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.

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

Neisseria meningitidis is an obligate human pathogen causing over 1000 cases of meningococcal disease within the U.K., 10 % of which result in long-term disability or fatality. 10-70 % of the population carry N. meningitidis in their nasopharynx, the natural reservoir of this bacterium, as a commensal. The host-pathogen interactions of this species are complex and a greater understanding of the molecular mechanisms involved in pathogenesis and immune evasion is required. Three aspects of N. meningitidis pathogenesis were explored in this study. One mechanism of immune evasion which promotes serum resistance of N. meningitidis is recuitment of complement factor H through domains 6 and 7 (fH₆₇) by factor H binding protein (fHbp). In this study, mouse fH₆₇ was recombinantly expressed and purified. fHbp did not bind mouse fH₆₇ at physiologically relevant protein concentrations. The structure of mouse fH₆₇ was solved, showing differences in domain orientation and surface chemistry compared to the human version of this protein, potentially accounting for the host specificity of this interaction. Type IV pili (T4P) are crucial adhesins of N. meningitidis, the fibre of which is composed of thousands of copies of PilE. A method was developed to recombinantly produce large quantities of this protein from a variety of meningococcal strains and the structure was solved of one PilE protein. Subsequent analysis was performed with the PilE proteins investigating their interaction with the putative pilus receptor CD46 and human epithelia as well as their immunogenicity. A method was also established to produce PilC, the proposed tip-assocoated adhesin of T4P. ZapE has recently been identified as an important protein in pathogen colonisation, functioning as an ATPase linked to Z-ring formation in bacterial cell fission. Both N. meningitidis and E. coli ZapE were recombinantly produced. The domain boundaries were mapped and ATPase activity was confirmed. No interaction was seen with FtsZ but DNA binding and modulation was observed by shift assays, the exact function of which remains to be elucidated in future studies.

No description available.

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.

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

O trabalho aqui desenvolvido teve como objeto o estudo estrutural e funcional de várias proteínas do fitopatógeno Xanthomonas axonopodis pv citri (Xac), dentre as quais se destacam as proteínas hipotéticas conservadas YaeQ e SufE, as proteínas RpfC, RpfF e RpfG envolvidas em quorum sensing e proteínas PilZ, FimX e PilB envolvidas na biogênese do pilus tipo IV. Para o desenvolvimento deste trabalho foram utilizadas diferentes técnicas incluindo: clonagem, expressão, purificação, desnaturação térmica, cristalografia, difração de raios-X, RMN, ensaios de 2-híbrido, produção de nocautes, mutação sítio dirigida, Western- e Far- Western, entre outras. Dentre os resultados mais importantes obtidos temos a determinação estrutural das proteínas YaeQ e PilZ pela técnica MAD. Em ambos os casos, as estruturas representaram topologias inéditas. Com base nos dados estruturais, mostramos que YaeQ pertence à família PD-(D/E)XK presente em endonucleases dependentes de magnésio, e a partir de ensaios funcionais obtivemos evidências que sugerem que YaeQ está envolvida em alguma via de reparo de DNA em Xac. A estrutura tridimensional de PilZ revelou uma inesperada variedade estrutural dentro da família PilZ e mostrou de forma clara porque ortólogos não interagem com o segundo mensageiro bacteriano, c-diGMP. A cadeia principal de PilZ foi assinalada por RMN e a estrutura secundária de PilZ em solução é consistente com aquela determinada por cristalografia. Duas proteínas que interagem com PilZ foram identificadas: PilB e FimX. Como PilZ, ambos exercem papéis na biogênese do pilus tipo IV (T4P). Mostramos que PilZ interage especificamente com o domínio EAL de FimX e que resíduos conservados na região do C-terminal de PilZ estão envolvidos na interação com PilB, mas não com FimX. Ensaios de mutação sítio dirigida mostraram que a Y22 de PilZ pode estar envolvida na regulação da interação de PilZ com FimX e com PilB. Apesar de PilZ não interagir com c-diGMP seu parceiro, FimX, interage. PilZ consegue interagir com PilB ao mesmo tempo em que interage com FimX, formando um complexo ternário que é independente da interação de FimX com c-diGMP. Com base em todos estes resultados propusemos possíveis mecanismos de ação de PilZ e FimX no controle da biogênese do T4P. Além dos resultados acima descritos, determinamos a estrutura de SufE e mostramos que esta aumenta a atividade cisteína dessulfarase de seu parceiro, SufS, em torno de 10 vezes, como ocorre com SufE-SufS de E.coli. Clonamos, expressamos, purificamos e fizemos ensaios de cristalização de algumas proteínas envolvidas no controle de quorum sensing em Xac. Tivemos êxito na cristalização do domínio HPT (histidina fosfotransferase) da proteína chave deste sistema, RpfC / The aim of the project was to perform structural and functional studies of different Xanthomonas axonopodis pv citri (Xac) proteins including the hypothetical proteins YaeQ and SufE; RpfC, RpfF and RpfG involved in the quorum sensing and PilZ, FimX and PilB that play roles in type IV pilus (T4P) biogenesis. Several experimental techniques were employed including cloning, expression and purification of recombinant proteins, thermal denaturation, protein crystallography, X-ray diffraction, NMR, two-hybrid assays, Western- and Far-Western Blotting assays, site direct mutagenesis, and the production of Xac knockouts strains. The most important results include the determination of the three-dimensional crystal structures of PilZ and YaeQ using the MAD technique. In both cases, the structures reveled new protein topologies. The comparison of the YaeQ structure with others deposited in public databases revealed that YaeQ proteins represent a new variation within the PD-(D/E)XK magnesium dependent endonucleases superfamily. Functional assays suggest that YaeQ may be envolved in DNA repair in Xac. The PilZ three-dimensional structure revealed an unexpected structural variation within the PilZ domain superfamily and showed why PilZ orthologs are not able to bind the important bacterial second messenger, c-diGMP. We assigned the PilZ main chain by NMR and used this information to demonstrate that the PilZ secondary structure in solution is consistent with the PilZ crystal structure. We identified two proteins that interact with PilZ: PilB and FimX. As with PilZ, both PilB and FimX are involved in T4P biogenesis. PilZ binds specifically to the EAL domain of FimX and the conserved residues located in the PilZ unstructured C-terminal region contribute to binding with PilB but not with FimX. Site direct mutagenesis studies showed that PilZ residue Y22 is necessary for its capability to interact with both PilB and FimX. Although PilZ does not bind c-diGMP, her partner, FimX, does. We present evidence that PilZ can bind simultaneously to FimX and PilB, forming a ternary complex that is independent of c-diGMP. These results allow us to propose possible mechanisms by which PilZ and FimX control T4P biogenesis. Other results obtained during this period include the resolution of the crystal structure of the SufE protein from Xac using the molecular replacement technique. We show that SufE induces a 10-fold increase in the cysteine desulfurase activity of SufS, similar to that observed for the SufE-SufS complex from E. coli. Several proteins involved in quorum sensing and c-di-GMP signaling were cloned, expressed and submitted to crystallization trials. Crystals of the HPT (histidine phophotransferase) domain) of the RpfC sensor histidine kinase were obtained