2 no T3SS de Xac, desenvolvendo experimentos para determinar a região da proteína imprescindível para sua função permanecer inalterada. Realizamos mutações sítio dirigidas, a fim de introduzir códons de terminação em diferentes regiões da proteína e testar a habilidade desses fragmentos de reverter os sintomas da doença na planta. Monitoramos a capacidade de proteínas mutantes de reverter fenótipos de patogenicidade em citrus, ausentes na cepa Xac ΔhrpB2 e revertidos na cepa de complementação Xac ΔhrpB2+pUFR047_hrpB2. Desta maneira, determinamos que os últimos seis aminoácidos de HrpB2 estão envolvidos no desenvolvimento da/s função/ões em Xac. / Citrus canker, caused by the bacterial pathogen Xanthomonas axonopodis pv citri (Xac), is a disease with significant economic consequences for the Brazilian and global citrus industry due to reductions in production and fruit quality. Our laboratory has initiated studies for the identification and functional analysis of protein-protein interactions involving Xac systems involved in pathogenicity (Alegria et. al., 2004). One objective has been to study functional and physiological interactions between proteins that make up the Xac Type III secretion system (T3SS). The focus of the present study is to unravel the biological significance in Xac pathogenicity of the following previously identified protein-protein interactions: HrpB2-HrcU; HpaA-HpaBHrcV; HrpD6-HrpB1-HrpW. With therefore cloned, expressed and purified the above-mentioned recombinant proteins. Specific polyclonal serum were produced and interactions between the proteins were studied in vitro using a variety of methods, including Far-Western Blot, Pull Down, fluorescence and circular dichroism. To monitor the individual contribution of these proteins in disease development in planta, we produced mutant Xac strains in which the hrpB2, hrcU, hpaA, hpaB, hrpB1 and hrpG genes were disrupted. The nonpolar knockouts as well as the corresponding complementation strains were infiltrated into Citrus sensensis plants and the development of citrus canker symtoms and bacterial proliferation in planta was evaluated. We also evaluated the T3SS-dependent secretion of proteins HpaA and HrpB2 by these Xac mutant strains. Structure-function relationships of the HrpB2 protein were studied in more detail. We developed experiments to determine the region of the protein essential for its function. We produced a series of hrpB2 mutants which were used to complement the hrpB2 knockout strain and evaluated their abilities to reverse the symptoms of the disease in the plant. The results demonstrate that the last six amino acids HrpB2 are important for its function in the development of disease symptoms by Xac.

Molecular dynamics (MD) simulation, which employs an empirical potential energy function to describe the interactions between the atoms in a system, is used to investigate atomistic motions of proteins. However, the timescale of many biological processes exceeds the reach of standard MD due to computational limitations. To circumvent these limitations, steered molecular dynamics (SMD), which applies external forces to the simulated system, can be used.Dynamical properties of the gonococcal type IV pilus (GC-T4P) from the bacteria Neisseria gonorrhoeae are first considered. T4 pili are long, filamentous proteins constructed from a subunit (pilin) found to emanate from the surface of pathogenic bacteria. They can withstand large forces (~100 pN), and are implicated in infection. SMD simulations are performed to study the response of the filament to an applied force. Our simulations reveal that stability of the pilus likely results from hydrophobic contacts between pilin domains buried within the filament core. Along the filament surface, gaps are formed between pilin globular head domains. These gaps reveal an amino acid sequence that was also observed to become exposed in the experimentally stretched filament. We propose two other regions initially hidden in the native filament that might become exposed upon stretching.The multidrug resistance transporter EmrD, found in the inner membrane of Escherichia coli is also the target of our studies. EmrD removes harmful drugs from the bacterial cell. We use MD to explore equilibrium dynamics of the protein, and MD/SMD to study drug interactions and transport along its central cavity. Motions supporting a previously proposed lateral diffusion pathway for substrate from the cytoplasmic membrane leaflet into the central cavity were observed. Additionally, interactions of a few specific residues with CCCP have been identified.Finally, we describe network analysis as an approach for analyzing conformational sampling by MD simulations. We demonstrate for several model systems that networks can be used to visualize both the dominant conformational substates of a trajectory and the connectivity between them. Specifically, we compare the results of various clustering algorithms to the network layouts and show how information from both methods can be combined.

Bacteria of the Neisseria are predominately commensal, though N. gonorrhoeae and N. meningitidis are capable of causing disease. Both of these species often asymptomatically colonize humans, a trait reminiscent of their commensal cousins. The factors that shift the balance between asymptomatic carriage and disease are unknown. Pathogenic Neisseria use retractile surface structures called Type IV pili to coordinate community behavior and to initiate and sustain infection. Previously, the contributions of pilus retraction have been studied by deleting the pilus retraction motor, PilT. Recent findings suggest the speed and force exerted by pilus retraction is responsive to environmental cues. By examining several PilT mutants that maintain the ability to retract pili, I show retraction, per se, is not required for N. gonorrhoeae social interactions with bacteria or with human cells. Furthermore, Type IV pilus retraction by the commensal N. elongata affects the host cell differently than retraction by N. gonorrhoeae. These observations collectively suggest pilus retraction properties shape the host cell response to Neisseria colonization and could tip the balance of asymptomatic colonization to symptomatic disease.

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 / This research was conducted to understand type IV pilus (T4P), a hair-like structure found on the surface of many bacteria groups. T4P is a versatile structure; it plays many vital roles in bacterial life such as in surface motility, surface attachment, gene transfer, and virulence factor. Pilus is a dynamic polymer composed of many small pilin proteins that can be assembled or disassembled. Structurally, pilus is supported by machinery that helps to extend and retract pilus by adding or removing pilin proteins. At the core of the machinery, two different proteins are responsible to power the assemble and disassemble process by converting the chemical energy in ATP into mechanical energy. This study focuses on the protein that powers pilus assembly, PilB. Understanding PilB will be very beneficial in elucidating how the strongest biological motor work in action. The structure of PilB was determined to be a hexamer consist of six identical copies of the same protein forming a ring structure with 2-fold symmetry. This structure suggests that PilB works using symmetric rotary mechanism. Previous studies of PilB have not been productive because the purified PilB did not behave well during the assay. In this study, PilB from Chloracidobacterium thermophilum (CtPilB) was determined to be a reasonable model for the study. CtPilB was successfully purified and it was identified to have a robust activity outside the cell allowing for further biochemistry studies. The profile of CtPilB kinetics was unique and it did not conform with the classical kinetic profile. The analysis of the profile suggests that CtPilB exhibit a complex mechanism in hydrolyzing ATP.

Helicobacter pylori est une bactérie qui colonise les cellules épithéliales gastriques humaines. Une des conséquences de cette infection est l'induction de cancers de l'estomac dans 1 à 3 % des cas, via l'injection d'une cytotoxine appelée CagA qui dérégule les voies de signalisation des cellules cibles. Cette injection, dont le mécanisme est encore inconnu, se fait grâce à un système de sécrétion de type IV (T4SS). Le pilus du cagT4SS est encore mal caractérisé. Les protéines CagI, CagL et CagH sont essentielles à la fonctionnalité du cagT4SS et à la biogenèse du pilus. De plus les trois protéines forment un sous-complexe dont les détails moléculaires n'ont pas encore été élucidés. Par conséquent mes études se sont focalisées sur ces trois protéines, leurs interactions et leur relation structure/fonction. J'ai mis en évidence que CagL interagissait directement avec CagI et CagH avec une affinité de l'ordre du micromolaire et que CagI et CagH n'interagissaient pas entre elles. La caractérisation de ces interactions a permis notamment d'identifier un complexe CagL-CagI. Afin de comprendre les détails structuraux de ce complexe, j'ai entrepris deux études structurales. La première consiste à déterminer les résidus de CagL impliqués dans l'interface d'interaction avec CagI par RMN. La seconde étude se focalise sur la détermination de la structure 3D du complexe CagI-CagL par microscopie électronique. Pour cela j'ai purifié le complexe CagI-CagL, monodisperse et stable en solution. Nous avons collecté des images du complexe par cryoEM et généré des classes 2D. Cette étude a permis pour la première fois de caractériser les interactions entre CagL-CagI-CagH et d'obtenir des informations structurales du complexe CagI-CagL / Helicobacter pylori is a bacterium that colonizes the human stomach in half of the world population. It is estimated that 20% and 3% of patients develop peptic ulcer and gastric cancer, respectively. For these reasons, H. pylori was identified as a group 1 carcinogen by the World Health Organization (WHO) in 1994. The most virulent strains of H. pylori carry a type IV secretion system (Cag-T4SS) responsible for the injection of the oncoprotein CagA into gastric epithelial cells. One remarkable feature of the cagT4SS is its external pilus which composition is not clear. CagL, CagH and CagI proteins are critical components of the Cag-T4SS because these proteins are necessary for CagA translocation and are involved in pilus formation. Moreover CagL forms a sub-assembly with CagI and CagH but the molecular details of the complex are still to be discovered. Our objective is to better understand the molecular basis of CagLIH complex by interaction and structural study. CagL interacts with CagI and CagH with a with Kds of 5 µM. CagI does not interact with CagH. The structural study of CagL/CagI complex has been investigated by a two-pronged approach. First I have purified the CagL/CagI complex and collected cyo-EM micrographs. In parallel I have collected NMR spectra of CagL in the presence of CagI and identify the changes in the spectra to determine the residues involved in the interaction. In this study we have, for the first time, characterize the CagL-CagI-CagH interactions and obtained structural informations of the CagI-CagL complex