The guanosine monophosphate synthetase (GMPS) is a class I glutamine amidotransferase, involved in the de-novo purine nucleotide biosynthesis. The enzyme catalyzes the biochemical transformation of xantosine (XMP) into guanosine monophosphate (GMP) in presence of ATP, Mg2+ and glutamine. All GMPSs consist of two catalytic sites 1) for GATase activity 2) for the ATPPase activity. The two catalytic sites may be housed in the same polypeptide (two-domain-type) or in separate polypeptides (two-subunit-type). Most of the studies have been performed on two-domain-type GMPSs, while only one study has been reported from two-subunit-type GMPS (Maruoka et al. 2009). The two-subunit-type GMPS presents an example where the component reactions of a single enzymatic reaction are carried out by two distinct subunits. In order to get better understanding of structural aspects and mechanistic principle that governs the GMPS activity in two-subunit-type GMPSs, we initiated the study by taking GMPS of Methanocaldococcus jannaschii as a model system. The GMPS of M. jannaschii (Mj) is a two-subunit-type protein. The GATase subunit catalyzes the hydrolysis of glutamine to produce glutamate and ammonia. The ATPPase subunit catalyses the amination of XMP to produce GMP using the ammonia generated in GATase subunit. Since the two component reactions are catalysed by two separate subunits and are coupled in the way that product of one reaction (ammonia) acts as a nucleophile in the second reaction. The cross-talk between these two subunits in order to maximise the efficiency of overall GMPS warrants investigation. The GATase activity is tightly regulated by the interaction with ATPPase domain/subunit, in all GMPS except in the case of P. falciparum. This interaction is facilitated by substrate binding to the ATPPase domain/subunit. Though, the conditions for the interaction between two subunits is known in a two-subunit-type GMP synthetase from P. horikoshii, the structural basis of substrate dependent interaction is not known. As a first step to understand the structural basis of interaction between the Mj GATase and Mj ATPPase subunits, we have determined the structure of Mj GATase (21 kDa) subunit using high resolution, multinuclear, multidimensional NMR spectroscopy. Sequence specific resonance assignments were obtained through analysis of various 2D and 3D hetero-nuclear multidimensional NMR experiments. NMR based distance restraints were obtained from assignment of correlations observed in NOE based experiments. Data were acquired on isotopically enriched samples of Mj GATase. The structure of Mj GATase (2lxn) was solved by using cyana-3.0 using NMR based restraints as input for the structure calculation. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35±0.06 Å for backbone atoms and 0.8±0.06 Å for all heavy atoms. Attempts were also made to obtain assignments for the 69.6 kDa dimeric ATPPase subunit. Partial assignments have been obtained for this subunit. The GATase subunit is catalytically inactive. So far, there has been only one published report on a two-subunit-type GMPS from P. horikashii. The study has shown that the catalytic activity of GATase is regulated by the GATase-ATPPase interaction which is facilitated by the substrate binding to the ATPPase subunit. For the first time, we have provided the structural basis of interaction between GATase-ATPPase (112 kDa) in a two-subunit-type GMPS. Observed line width changes were used to identify residues in GATase residues that are involved in the Mj GATase-ATPPase interaction. Our data provides a possible explanation for conformational changes observed in the Mj GATase subunit upon GATase-ATPPase interaction that lead to GATase activation. Ammonia is generated in GATase subunit and is very reactive and labile. Thus, the faithful transportation of ammonia from GATase to ATPPase subunit is very crucial for optimal GMPS activity. Till date, a PDB query for GMPS retrieves only one structure which belongs to two-subunit-type GMPS, where authors have determined the structures of GATase and ATPPase subunits separately. However, the structure of holo-GMPS is not determined yet. Using interface information from experimental data and HADDOCK, we have constructed a model for the holo-GMPS from M. jannaschii. A possible ammonia channel has been deduced using the programs MOLE 2.0 and CAVER 2.0. This ammonia channel has a length of 46 Å, which is well within the range of the lengths calculated for similar channels in other glutamine amidotransferase. It had been suggested earlier that in addition to the magnesium required for charge stabilization of ATP, additional binding sites were present on GMPS. The effect of excess Mg2+ requirement on the GMPS activity has been studied in two-domain-type GMPS. However, the interaction between GATase and Mg2+ has been not investigated in any GMPS. This prompted us to investigate the effect of MgCl2 on Mj GATase subunit. For the first time, using chemical shift perturbation, we have established interaction between Mj GATase and Mg2+. The dissociation constant (Kd) of the Mj GATase-Mg2+ interaction was determined. The Kd value was found to be 1 mM, which indicates a very weak interaction. The substrate of the GATase subunit is glutamine. The condition of the hydrolysis of the glutamine is known in GMPS. However, the binding of the glutamine and associated conformational changes in GATase have been not studied in GMPS. Furthermore, till date there is no structure available for the glutamine bound GMPS/GATase. Using isotope edited one dimensional and two-dimensional NMR spectroscopy; we have shown that the Mj GATase catalytic residues are not in a compatible conformation to bind with glutamine. Thus, a conformational change in Mj GATase subunit is a pre-requisite condition for the binding of glutamine. These conformational changes are brought by the Mj GATase-ATPPase interaction.

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.

Neben den cAMP- und cGMP-abhängigen Proteinkinasen (PKA bzw. PKG) sind als zyklonukleotid-regulierte Effektorproteine die Ionenkanäle CNG1-4, der "guanine nucleotide exhange factor“ Epac sowie die zyklonukleotid-spaltende Familie der Phosphodiesterasen (PDEs) von Bedeutung. Industriell synthetisierte cGMP- und cAMP-Analoga besitzen zwar meist eine hohe Affinität für ihr Zielprotein, über ihre Hydrolysestabilität gegenüber PDEs in der Zelle ist jedoch wenig bekannt. In dieser Arbeit wurden die kinetischen Konstanten von elf der am häufigsten genutzten cAMP-und cGMP-Analoga an verschiedenen Vertretern der PDE-Familien mittels Mikrokaloriemetrie bestimmt. Zudem konnte in den Messungen der inhibitorisch Effekt hydrolysestabiler Derivate auf die PDEs qualitativ und quantitativ ermittelt werden kann. Die Ergebnisse zeigen, dass Phosphodiesterasen in der Lage sind, auch chemisch modifizierte Analogsubstanzen der Cyclonukleotide cAMP und cGMP zu hydrolysieren. Hydrolysestabile Derivate dagegen entwickeln häufig inhibitorische Wirkung auf die PDEs und verursachen dadurch Veränderungen der intrazellulären cAMP und cGMP Konzentrationen. So vermag z. B. die Epac-spezifische Substanz Sp-8-pCPT-2’-O-Me-cAMPS in den in vitro Experimenten die PDEs mit ki-Werten im einstelligen mikromolaren Bereich zu inhibieren. In mit Sp-8-pCPT-2’-O-Me-cAMPS stimulierten Thrombozyten steigt als Folge dieser PDE-Hemmung die cGMP Konzentration in der Zelle an und man beobachtet eine als Folge eine PKG-vermittelte Phosphorylierung des Substratproteins VASP – eine unerwünschte Nebenreaktion. Die erhobenen Daten lassen außerdem Rückschlüsse auf den Inhibitionsmechanismus zu. Einige Analoga inhibieren die cGMP-bindenden GAF-Domänen in den PDEs 2A, 5A, 6cone und 10A sowie die PDE 4D3 nach dem linear-mixed-Typ und beeinflussen daher, neben der katalytischen Aktivität, vermutlich auch regulatorische Zentren dieser Enzyme. Zusammenfassend erleichtern die erhobenen Daten Wissenschaftlern die Auswahl des für ihre Fragestellung am besten geeigneten Derivates. / cAMP and cGMP are critical second messengers that regulate multiple targets including different cAMP/cGMP-dependent protein kinases (PKA/PKGs), exchange proteins directly activated by cAMP (Epacs), phosphodiesterases (PDEs) and cyclic nucleotide-gated ion channels (CNGs). Second and third generation cyclic nucleotide analogs are widely used to elucidate specificity of cellular signaling, mediated by these target proteins. However, the selectivity and stability of these analogs need to be fully understood in order to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs I measured the activation or inhibitory activity of 13 commonly-used cyclic nucleotide analogs 8 different PDEs. To measure their stability to hydrolysis I utilized isothermal microcalorimetry, a method that allows to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. I demonstrate that indeed some of these analogs can be hydrolyzed by multiple PDEs and others are competitive inhibitors. Herein I provide Ki data for all of the non-hydrolyzable analogs and Km and Vmax values for all of the hydrolyzable analogs. Each of these values, as well as their mode of inhibition can be determined in a single experiment. The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could of course cloud interpretation of the use of these analogs. Similarly, those that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we show that Sp-8-pCPT-2’-O-Me-cAMPS, a highly specific non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP/PKG and cAMP/PKA signaling pathways in intact platelets. Specifically we found enhanced VASP phosphorylation at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2’-O-Me-cAMPS. These data indicate that this “selective Epac activator” is able to indirectly activate the cAMP/PKA and cGMP/PKG signalling pathways presumably through inhibition of platelet PDE5 and/or PDE3. The data together allow to provide recommendations for how best to probe the different cyclic nucleotide signalling pathways using cyclic nucleotide analogs. In summary, the data provide evidence that most cAMP and cGMP analogs have multiple targets. Therefore, interpretation of any effects these analogs have in cells should take into consideration their possible cross-target reactivities.

Segundos mensageiros nucleotídicos são amplamente utilizados por bactérias para se adaptar às mudanças ambientais e fisiológicas. Neste cenário destaca-se o c-di-GMP, um segundo mensageiro praticamente universal em bactérias responsável por controlar a transição do estilo de vida bacteriano. Em geral, altos níveis celulares de c-di-GMP promovem um estado séssil, de formação de biofilme, enquanto baixos níveis induzem a motilidade. Xanthomonas citri subsp. citri (Xac), um fitopatógeno de grande importância econômica no Brasil, possui uma complexa regulação da sinalização de c-di-GMP, possuindo mais de 30 proteínas envolvidas na síntese, na degradação e na detecção deste segundo mensageiro. Dentre essas proteínas, destacam-se as que possuem os domínios de síntese e degradação presentes na mesma cadeia polipetídica, os domínios GGDEF e EAL respectivamente. A análise das estruturas primárias das 11 proteínas GGDEF-EAL codificadas pelo genoma de Xac revelou que a maior parte delas (6) provavelmente possui o domínio GGDEF inativo, enquanto o EAL é ativo. Três possivelmente possuem ambos os domínios ativos enquanto outras duas possuem ambos os domínios inativos. O nocaute do gene xac2382 que codifica uma dessas proteínas (que possui um domínio periplasmático seguido dos domínios citoplasmáticos HAMP-GGDEF-EAL), demonstra um aumento de motilidade e uma diminuição na formação de biofilme. Construções de fragmentos da proteína revelaram que XAC2382 necessita pelo menos dos domínios HAMP-GGDEF para complementar a cepa nocaute e que a atividade de diguanilato ciclase é essencial para isto. O domínio periplasmático de XAC2382 se mostrou interagir com XAC2383, uma proteína codificada por um gene presente no mesmo cluster do gene de XAC2382, e essa interação parece importante para o controle da motilidade de Xac. A estrutura de XAC2383 foi resolvida por cristalografia de raios X na qual foi revelada uma topologia típica de proteínas da família das periplasmic binding proteins (PBPs) possuindo ainda uma cavidade carregada positivamente contendo um motivo Ser-Thr-Ser (amnioácidos 152-154) importante para a ligação de compostos com grupos fosfatos ou fosfonatos. A mutação sítio dirigida nesse motivo aboliu os efeitos na motilidade dependentes dessa proteína. Esses resultados sugerem que XAC2383 é um sensor periplasmático de um composto eletronegativo e esta proteína interage com XAC2382 regulando a motilidade bacteriana. XAC0495, uma proteína com ambos os domínios GGDEF-EAL provavelmente inativos, pode fazer parte de um sistema de dois componentes com a histidina quinase XAC0494. XAC0495 se comporta como um monômero em solução e possui um formato alongado, como revelado por experimentos de SAXS. / Nucleotide based second messengers are widely used by bacteria in signaling pathways that mediate adaptations to environmental and physiological changes. c-di-GMP is a nucleotide second messenger ubiquitous in Gram-negative bacteria, where it plays a role in many important behaviors that define bacterial lifestyle. In general, high cellular levels of c-di-GMP promote biofilm formation, while low levels induce bacterial motility. Xanthomonas citri subsp. Citri (Xac), a pathogen of great economic importance in Brazil, has a complex repertoire of c-di-GMP signaling molecules, with more than 30 genes coding for proteins involved in the synthesis, degradation and detection of this second messenger. Among these proteins, many have both GGDEF and EAL domains (often associated with c-di-GMP synthesis and degradation, respectively) present in the same polypeptide chain. Analysis of the primary structure of 11 GGDEF-EAL proteins coded by the Xac genome revealed that six most likely possess an inactive GGDEF domain plus an active EALdomain. Another three proteins have both domains active while the other two have both domains inactive. The knockout of the xac2382 gene, coding for a protein which contains a periplasmic domain followed by cytoplasmic HAMP, GGDEF (active) and EAL (active) domains, shows an increase in motility and a decrease in biofilm formation. Constructions containing fragments of this protein revealed that constructs containing at least the HAMP and GGDEF domains are able to complement the knockout strain and that diguanilate cyclase activity is essential for this. The XAC2382 periplasmic domain was shown to interact with a protein encoded by a gene situated in the same cluster, XAC2383, and that this interaction seems crucial for the control of Xac motility. The structure of XAC2383 was solved by X-ray crystallography and was shown to adopt a topology typical of the periplasmic binding proteins (PBP) family. The protein possesses a positively charged groove that contains a Ser-Thr-Ser motif (152STS154) important for the binding of compounds with phosphate or phosphonate groups. Site-directed mutagenesis of this motif abolished the effects on motility caused by this protein. These results suggest that XAC2383 is a periplasmic protein responsible for sensing a compound with electronegative characteristics and which interacts with XAC2382, thereby regulating the bacterial motility. Another protein, XAC0495 (with both GGDEF-EAL domains probably inactive) may be part of a two-component system with the histidine kinase XAC0494. Small-angle X-ray scattering (SAXS) experiments reveal that XAC0495 exists as an elongated monomer in solution.

Bei den meisten Bakterien wird die Biofilmbildung durch das Botenmolekül c-di-GMP stimuliert. Durch die enzymatische Aktivität von c-di-GMP-synthetisierenden Diguanylatzyklasen/DGC und c-di-GMP-abbauenden Phosphodiesterasen/PDE wird der c-di-GMP-Gehalt als eine Antwort auf diverse Stress- und suboptimale Umweltbedingungen reguliert. Vor allem Gram-negative Bakterien haben multiple DGC/PDE. So besitzt Escherichia coli K-12 29 solche Proteine, darunter 12 DGC, 13 PDE sowie 4 degenerierte Proteine ohne enzymatische Funktion. Dieses komplexe c-di-GMP-Kontrollsystem reguliert die Produktion der extrazellulären Biofilmmatrix, die in E. coli während des Übergangs in die stationäre Wachstumsphase stattfindet. Das Ziel dieser Arbeit war es zu untersuchen, ob die 29 DGC/PDE von E. coli ein spezifisches Interaktom bilden, das DGC/PDE-Pärchen enthält. Durch umfangreiche Two-Hybrid-Untersuchungen konnte gezeigt werden, dass es ein solches Interaktom in der Biofilmregulationskaskade tatsächlich gibt, das allerdings nicht in Pärchen organisiert ist. Vielmehr wird die Biofilmbildung von einer Kerngruppe von Enzymen, welche multiple Interaktionen untereinander und mit anderen DGC/PDE aufweisen, kontrolliert. Die Funktionsweise der Kerngruppe von Enzymen könnte jedoch möglicherweise unter bestimmten Wachstumsbedingungen durch Interaktionen mit weiteren Proteinen moduliert werden. Die Dynamik des Interaktionsnetzwerks ermöglicht vermutlich eine rationelle Ressourcenverwaltung in den verschiedenen Zonen des Biofilms, was zum Aufbau der komplexen Matrixarchitektur beitragen könnte, und eine hohe Anpassungsfähigkeit der Bakterien und der von ihnen aufgebauten Biofilmstrukturen gewährleisten könnte. Insgesamt führt diese Arbeit aus einer systemischen Perspektive zu einem neuen Modell der lokalen Biofilmbildungsregulation durch den Botenstoff c-di-GMP und legt die Basis für weitere Untersuchungen der daran beteiligten Mechanismen einzelner GGDEF/EAL-Domäne-haltiger Proteine in E. coli. / The messenger molecule c-di-GMP stimulates the formation biofilms in most bacteria species. The enzymatic activities of the diguanylate cyclases/DGC and the phosphodiesterases/PDE adjust the c-di-GMP content in response to diverse stress and suboptimal environmental conditions. Above all, Gram-negative bacteria have multiple GGDEF/EAL domain proteins. Escherichia coli K-12 possesses 29 of such proteins: 12 DGCs, 13 PDEs and 4 so called degenerate proteins without any enzymatical function. Mainly, this complex c-di-GMP control system regulates the production of the extracellular biofilm matrix, which in E. coli takes place during the switch into the stationary growth phase. The main compounds of the matrix are amyloid curli-fibers and exoplysaccaride cellulose. The goal of this work was to investigate, whether the 29 DGC/PDE from E. coli develop a specific interactome containing additional DGC/PDE pairs. In a comprehensive two-hybrid study, it could be demonstrated that there is indeed a specific interactome in the biofilm formation cascade. However, this interactome does not contain additional DGC/PDE pairs. Mainly, the core group of enzymes, which have multiple interactions among each other and with other DGC/PDE, controls biofilm formation. Under certain growth conditions the mode of action of the core enzymes might be adjusted through the interaction with other proteins. Presumably, the dynamics of the interaction network allows managing the resources in the different biofilm zones efficiently, which could conribute to the complex organisation of the matrix architecture. Therefore, the rapid adaptation of bacteria and the formed biofilm structures could be better organized. Altogether, this work provides a new model for the local regulation of the biofilm formation by the secondary messenger c-di-GMP guided from a systemical perspective. Hereby, the basis for further investigations on regulation mechanisms of individual DGC/PDE was set. / Переход от подвижного и планктонообразного образа жизни к формированию биоплёнок является важной и интересной особенностью различных микроорганизмов. Кишечная палочка (Escherichia coli) представляет собой удобный модельный организм для изучения подобных трансформаций. У этой грамотрицательной бактерии образование биоплёнки обусловлено внутриклеточной аккумуляцией циклического дигуанилата (цикло-диГМФ). Известно, что активность ферментов, синтезирующих (дигуанилатциклазы/ДГЦ) и разлагающих (диэстеразы/ДЭ) эти сигнальные молекулы, меняется в ответ на стрессовые и субоптимальные раздражители. Кишечная палочка имеет 12 ДГЦ, 13 ДЭ и четыре дегенерированных протеина. Цель данной работы – изучить специфический, важный при формировании биоплёнки интерактом и выяснить, способны ли другие ДГЦ/ДЭ образовывать дополнительные ДГЦ/ДЕ модули и вносить свой вклад в формирование биоплёнки. В работе были изучены молекулярные взаимодействия, ответственные за формирование биоплёнок у кишечной палочки. Так, было доказано отсутствие в интерактоме дополнительных локальных ДГЦ/ДЕ модулей, участвующих при каскадных процессах регуляции роста биоплёнки. Установлено, что процесс формирования биоплёнки в большей степени контролируется основной группой ферментов, которые имеют множественные взаимодействия между собой и с другими ДГЦ/ДЭ. Вероятнее всего, такие взаимодействия способны модулировать работу основных ферментов при определенных условиях культивирования. Динамика подобной сети взаимодействий позволяет микроорганизмам целесообразно использовать свои клеточные ресурсы при образовании биоплёнок и вносит свой вклад в её сложную архитектуру, повышая тем самым приспособляемость бактерий и созданных ими сложных биоплёночных структур к внешним условиям. В целом, в данной работе предложена новая модель локальной регуляции образования биоплёнки с помощью сигнальной молекулы цикло-диГМФ и заложен фундамент для дальнейших исследований механизмов действия отдельных ДГЦ/ДЭ.

Recentemente, o bis-(3\',5\')-di-guanosina monofosfato cíclico (c-di-GMP) surgiu como uma importante molécula sinalizadora nas bactérias. Essa molécula foi identificada como uma das responsáveis pelo controle do comportamento bacteriano e está relacionada com a patogenicidade e a adaptação de diversas bactérias, coordenando a expressão de genes envolvidos com virulência, motilidade e formação de biofilme. O mecanismo pelo qual c-diGMP atua vem sendo motivo de estudo de vários grupos de pesquisa nos últimos anos. Já foi demonstrado o papel dessa molécula em diferentes etapas do controle da expressão gênica. Acredita-se que a manipulação dos níveis de c-di-GMP pode ser uma nova abordagem terapêutica contra bactérias patogênicas. Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que atua como um patógeno oportunista, causando infecções em pacientes imunocomprometidos, sendo o maior causador de infecções crônicas em pacientes portadores de fibrose cística. O genoma de P. aeruginosa PA14 apresenta vários genes que codificam proteínas envolvidas no metabolismo e/ou ligação de c-di-GMP, o que pode indicar um amplo papel regulatório deste nucleotídeo nessa bactéria. Uma associação infundada entre níveis elevados de c-di-GMP e a resistência aos antibióticos é geralmente assumida, já que altos níveis de c-di-GMP levam à formação de biofilme, que é comprovadamente um modo de crescimento mais resistente. Nesse trabalho, utilizando uma abordagem proteômica, mostramos que Pseudomonas aeruginosa PA14 regula a expressão de cinco porinas em resposta a variações nos níveis de c-di-GMP, independentemente dos níveis de mRNA. Uma dessas porinas, OprD, é responsável pela entrada do antibiótico β-lactâmico imipenem na célula e é menos abundante em condições de alto c-di-GMP. Também demonstramos que linhagens com altos níveis de c-di-GMP apresentam uma vantagem competitiva de crescimento em relação a linhagens com níveis mais baixo de c-di-GMP quando crescidas em meio contendo imipenem. Em contraste, observamos que células planctônicas com elevados níveis c-di-GMP são mais sensíveis a tobramicina. Em conjunto, estes resultados mostram que c-di-GMP pode regular a resistência a antibióticos em sentidos opostos, e independentemente do crescimento em biofilme / Following the genomic era, a large number of genes coding for enzymes predicted to synthesize and degrade 3\'-5\'-cyclic diguanylic acid (c-di-GMP) was found in most bacterial genomes and this dinucleotide emerged as an important intracellular signal molecule controlling bacterial behavior. Diverse molecular mechanisms have been described as targets for c-di-GMP, but several questions remain to be addressed. An association between high c-di-GMP levels and antibiotic resistance is largely assumed, since high c-di-GMP upregulates biofilm formation and the biofilm mode of growth leads to enhanced antibiotic resistance; however, a clear understanding of this correlation is missing. Pseudomonas aeruginosa is a versatile gamma-proteobacterium that behaves as an opportunistic pathogen to a broad range of hosts. The ability of P. aeruginosa to form biofilms contributes to its virulence and adaptation to different environments. The P. aeruginosa PA14 genome presents several genes encoding proteins involved in metabolism or binding to c-di-GMP, which may indicate a wide regulatory role of this nucleotide in this bacterium. Here, using a proteomic approach, we show that Pseudomonas aeruginosa PA14 regulates the amount of five porins in response to c-di-GMP levels, irrespective of their mRNA levels. One of these porins is OprD, decreased in high c-di-GMP conditions, which is responsible for the uptake of the β-lactam antibiotic imipenem. We also demonstrate that this difference leads strains with high c-di-GMP to be more resistant to imipenem even when growing as planktonic cells, giving them a competitive advantage over cells with low c-di-GMP. Contrastingly, we found that planktonic cells with high c-di-GMP levels are more sensitive to aminoglycosides antibiotics. Together, these findings show that c-di-GMP levels can regulate the antibiotic resistance to different drugs in opposite ways and irrespective of a biofilm mode of growth.

Le virus de l’hépatite B (HBV) est l’agent étiologique de l’hépatite B. Ce virus est responsable d’hépatite chronique B, de cirrhose et de cancer du foie au niveau mondial. L’absence d’activation de la voie Interféron (IFN) suite à l’infection par HBV est encore mal comprise. Récemment, le senseur cellulaire cytosolic GMP-AMP synthase (cGAS) a été décrit comme un senseur efficace de DNA double brin possédant également une activité antivirale envers des virus à ADN et à ARN. Le but de mes travaux de thèse a été de contribuer à la compréhension des relations existants entre le HBV et cGAS, à des stades précoces et tardifs de l’infection HBV en utilisant des expériences de perte- et gain- de function ainsi que du profilage génomique des génes apparentés à cGAS dans un modéle cellulaire permissif au HBV. Mes travaux ont démontré (1) que cGAS exerce une forte activité antivirale envers le HBV incluant une réduction de la forme nucléaire du génome, le cccDNA; (2) alors que le rcDNA génomique nu est reconnu par la voie cGAS/STING et induit une réponse IFN efficace, la nucléocapside virale protège le DNA génomique viral et l’empêche d’être détecté par la réponse immunitaire innée; et (3) que l’infection par HBV diminue l’expression des acteurs de la voie cGAS-STING et des gènes impliqués dans la réponse immunitaire innée in vitro et in vivo. Ce dernier point met en lumière le rôle de cGAS dans un nouveau mécanisme d’échappement du HBV au système immunitaire inné dans les cellules hépatocytaires et dans ce mécanisme. / Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this PhD work, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an HBV infection-susceptible cell culture model. Collectively, our data show that (1) the cGAS-STING pathway exhibits robust antiviral activity against HBV infection including reduction of viral cccDNA levels; (2) naked HBV genomic rcDNA is sensed in a cGAS-dependent manner whereas packaging of the viral genome during infection abolishes host cell recognition of viral nucleic acids; (3) HBV infection down-regulates the cGAS/STING pathway actors as well as innate immune effector gene expression in vitro and vivo. Overall, this work led to describing new aspects of the complex interaction between HBV and the DNA sensor cGAS in hepatocytes.

Segundos mensageiros nucleotídicos são amplamente utilizados por bactérias para se adaptar às mudanças ambientais e fisiológicas. Neste cenário destaca-se o c-di-GMP, um segundo mensageiro praticamente universal em bactérias responsável por controlar a transição do estilo de vida bacteriano. Em geral, altos níveis celulares de c-di-GMP promovem um estado séssil, de formação de biofilme, enquanto baixos níveis induzem a motilidade. Xanthomonas citri subsp. citri (Xac), um fitopatógeno de grande importância econômica no Brasil, possui uma complexa regulação da sinalização de c-di-GMP, possuindo mais de 30 proteínas envolvidas na síntese, na degradação e na detecção deste segundo mensageiro. Dentre essas proteínas, destacam-se as que possuem os domínios de síntese e degradação presentes na mesma cadeia polipetídica, os domínios GGDEF e EAL respectivamente. A análise das estruturas primárias das 11 proteínas GGDEF-EAL codificadas pelo genoma de Xac revelou que a maior parte delas (6) provavelmente possui o domínio GGDEF inativo, enquanto o EAL é ativo. Três possivelmente possuem ambos os domínios ativos enquanto outras duas possuem ambos os domínios inativos. O nocaute do gene xac2382 que codifica uma dessas proteínas (que possui um domínio periplasmático seguido dos domínios citoplasmáticos HAMP-GGDEF-EAL), demonstra um aumento de motilidade e uma diminuição na formação de biofilme. Construções de fragmentos da proteína revelaram que XAC2382 necessita pelo menos dos domínios HAMP-GGDEF para complementar a cepa nocaute e que a atividade de diguanilato ciclase é essencial para isto. O domínio periplasmático de XAC2382 se mostrou interagir com XAC2383, uma proteína codificada por um gene presente no mesmo cluster do gene de XAC2382, e essa interação parece importante para o controle da motilidade de Xac. A estrutura de XAC2383 foi resolvida por cristalografia de raios X na qual foi revelada uma topologia típica de proteínas da família das periplasmic binding proteins (PBPs) possuindo ainda uma cavidade carregada positivamente contendo um motivo Ser-Thr-Ser (amnioácidos 152-154) importante para a ligação de compostos com grupos fosfatos ou fosfonatos. A mutação sítio dirigida nesse motivo aboliu os efeitos na motilidade dependentes dessa proteína. Esses resultados sugerem que XAC2383 é um sensor periplasmático de um composto eletronegativo e esta proteína interage com XAC2382 regulando a motilidade bacteriana. XAC0495, uma proteína com ambos os domínios GGDEF-EAL provavelmente inativos, pode fazer parte de um sistema de dois componentes com a histidina quinase XAC0494. XAC0495 se comporta como um monômero em solução e possui um formato alongado, como revelado por experimentos de SAXS. / Nucleotide based second messengers are widely used by bacteria in signaling pathways that mediate adaptations to environmental and physiological changes. c-di-GMP is a nucleotide second messenger ubiquitous in Gram-negative bacteria, where it plays a role in many important behaviors that define bacterial lifestyle. In general, high cellular levels of c-di-GMP promote biofilm formation, while low levels induce bacterial motility. Xanthomonas citri subsp. Citri (Xac), a pathogen of great economic importance in Brazil, has a complex repertoire of c-di-GMP signaling molecules, with more than 30 genes coding for proteins involved in the synthesis, degradation and detection of this second messenger. Among these proteins, many have both GGDEF and EAL domains (often associated with c-di-GMP synthesis and degradation, respectively) present in the same polypeptide chain. Analysis of the primary structure of 11 GGDEF-EAL proteins coded by the Xac genome revealed that six most likely possess an inactive GGDEF domain plus an active EALdomain. Another three proteins have both domains active while the other two have both domains inactive. The knockout of the xac2382 gene, coding for a protein which contains a periplasmic domain followed by cytoplasmic HAMP, GGDEF (active) and EAL (active) domains, shows an increase in motility and a decrease in biofilm formation. Constructions containing fragments of this protein revealed that constructs containing at least the HAMP and GGDEF domains are able to complement the knockout strain and that diguanilate cyclase activity is essential for this. The XAC2382 periplasmic domain was shown to interact with a protein encoded by a gene situated in the same cluster, XAC2383, and that this interaction seems crucial for the control of Xac motility. The structure of XAC2383 was solved by X-ray crystallography and was shown to adopt a topology typical of the periplasmic binding proteins (PBP) family. The protein possesses a positively charged groove that contains a Ser-Thr-Ser motif (152STS154) important for the binding of compounds with phosphate or phosphonate groups. Site-directed mutagenesis of this motif abolished the effects on motility caused by this protein. These results suggest that XAC2383 is a periplasmic protein responsible for sensing a compound with electronegative characteristics and which interacts with XAC2382, thereby regulating the bacterial motility. Another protein, XAC0495 (with both GGDEF-EAL domains probably inactive) may be part of a two-component system with the histidine kinase XAC0494. Small-angle X-ray scattering (SAXS) experiments reveal that XAC0495 exists as an elongated monomer in solution.

Les Porifères et les Cténophores sont probablement les deux lignées animales les plus anciennes. Leur étude permet de retracer l’histoire précoce des métazoaires, et d’aborder l'origine de la distinction entre les lignées somatique et germinale. En effet, chez les éponges cette distinction n’existe pas: les archéocytes et choanocytes pouvant donner les cellules somatiques et les gamètes.Après avoir établi la liste des gènes considérés comme impliqués dans la gamétogenèse chez les métazoaires, j’ai caractérisé leurs séquences et retracé leur histoire grâce à des analyses comparatives intégrant les principales lignées animales. Enfin, un suivi in situ d'Oscarella lobularis m’a permis d’affiner son cycle de vie et d’accéder à toutes les étapes du développement pour étudier l’expression de ces gènes et de tester leur implication dans la gamétogenèse.Ainsi, j'ai montré que 18 gènes du GMP (Germline Multipotency Program) sont présents ancestralement chez les animaux. Parmi ceux-ci 11 s’expriment pendant la gamétogenèse, au cours de l’embryogenèse, de la reproduction asexuée et de la régénération. Enfin, le suivi in situ a montré l’influence de la variation de la température et de la matière organique sur le déclenchement de la gamétogenèse.Mon travail suggère d’une part, que la spécification des cellules germinales est régie par des mécanismes génétiques communs à l’échelle de métazoaires, et d’autre part que ces gènes pourraient être impliqués dans la multipotence. Ces résultats renforcent l’hypothèse proposant une origine commune de la lignée germinale et des cellules souches somatiques. / Porifera and Ctenophora are probably the two most ancient animal lineages. Their study therefore allows to trace back the early history of metazoans and to address the origin of the distinction between somatic and germ lines. Indeed, in sponges this distinction does not exist: archeocytes and choanocytes can give rise to both somatic cells and gametes.After establishing the list of genes considered to be involved in gametogenesis in metazoans, I searched for these candidate genes (by local blast) in the transcriptomes of two sponge species (Oscarella lobularis and Oopsacas minuta). I thereby managed to characterize their sequences (phylogenetic and protein domain analyzes) and to trace their evolution through comparative analyzes including all main animal phyla. Finally, the in situ monitoring of O. lobularis enabled me to refine its life cycle and access all key developmental stages in order to study the expression of candidate genes in order to test their possible involvement in gametogenesis in this species.I have shown that 18 GMP (Germline Multipotency Program) genes are present ancestrally in animals. Among them, at least 11 are expressed not only during gametogenesis but also during embryogenesis, asexual reproduction and regeneration. Finally, in situ monitoring showed the influence of temperature variations and organic matter availability on gametogenesis.My work suggests, firstly that the specification of germ cells is controlled by common genetic mechanisms across metazoans, and secondly that these ancestral genes might be involved in pluripotency. These results reinforce the hypothesis suggesting a common origin of the germline and somatic stem cells.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.