1 Abstract Adenylate cyclase toxin (CyaA) is one of the major virulence factors of bacterium Bordetella pertussis, which is a causative agent of whooping cough. CyaA belongs to the family of RTX toxin-hemolysins. The toxin targets primarily cells expressing integrin receptor CD11b/CD18 but it can also penetrate cells lacking this receptor. CyaA acts on host cells by two independent activities. One is formation of small cation-selective channels, which can lead to colloid osmotic lysis of target cells. The second is disruption of cell signaling through the translocation of the adenylate cyclase (AC) domain to host cell cytosol, which leads to the conversion of ATP into cyclic AMP. It was recently shown that cholesterol affects endocytosis of CyaA. CyaA translocates it's AC domain after relocation of CyaA molecule to the cholesterol-rich lipid raft (Bumba et al. 2010). In this work I examined the effect of cholesterol on channel- forming activity and selectivity of ion channels created by CyaA. For measurements I used artificial membranes enriched with cholesterol. CyaA channels are voltage-dependent. The positive membrane potential on the side of toxin is rquired for incorporation of CyaA molecule into cell membrane. I tried to find out whether the value of voltage has effect on channels opening time....

L'objectif principal de ces travaux de thèse est de développer des modèles membranaires biomimétiques pour la reconstitution et l'étude d'interactions protéine/membrane. Dans ce but, deux approches sont adoptées : l'une mettant en œuvre une plateforme basée sur des nanoparticules de silice/Au recouvertes de lipides et l'autre comprenant la formation de bicouches lipidiques découplées d'un support solide d'or. Dans la première approche, nous avons synthétisé des particules de silice de taille nanométrique contenant des grains d'or inclus dans la matrice silicique. Ces nanoparticules sont ensuite recouvertes par différents phospholipides. Les propriétés plasmoniques acquises grâce aux grains d'or sont caractérisées puis utilisées pour suivre l'interaction avec les lipides et/ou les protéines. Le suivi de ces interactions est également visualisé par analyse de la mobilité électrophorétique des particules. La deuxième stratégie développée, consiste à assembler un système membranaire sur une surface solide d'or. Dans un premier temps, une couche de calmoduline est liée à la surface de manière stable. Dans un deuxième temps, une bicouche est formée au-dessus de la couche de calmoduline par deux méthodes. La première méthode consiste à ancrer la bicouche directement sur la couche de protéine par un mécanisme faisant intervenir des lipides chélateurs. Alors que dans la deuxième méthode les lipides sont liés à la surface et découplés grâce à l'utilisation d'une surface d'or modifiée par de la cystéamine et à des lipides fonctionnalisés. L'ancrage est assuré par des groupements succinimidyl et le découplage par des polymères de polyéthylène glycol porté sur un même lipide. Dans les deux stratégies, un réservoir sub-membranaire est créé entre la bicouche étanche et le support. Le suivi des constructions moléculaires est réalisé par résonance plasmonique de surface et analyse du retour de fluorescence. De plus le système est implémenté par des électrodes afin d'étudier l'effet d'application de potentiel sur la bicouche. Après caractérisation, le modèle membranaire est validé par la reconstitution de la translocation de la toxine CyaA de Bordetella pertussis. Cette protéine dispose en effet d'un mécanisme d'internalisation singulier qui permet d'explorer tout le potentiel de notre modèle membranaire. / The main objective of this work is to develop biomimetic membrane models for the reconstitution and study of protein/membrane interaction. Two devices were designed: one operate a nanometric platform composed of phospholipids coated lipid silica/Au nanoparticles, while the other including tethered lipid bilayer reconstitution on a gold surface. The first approach needs the synthesis of nanometer sized gold/silica particles and that are subsequently coated with different phospholipids. The plasmonic properties provided by gold seeds are characterized and they are of utility to follow the interaction between lipids and/or proteins at the surface. Following of these interactions was also realized with electrophoretic mobility analysis. The second biomimetic device involves a membrane assembly on a gold surface. In a first time, a calmodulin layer is bound on the surface. In a second time, a lipid bilayer is assembled above the calmodulin layer by two approaches. In the first approach the lipid bilayer is anchored on the protein layer with chelators lipid and His-Tag bearing by the proteins. While, in the second approach, lipids are bound on the surface and tethered with the use of a cysteamin modified gold surface and functionalized lipids. The anchorage is realized by succinimidyl group and the tethering by polyethylene glycol group wearing by one kind of lipid. A sub-membrane reservoir is created under the lipid bilayer. The biomimetic model formation was followed by plasmonic resonance and fluorescence recovery after photobleaching. After their characterization the tethered model is validated by reconstitution of a particular mechanism: the CyaA toxin from Bordetella pertussis translocation.

Human schistosomiasis popularly known as bilharzias in many regions of Africa is a freshwater snail-transmitted disease caused by parasitic flatworms known as schistosomes. The growth and development of schistosomes typically requires developmental stages in multiple hosts and transmission stages in freshwater. These life cycle environments present a plethora of stressors. Certain gene families including heat shock proteins (HSPs/Hsps) and universal stress proteins (USPs) help schistosomes to respond to unfavourable conditions. The availability of genomes sequences information for Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium provide unique research resources to apply bioinformatics analysis of its associated USPs to predict regulatory features from sequence analysis. The objectives of the research were to (i) Infer the biochemical and environmental regulation of universal stress proteins of Schistosoma species; (ii) Identify biological function relevant protein sequence and structure features for prioritized universal stress proteins from Schistosoma species; (iii) Determine the distinctive structural features of a predicted regulator of Schistosoma adenylate cyclase activity that has possible influence on the functioning of universal stress proteins. The findings revealed that (i) schistosomes USPs are hydrophilic and very reactive in the water environment or in aqueous phase, which seems adaptive with their immediate environment and developmental stages; (ii) The functions of Smp_076400 and Sjp_0058490 (Q86DW2) are regulated by conserved binding site residues and metallic ions ligands (Ca2+, Mg2+ and Zn2+), particularly Ca2+ predicted to bind to both USPs; (iii) The S. mansoni life cycle and stress resistance pathway protein (Smp_059340.1) is regulated by Ser53, Thr188, Gly210 and Asp207 residues. The overall scope has highlighted the role of bioinformatics in predicting exploitable regulatory features of schistosome universal stress proteins and biological pathways that might lead to identification of putative functional biomarkers of common environmental diseases. The findings of this research can be applicable to other areas of environmental health and environmental genomics. / Environmental Sciences / (D. Litt et Phil. (Environmental Sciences)

The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...

The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in virulence of the whooping cough agent Bordetella pertussis. It translocates an AC enzyme into cytosol of CD11b+ phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. In parallel, CyaA permeabilizes cellular membrane by forming cation-selective pores. The goal of my diploma thesis was an analysis of the mechanism of interaction of the segment linking the invasive adenylate cyclase domain and the RTX hemolysin moiety of CyaA with target membrane. Our data show that the segment linking the AC to the hydrophobic domain of CyaA is directly involved in the interaction of the toxin with the membrane and controls the formation of small cationt-selective pores. Our results generate new knowledge that will be of relevance to the entire field of toxin biology and will enable the design of improved CyaA- based vaccines. Keywords: Bordetella pertussis, adenylate cyclase toxin, membrane translocation, pore- forming activity, black lipid bilayers, liposomes

Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....

The soluble form of ADCYs, ADCY10, is ubiquitously expressed in the cytoplasm and distinct organelles including cell nucleus. In contrast to its membrane-associated isoforms (ADCY1-9) which are stimulated by G-protein-coupled receptors, ADCY10 is activated by bicarbonate (HCO3-) and can form cAMP in nearly all cell compartments. ADCY10 is involved in a variety of physiological as well as pathological processes including cell cycle control in tumor cells. However, the underlying mechanism is still unclear. Here the role of ADCY10 in cell cycle control and cell proliferation is studied in endothelial cells from human umbilical veins (HUVECs). The current study reveals that ADCY10 and α-Tubulin translocate and colocalize during mitosis suggesting a role of ADCY10 in cell division. In addition, FACS analysis demonstrated that ADCY10 plays a role in cell proliferation by modulating cell cycle control. Inhibition of ADCY10 by 0 mM HCO3- or 10 μM KH7 (specific ADCY10 inhibitor) induced cell accumulation in G2 phase rather than M phase determined by decreased mitotic indicator cyclin B1 level. Thus, ADCY10 inhibition leads to decreased cell proliferation. The known cAMP effectors, Epac and PKA, were assessed as possible downstream targets of ADCY10 in cell proliferation. It was shown that ADCY10 and Epac induce cell proliferation via ERK1/2-MAPK pathway. Inhibition of Epac was associated with suppressed cell proliferation. However, an arrest of cell cycle after Epac inhibition was observed in G0/G1 phases rather than S or G2/M phases. Thus, Epac inhibition causes a different arrest of cell cycle compared to ADCY10 inhibition. Regarding PKA, it was demonstrated that deficiency of PKA might play a role in either activation or inhibition of cell proliferation. However, direct inhibition of PKA by PKI and H-89 did not lead to cell accumulation in G2. This effect might be associated with broadened roles of PKA in different pathways. In contrast, direct stimulation of PKA under ADCY10 inhibition revealed that PKA is a downstream molecule of ADCY10 as a regulator of cell cycle transition from G2 to mitotic phase. However, the underlying pathway remains to be investigated. The cell cycle transition of G2/M phase is regulated by an auto-amplification loop of cyclin B1/CDK1, which is controlled by the kinase WEE1 and the phosphatase PP2A. WEE1 content was regulated via ADCY10 but was independent of PKA or Epac. Direct inhibition of PP2A showed a suppression of cell proliferation and induced cell cycle arrest in G2. These results were in accordance with those observed after the ADCY10. Furthermore, inhibition of ADCY10 had no effect on PP2A expression level but rather affected PP2A activity and was independent of Epac and PKA. Therefore, this data provides evidence that ADCY10 controls cell proliferation and cell cycle regulation via PP2A. Taken together, ADCY10 coordinates the cell cycle progression in a complex framework. Downstream of ADCY10, Epac promotes G1/S transition, whereas PKA mediates cell cycle transition of G2/M.

Adenylate cyclase toxin (CyaA) produced by the causative agent of whooping cough Bordetella pertussis, is a key virulence factor important for colonization of the host. CyaA targets preferentially myeloid phagocytes expressing CD11b/CD18 integrin. By elevating cytosolic cAMP in the host cells, CyaA interferes with their phagocytic, chemotactic and oxidative burst capacities. Furthermore, CyaA modulates the secretion of cytokines and the maturation state in LPS-stimulated dendritic cells (DC) by affecting the expression of costimulatory molecules. In this study, we investigated the effects of CyaA on the capacity of murine bone-marrow DC to prime CD4+ and CD8+ T cells in response to ovalbumin epitopes delivered by the CyaA-AC- toxoid, as a model antigen. Further, we examined the possible impact of CyaA on the antigen uptake and processing for MHC class I and II-restricted presentation by DC, as we previously observed a decreased T cell stimulatory capacity of CyaA-treated DC in response to soluble ovalbumin. We found out that the high levels of cAMP generated by CyaA in LPS-stimulated DC account for the decreased presentation of ovalbumin epitopes carried by CyaA-AC- toxoid on MHC class I and II molecules, thereby impairing the CD8+ and CD4+ T cell responses. Whereas CyaA did not influence the...

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.

Foram estudadas as enzimas implicadas no metabolismo de cAMP, bem como as variações na concentração deste nucleotídeo cíclico e na atividade de adenilato ciclase durante o ciclo biológico de B. emersonii. Demonstrou-se que os zoósporos contêm enzimas específicas e distintas para a hidrólise de cAMP e cGMP. Existe apenas uma espécie molecular da cAMP fosfodiesterase, que hidrolisa cAMP a 5\'-AMP com um Km aparente de 2-4 µM; a presença de cGMP nas misturas de reação, não altera as propriedades cinéticas da enzima. A adenilato ciclase de B. emersonii é uma enzima particulada, provavelmente ligada à membrana plasmática do zoósporo, que exige especificamente Mn2+ para sua atividade. A enzima não é ativada por NaF, catecolaminas ou outros compostos de estrutura semelhante. O estudo das propriedades cinéticas da adenilato ciclase sugere um modelo simples no qual o verdadeiro substrato da enzima é o complexo MnATP2- e tanto ATP corno Mn2+ , nas suas formas livres, competem com o complexo pelo sítio catalítico da enzima, que apresenta uma afinidade maior pelas formas livres do que pelo complexo MnATP2-. A atividade especifica da adenilato ciclase, determinada durante o ciclo biológico do fungo, mostra-se elevada nos zoósporos, cai lentamente durante a germinação e permanece baixa em todo o período de crescimento, só voltando a apresentar um aumento na atividade após a indução da esporulação. Quando este processo e induzido na presença de cicloheximida, a atividade permanece baixa, sugerindo que a enzima é sintetizada \"de novo\" nesta fase do ciclo evolutivo. A concentração intracelular de cAMP foi também determinada nas várias fases do ciclo biológico de B. emersonii. No zoósporo encontrou-se um valor médio de 33 pmoles cAMP/mg proteína. Durante a germinação, os níveis de cAMP aumentam, atingindo um máximo (~ 100 pmoles/mg proteína)quando a quase totalidade dos zoósporos se transformou em esferócitos. A partir daí observou-se um declínio gradual nos níveis de cAMP, que permanecem baixos durante toda a fase de crescimento, voltando a elevar-se na fase final da esporulação até alcançar o nível de zoósporo. O grande aumento na concentração intracelular de cAMP na fase de esferócitos é parcialmente explicado pela predominância da atividade de adenilato ciclase sobre a atividade de cAMP fosfodiesterase neste período; a possibilidade de uma ativação \"in vivo\" da adenilato ciclase, neste estágio do ciclo, não pode ser excluída. A queda nos níveis de cAMP que ocorre na passagem de esferócito a gérmen, numa fase onde a atividade de cAMP fosfodiesterase já e muito baixa, é devido principalmente a excreção deste nucleotídio cíclico para o meio extracelular. O grande aumento nos níveis de cAMP durante a transição de zoósporo a esferócito pode estar relacionado com a ativação metabólica ocorrendo nesta fase e pode também refletir uma característica de sistemas em diferenciação, isto é, a necessidade de altos níveis de cAMP para a transição entre dois estados celulares diferenciados. / The enzymes involved in the metabolism of cAMP have been studied, as well as the fluctuations in the concentration of this cyclic nucleotide and in the adenylate cyclase activity during the life cycle of B. emersonii. Zoospores were shown to contain independent specific enzymes involved in the hydrolysis of cAMP and cGMP. A single molecular species was found for the cAMP phosphodiesterase activity, which catalyses the hydrolysis of cAMP to 5\'-AMP. This enzyme displays normal Michaelis kinetics with an apparent Km of 2-4 µM; the addition of cGMP to the reaction mixtures does not modify the kinetic properties of the enzyme. Adenylate cyclase activity in B. emersonii is associated with particulate subcellular fractions, most probably bound to the zoospore plasma membrane. The activity requires Mn2+ and it is not activated by NaF, cathecolamines or other related compounds. The enzyme substrate is the MnATP2- complex and the kinetic data obtained studying the adenylate cyclase activity can be explained by a simple model where free ATP and Mn2+ compete with MnATP2- for the catalytic site of the enzyme, the affinity for MnATP2- being lower than for free Mn2+ and ATP. The specific activity of adenylate cyclase has been determined throughout the fungus life cycle. The enzyme activity is high in zoospores, falls slowly during germination remaining low at the growth phase and rising again during the later stage of sporulation. When this process is induced in the presence of cycloheximide, there is no increase in adenylate cyclase activity, suggesting that \"de novo\" synthesis of the enzyme occurs at this stage. Fluctuations in the intracellular levels of cAMP during the cell cycle of B. emersonii have also been shown. Zoospores contain an average concentration of 33 pmoles cAMP/mg protein. During germination, a significant increase in the cAMP levels is observed, reaching a maximum (ca. 100 pmoles/mg protein) when the majority of the zoospores have changed into round cells. From then on a gradual decline in the cAMP levels is observed. During the growth phase the cAMP contents of the cells remain low, increasing again late in the sporulation stage. The large increase in the intracellular concentration of cAMP in the round cell phase is partially explained by the predominance of adenylate cyclase activity over cAMP phosphodiesterase activity (during this stage); the possibility of an \"in vivo\" activation of the adenylate cyclase during this period, however, cannot be excluded. The decrease in the cAMP levels occurring during the passage of round cells to germlings, in a stage where cAMP phosphodiesterase activity is negligible, is mainly due to the excretion of this cyclic nucleotide to the extracellular medium. The rise in cAMP contents during encystment might be related to the activation of metabolism occurring in this phase and may also reflect a characteristic of differentiating systems, that is, high cAMP levels being necessary for a differentiative transition.