Symbiosis is the close and protracted interaction between organisms. The molecular interactions that occur during symbiosis are complex with multiple barriers that must be overcome. Many Gram-negative, host-associated bacteria use a type III secretion system to mediate associations with their eukaryotic hosts. This secretion system is a specialized apparatus for the injection of type III effector proteins directly into host cells, which in the case of plant pathogens, are collectively necessary to modulate host defense. The type III secretion system is not a mechanism exclusive to pathogens, however, as many strains of commensal Pseudomonas fluorescens and mutualistic rhizobia demonstrably require a type III secretion system to interact with their host plants. The work presented in this thesis describes genome-enabled approaches for characterizing type III effector genes across the range of plant symbiosis. Using high-throughput sequencing technology, draft genome sequences were generated for the plant pathogen, Xanthomonas hortorum pv. carotae M081, the plant commensal, Pseudomonas fluorescens WH6, and six strains from the plant mutualists Sinorhizobium fredii and Bradyrhizobium japonicum. Analyses of the draft genome sequences and publicly available finished sequences contributed insights into mechanisms of host-association and to increasing the inventory of type III effector sequences as well as developing methods directly applicable for agriculture. Finally, characterization of the genetic diversity of type III effectors from rhizobia shows that collections of type III effectors of mutualists are static, with little diversity in content and sequence variation. This represents the first comprehensive cataloging of type III effector from species of mutualistic bacteria and the first to provide evidence for purifying selection of this important class of genes. / Graduation date: 2012

In this thesis study, a two-fingered gripper and a four-fingered multipurpose gripper are developed and manufactured. In addition to development of robotic hands, computer control hardware and software are also developed for computer control of both hands. The two-fingered gripper is designed for a specially defined pick and place operation. Its task is to pick a cylindrical work piece and place it in the appropriate position in a flexible manufacturing cell. Pneumatic actuator is used for power generation and mechanical links are used for power transmission. Fourfingered gripper is designed as a multipurpose gripper. The task is not predefined for this gripper, so, human hand and previous dexterous hands are taken as model during design. It consists of 3 fingers and a thumb. It has 1 degree of freedom for every finger and thumb. Pneumatic actuators are also used for this gripper. Rope and pulley system is used for the power transmission mechanism. Structures of both hands are manufactured from 5083 series aluminum. Gripping force can be controlled by the pressure regulator of the pneumatic system for both hands. Computer software is developed for the control of open and close motion of the fingers. Also, a motion control card is designed and manufactured for control of the pneumatic valves.

The flavin-containing monooxygenases (FMO / E.C.1.14.13.8) are microsomal NADPH and oxygen-dependent flavoprotein enzymes that catalyze the oxidation of a wide variety of xenobiotics, including drugs and environmental toxicants. Nucleophiles containing nitrogen, sulfur, phosphorus and selenium heteroatoms are the substrates of FMO. Bovine liver microsomal FMO enzyme activity was characterized using methimazole as substrate, which is a highly specific substrate for FMO. From 12 different bovine liver samples, microsomes were prepared and the average specific activity of bovine liver microsomal FMO was found to be 2.37 &amp / #61617 / 0.30 nmol/min/mg (Mean &amp / #61617 / SE, n=12). The rate of reaction was linear up to 0.5 mg of bovine liver microsomal protein. The maximum FMO enzyme activity was detected at 37 &amp / #61616 / C and at pH 8.0. Effects of detergents / Triton X-100 and Emulgen 913, on FMO activity were determined and found that enzyme activity increased by the addition of either detergent at all concentrations (0.1%-1.0%). The apparent Vmax and Km values of bovine liver microsomal FMO for methimazole substrate were found as 1.23 nmol/min/mg and 0.11 mM, respectively. Thermostability of bovine liver microsomal FMO was studied at four different temperatures / 24 &amp / #61616 / C, 37 &amp / #61616 / C, 50 &amp / #61616 / C and 65 &amp / #61616 / C. The incubation time required for the complete loss of enzyme activity was 5 minutes at 65 &amp / #61616 / C, 10 minutes at 50 &amp / #61616 / C and 6.5 hours at 37 &amp / #61616 / C. 68 % of the activity was still detectable at the end of 53 hours at 24 &amp / #61616 / C. Bovine liver microsomal activity towards two drug substrates, imipramine and chlorpromazine, was also determined and found to be 3.73 and 3.75 nmol NADPH oxidized/min/mg, respectively. Effects of two drug substrates, imipramine and chlorpromazine, on bovine liver microsomal FMO-catalyzed methimazole oxidation activity was also studied and found that they inhibit FMO activity at all concentrations studied. Modulation of bovine liver microsomal FMO activity was studied using three different heavy metal ions / Ni+2, Cd+2 and Hg+2. At all other concentrations studied for each heavy metal ion and at all substrate methimazole concentrations (0.1, 0.2, 0.5, 1.0 mM), FMO-catalyzed methimazole oxidation activity decreased compared to control activity. KI values for Ni+2, Cd+2 and Hg+2 were found to be 0.5 mM, 0.085 mM, 4.6 &amp / #61549 / M, respectively. From the Dixon plot, the pattern of inhibition for three heavy metal ions was observed to be noncompetitive.

Apicomplexan parasites are leading causes of human and livestock diseases such as toxoplasmosis and malaria caused by Toxoplasma gondii and Plasmodium falciparum respectively. These organisms are varied in their morphologies and astoundingly complex on their life cycles that include infections of more than one host organism, differentiation through several morphologically distinct forms, and both sexual and asexual replication. What we and others have initially proposed was that the control of gene expression and cellular differentiation are particularly interesting in these organisms, as the apparent lack of large families of recognizable transcription factors typically found in other eukaryotic organisms suggests that they may be unusually reliant on epigenetic mechanisms. The initial hypothesis had to be re-assessed in light of the discovery in Apicomplexa of an expanded family of plant-like transcription factors (TFs) harbouring APETALA2 (AP2)-like domains. Yet, a growing body of evidence tends to favor epigenetic as one of the main contributor to parasite developmental programs and adjustments to fluctuant environment. One way to examine dynamic changes in post-translations modifications (PTMs) patterns is to alter the histone code writing. We therefore took advantage of HDAC inhibitors and showed that specific inhibition of TgHDAC3 by the cyclopeptide FR235222 disrupts the genome wide steady-state level of histone H4 acetylation inducing derepression of stage-specific genes. Yet, many questions about TgHDAC3 modus operandi remain unanswered. During my thesis, I uncovered the TgHDAC3-regulated proteome-wide acetylome typified by the presence of non-histone proteins including AP2 TFs and novel PTMs, e.g. the acetylation at Lys31 within the globular domain of histone H4. H4K31ac promotes a relaxed chromatin state at the promoter of active genes through nucleosome disassembly in both parasites. We identified TgGCN5B and TgHDAC3 as two antagonist enzymes regulating H4K31 acetylation in T. gondii. In contrast, H4K31monomethylation is enriched throughout the gene body of T. gondii active genes and contributes to transcription, whereas it is enriched at transcriptionally inactive pericentromeric heterochromatin regions in P. falciparum, a region that is lacking H3K9me3 and heterochromatin protein 1 in this parasite. We also showed that treating T. gondii cystogenic strains with a low dose of FR235222 induces the levels of proteins known to be expressed exclusively in cat (sporozoite and merozoite) or in murine chronic stage (bradyzoite). Lastly, we determined the specific interactome of TgHDAC3 and found as partners a MORC protein (CR230), several AP2 TFs, and ELM2 domain-containing scaffolding proteins. Collectively, these data established TgHDAC3 family as a central regulator of gene expression and stage conversion in T. gondii and, likely, other Apicomplexa. / Apicomplexan genome architecture is typified by a binary chromatin structure, with a major fraction of the bulk genome packaged as transcriptionally permissive euchromatin while few loci are embedded in silenced heterochromatin. There is evidence that histone modifications occurring at the lateral surface of the nucleosome play a substantial role in shaping chromatin structure, yet our understanding of the exact mechanism of action is poor. Here, we address how versatile modifications at Lys31 within the globular domain of histone H4 contribute to genome organization and expression in Apicomplexa. H4K31 acetylation was found at the promoter of active genes. The residue lies where the DNA wraps around the histone and its acetylation may enhance nucleosome disassembly, thereby favoring a more relaxed, open chromatin state. This residue tends also to be monomethylated and depending of the parasite examined different patterns were found. H4K31me1 was enriched in the core body of Toxoplasma active genes, yet its occupancy was inversely correlated with transcripts levels likely because the mark by reducing histone turnover impedes RNA polymerase progression across transcribed units. In contrast to the methylation of H3, it is the first time that a methylated residue of H4 has been clearly associated with transcriptional regulation. In Plasmodium, H4K31me1 was exclusively enriched at transcriptionally inactive genomic regions and peculiarly at pericentromeric heterochromatin, likely to replace the missing H3K9me3 that commonly decorated pericentric nucleosomes in other species.

Made available in DSpace on 2015-03-26T13:37:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 992783 bytes, checksum: 865bebc902b66a9a2f40688ff9d50a96 (MD5) Previous issue date: 2012-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Brazil is the second world soybean producer. Currently, the main limiting factor in this crop production is the Asian soybean rust (ASR) whose etiologic agent is the fungus Phakopsora pachyrhizi. The rust fungi are obligate parasites that during their interaction with the plant, they secrete effector proteins that manipulate host metabolism and interfere with their defense responses. Some of these effector proteins, called Avr proteins, are recognized by encoded proteins by resistant R genes, which usually trigger a hypersensitivity response (HR) and resistance phenotype. At least, there are five described R genes (Rpp1 to Rpp5) that confer resistance to ASR and several genes that encode secreted proteins by this fungus were recently identified. However, the effector proteins (Avr) recognized by encoded proteins by Rpp genes were not identified yet. Since there is not a transformation assay protocol for P. pachyrhizi, a strategy to identify this fungus Avr proteins is the transient expression of R proteins in resistant varieties cytoplasm and the observation of a possible HR response. Thus, the specific objectives of this work were: to try to establish a methodology for transient expression in soybean by agroinfiltration using the gene GUSPlus as reporter gene; to establish a protocol for translocation of effector proteins by the type III secretion system (SST3) of Pseudomonas syringae pv. glycinea race 4 (Psg4), and also to evaluate the effector activity of candidate genes in soybean resistant genotypes to the isolate PPUFV02 of P. pachyrhizi. There was no expression of the gene GUSPlus in Agrobacterium tumefaciens strain EHA105 infiltrated soybean leaves, while using the same inoculum preparation and concentration of bacterial cells, there was a consistent expression of the gene GUSPlus in tobacco. This result derailed the use of agroinfiltration in the functional study of candidate genes in soybean effectors. All soybean genotypes evaluated were susceptible to Psg4, demonstrating that the viability to use this bacterial on functional analysis of candidate effector proteins mediated by SST3. Better symptoms reproducibility was observed with inoculation by vacuum infiltration of Psg4 in a bacterial concentration of OD600 = 0,01, for allowing a gradually symptoms analysis. The encoded protein by avrB gene is recognized by the RPG1 gene product, which is present in some genotypes of soybean. The construction pVSP61-avrB was able to induce HR in the genotype Williams 82, that contains the corresponding gene RPG1 and to induce it in the genotypes Conquista and PI 459025. This result allowed the use of this construction as a positive control for functional analysis of P. pachyrhizi putative effector proteins. Because of this, 12 sequences were cloned into vector pEDV6. This vector allows the expression of proteins of interest fused to secretion signal sequences by SST3, aiming its subsequent translocation into the cytosol. Nine from the constructions with pEDV6-PHPA_RSP transformed into Psg4 were submitted to functional analysis. The inoculated plants varied in severity of observed symptoms and no HR phenotype was observed. Instead, it was observed reduction, increase or absence of a significant change in the symptoms evolution of genotype-dependent manner in treated plants. These studies allowed a first screening of P. pachyrhizi effector candidates, selecting the candidates PHPA_RSP_71 and PHPA_RSP_78 as the most promising candidates for further detailed analysis. / O Brasil é o segundo maior produtor mundial de soja. Atualmente, o principal fator limitante na produção desta cultura é a ferrugem asiática da soja (FAS), cujo agente etiológico é o fungo Phakopsora pachyrhizi. Os fungos causadores das ferrugens são parasitas obrigatórios que durante sua interação com a planta secretam proteínas efetoras que manipulam o metabolismo do hospedeiro e interferem com suas respostas de defesa. Algumas dessas proteínas efetoras, denominadas proteínas Avr, são reconhecidas pelas proteínas codificadas por genes de resistência R, o que desencadeia a resposta de hipersensibilidade (HR) e fenótipo de resistência. Já foram descritos pelo menos cinco genes R (Rpp1 a Rpp5) que conferem resistência a FAS e vários genes que codificam proteínas secretadas por esse fungo foram recentemente identificados. Todavia, ainda não foram identificadas as proteínas efetoras (Avr) reconhecidas pelas proteínas codificadas pelos genes Rpp. Como não existe ainda um sistema de transformação para P. pachyrhizi, uma estratégia para identificar as proteínas Avr desse fungo é a expressão transiente das proteínas efetoras candidatas no citoplasma de variedades resistentes e a observação do possível desencadeamento de resposta de HR. Desta forma, os objetivos específicos deste trabalho foram tentar estabelecer uma metodologia de expressão transiente em soja via agroinfiltração utilizando como gene repórter o gene GUSPlus; estabelecer um protocolo de translocação de proteínas efetoras via sistema de secreção tipo III (SST3) de Pseudomonas syringae pv. glycinea raça 4 (Psg4), e também avaliar a atividade efetora de genes candidatos em genótipos de soja resistentes ao isolado monopustular PPUFV02 de P. pachyrhizi. Não se observou a expressão do gene GUSPlus em folhas de soja agroinfiltradas com Agrobacterium tumefaciens estirpe EHA105, enquanto que utilizando do mesmo preparo de inóculo e concentração de células bacterianas, observou-se a expressão consistente do gene GUSPlus em tabaco. Este resultado invibializou o uso de agroinfiltração no estudo funcional de genes efetores candidatos na soja. Todos os genótipos de soja avaliados foram suscetíveis a Psg4, demonstrando a vialibilidade do uso desta bactéria na análise funcional de proteínas candidatas a efetores mediada por SST3. Melhor reprodutibilidade de sintomas foi observada com a inoculação por infiltração a vácuo de Psg4 numa concentração bacteriana de OD600=0,01, por permitir uma análise dos sintomas de forma gradual. O produto do gene avrB é reconhecido pela produto do gene RPG1, presente em alguns genótipos de soja. A construção pVSP61-avrB, foi capaz de induzir HR no genótipo Williams 82, que contêm o gene RPG1 correspondente, e nos genótipos Conquista e PI 459025. Este resultado permitiu o uso desta construção como controle positivo para a análise funcional de proteínas efetoras putativas de P. pachyrhizi. Assim, 12 sequências foram clonadas no vetor pEDV6. Este vetor permite a expressão de proteínas de interesse fusionadas a sequências-sinais de secreção via SST3, visando a sua posterior translocação para o citosol. Nove das construções com pEDV6-PHPA_RSP transformadas em Psg4 foram submetidas à análise funcional. As plantas inoculadas variaram quanto à severidade dos sintomas observados e não foi constatado fenótipo de HR. Ao invés disso, nos tratamentos foi observado redução, aumento ou ausência de alteração significativa na evolução dos sintomas, de maneira genótipo-dependente. Esses estudos permitiram uma primeira triagem de candidatos a efetores de P. pachyrhizi, selecionando os candidatos PHPA_RSP_71 e PHPA_RSP_78 como os mais promissores para estudos futuros mais detalhados.

In order to fulfill increasing production rates, new automated production technologies are required for manufacturing carbon fiber reinforced plastic components for the aerospace industry. Currently, large, double curved composite components have to be manufactured manually, which leads to high process times and poor scalability. As a consequence, a team of cooperating robots with passively adjustable end-effectors was developed, that is capable of handling dry carbon textiles and can be used for layups in double curved molds. This thesis deals with the implementation of a robot program, that performs an automated adjustment of each end-effector to the surface geometry of the manufactured part. The functional principle and the accuracy of the process are evaluated. Further, the automatically adjusted end-effectors are utilized to cooperatively layup carbon plies. The results show, that the accuracy of the automated adjusting process is sufficient to drape carbon fabrics during pick-up and automated layup is possible with this approach. In conclusion, the developed process can be integrated into a fully automated process for future experiments, but hardware inaccuracies should be improved, in order to further enhance the accuracy of the system.

Les cellules dendritiques (DC) sont une charnière entre immunité innée et adaptative. Les DC du sous-type CD8a+ excellent à la présentation croisée d’antigènes exogènes pour activer les lymphocytes T (LT) CD8+. Mon projet de thèse consistait à analyser la contribution de gènes sélectivement exprimés par les DC CD8a+ à leur spécialisation fonctionnelle. Nous avons d’abord analysé le rôle du récepteur de chimiokine XCR1 que les DC CD8a;+ expriment spécifiquement. XCR1 reconnait XCL1, une chimiokine qui a un pouvoir attractant sur les DC CD8a+ in vitro, et qui est exprimée par les cellules NK, les lymphocytes innés de type 1 et les LT CD8+ activés. Nous avons montré que XCR1 était indispensable à l’hôte pour résister à une infection par le cytomégalovirus murin, en permettant l’activation optimale des cellules NK. L’axe XCL1-XCR1 est donc un moyen privilégié de communication entre les cellules NK et les DC CD8a+. Ensuite, nous avons utilisé un modèle murin ciblant spécifiquement les DC CD8a+ pour montrer leur rôle majeur dans la réactivation des LT CD8+ mémoires au cours d’infections secondaires. Enfin, nous avons tenté d’analyser si une autre molécule préférentiellement exprimée par les DC CD8a+, la GTPase Rab7b, était impliquée dans la présentation croisée des antigènes. Dans d’autres cellules, Rab7b interviendrait dans l’échange de matériel entre le réseau trans-Golgien et les endosomes tardifs. Dans les DC CD8a+, cette activité pourrait favoriser le trafic intracellulaire des antigènes vers les endosomes où s’effectue la présentation croisée. Mon travail de thèse a permis de mieux comprendre comment les DC CD8a+ activent les lymphocytes effecteurs innés et adaptatifs. / Dendritic cells (DC) link innate and adaptive immunity. The CD8α+ DC subtype is particularly efficient in activating CD8+ T cells through cross-presentation of exogenous antigens. The aim of my PhD project was to analyze the role of genes specifically expressed by CD8a+ DC in the functional specialization of these cells. First we investigated the role of the chemokine receptor XCR1 in the biology of CD8a+ DC. In mice, XCR1 is a powerful chemo-attractive molecule for CD8a+ DC in vitro. It recognizes XCL1, a chemokine expressed by NK cells, type 1 innate lymphoid cells, and activated CD8+ T cells. We have shown that XCR1 is crucial for the resistance to murine cytomegalovirus (MCMV) infection, allowing optimal activation of NK cells. Thus, the XCL1-XCR1 axis is a privileged way of communication between NK cells and CD8a+ DC. We then used a mouse model which specifically targets CD8a+ DC, to show their major role in the reactivation of memory CD8+ T cells upon diverse secondary infections. Finally, we tried to investigate the potential role in antigen cross-presentation of another molecule selectively expressed by CD8a+ DC, the Rab7b GTPase. ,. Indeed, in another cell type, Rab7b could be involve in material exchange between the trans-golgi network and late endosomes. In CD8a+ DC, this process could favor the intracellular trafficking of endocytosed antigens towards the endosomes where cross-presentation occurs. In summary, my PhD work advanced our understanding of how CD8a+ DC activate innate and adaptive effector lymphocytes

Mon travail a porté sur la caractérisation des effecteurs toxiques et protéines d’immunité du T6SS Sci-1 d’Escherichia coli Entero-agrégatif, éléments de la lutte inter-bactérienne. Nous avons identifié en outre Tle1, un effecteur de toxine codé par ce groupe et montré que Tle1 possède des activités de phospholipase A1 et A2 requises pour détruire la cellule proie dans la compétition interbactérienne. L'auto-protection de la cellule attaquante est assurée par une lipoprotéine de membrane externe, Tli1, qui lie Tle1 dans un rapport stoechiométrique 1: 1 avec une affinité nanomolaire et inhibe son activité phospholipase. Il a été prédit que la protéine 435 provenant à partir d'un groupe de gènes T6SS1 de l'agent pathogène AIEC LF82 est une phospholipase de la famille d'effecteurs Tle3 avec une activité PLA1. Sa toxicité peut être neutralisée par la protéine d'immunité cognate 434 qui est un Tli3 putatif, en formant le complexe de protéine Tle3 - Tli3. Les deux protéines séparées et leur complexe ont ensuite été appelées protéines complexes Tle3AIEC, Tli3AIEC et Tle3AIEC - Tli3AIEC, respectivement. Afin d'étudier plus en détail le mécanisme de Tle3-AIEC et de Tli3-AIEC, nous avons réalisé l'expression, la purification, la caractérisation, la cristallisation des deux protéines et des études cristallographiques de rayons X préliminaires du complexe Tle3-AIEC/Tli3-AIEC afin de comprendre comment la protéine Tle3-AIEC reconnaît et se lie à son effecteur apparenté Tli3-AIEC et inhibe son activité. Les données préliminaires de diffraction des rayons X ont été recueillies à partir de cristaux Tle3AIEC-SeMet/Tli3AIEC à une résolution de 3,8 Å. / Here, we analyzed the Entero-aggregative Escherichia coli Sci-1 T6SS toxin effectors. We identified Tle1, a toxin effector encoded by this cluster and show that Tle1 possesses phospholipase A1 and A2 activities required for the inter-bacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity.The protein 435 from the pathogen AIEC LF82 has been predicted to be a phospholipase of the Tle3 effector family with PLA1 activity from a T6SS1 gene cluster. Its toxicity can be neutralized by the cognate immunity protein 434 that is a putative Tli3, by forming Tle3 - Tli3 protein complex. The two separated proteins and their complex were then called Tle3AIEC, Tli3AIEC and Tle3AIEC - Tli3AIEC complex proteins, respectively. In order to further investigate the related mechanism of Tle3AIEC and Tli3AIEC, we performed expression, purification, characterization, crystallization of the two proteins and preliminary X-ray crystallographic studies of the Tle3AIEC - Tli3AIEC complex in order to understand how Tle3AIEC protein recognizes and binds to its cognate Tli3AIEC effector and inhibits its activity. X-ray diffraction data were collected from selenomethionine-derivatize Tle3AIEC SeMet - Tli3AIEC crystals to a resolution of 3.8 Å.

L. pneumophila est une bactérie ubiquitaire des environnements aquatiques, responsable de la légionellose. Elle est principalement retrouvée au sein de protozoaires, mais aussi dans les biofilms. Il est admit que le fer est l'un des éléments indispensable à la croissance de ce pathogène. En 2008, une étude a été réalisée au sein de notre équipe montrant une modulation de l'expression des gènes entre L. pneumophila à l'état planctonique, et à l'état de biofilm. Dans cette même étude, l'ajout de forte concentration en fer (1,25 g/l), dans le milieu de culture des biofilms, a révélé une inhibition de leur développement. Le fer présente donc un rôle dans l'établissement de biofilms mono espèces de L. pneumophila. Nous avons développé un modèle de biofilms naturels, formés à partir d'eau de rivière, afin de tester l'établissement de L. pneumophila, dans des conditions où l'eau de rivière est supplémentée en fer ou au contraire appauvrit, par l'ajout de chélateurs, le deferoxamine mesylate (DFX) ou le dipyridyl (DIP). Les ajouts de fer et de DFX n'ont eu aucun impact sur l'établissement de L. pneumophila contrairement au DIP, qui a induit une augmentation de l'implantation de ces bactéries.Par ailleurs, nous avons effectué une analyse transcriptomique sur L. pneumophila cultivées en milieu liquide supplémenté en DFX. L'ajout du chélateur a entrainé une induction de l'expression de 113 gènes et la répression de 246 gènes. Parmi les gènes induits, certains sont déjà connus comme étant impliqués dans le métabolisme du fer ou contrôlés par le fer. Parmi eux, un gène a été surexprimé, il n'a jamais été associé au fer et sa fonction est encore inconnue à ce jour. Il s'agit du gène lpp2867. Des investigations ont été réalisées afin de caractériser et de comprendre le rôle de la protéine pour laquelle il code. Elle est notamment impliquée dans l'infection des amibes et des macrophages. Son rôle dans le transport du fer ferreux a également été mis en avant. Cette protéine a été nommée IroT pour « iron transporter ». / L. pneumophila is a ubiquitous bacterium found in aquatic environments, and responsible for legionellosis. It is mainly found into both protozoa and biofilms. Iron is a key nutrient for an optimal growth of this pathogen. In 2008, a transcriptomic analysis was carried out within our team, revealing a differential expression of genes involved in iron metabolism between sessile and planktonic bacteria. Also, this study showed that, for high iron concentrations (1.25 g/l), biofilm formation by L. pneumophila was inhibited. It suggested that iron is important for biofilm formation by L. pneumophila. To extend these observations in more natural conditions, a model of biofilm formation, using natural river water, was developed. Water was spiked with L. pneumophila and supplemented with iron or iron chelator, deferoxamine mesylate (DFX) or dipyridyl (DIP). Addition of iron and DFX did not have any effect on L. pneumophila establishment unlike the DIP which induced an increase of L. pneumophila concentration into the biofilms.Otherwise, we performed transcriptomic analysis on L. pneumophila grown in liquid medium supplemented with DFX. The addition of this chelator led to the induction of 113 genes and 246 genes were repressed significantly. Among the induced genes, some are involved in iron metabolism or controlled by iron. There was an induced gene, lpp2867, never associated with iron metabolism and whose function was still unknown. Investigations were realized to characterize and understand the role of the protein encoded by this gene. It is involved in L. pneumophila virulence and in ferrous iron assimilation. This new protein was named IroT for iron transporter.

La symbiose ectomycorhizienne associe les racines d’un arbre aux hyphes d’un champignon, conduisant à un échange réciproque de nutriments entre les deux partenaires. La colonisation fongique massive du cortex racinaire est caractérisée par la formation d’une interface symbiotique, le réseau de Hartig. L’acquisition du génome du symbionte ectomycorhizien Laccaria bicolor a permis d’identifier des petites protéines prédites sécrétées, les MiSSPs (Mycorrhiza-induced Small Secreted Proteins). Mon projet de thèse avait pour objectifs la comparaison des sécrétomes, notamment les petites protéines sécrétées (SSPs), de champignons ectomycorhiziens et saprotrophes, la localisation subcellulaire in planta et l’analyse fonctionnelle de MiSSPs de L. bicolor. L’analyse bioinformatique a notamment permis de révéler des clusters de SSPs conservées entre champignons ectomycorhiziens et saprotrophes ou spécifiques de champignons ectomycorhiziens, mettant en lumière que les champignons ectomycorhiziens partagent des SSPs avec leurs ancêtres saprotrophes mais possèdent aussi d’autres SSPs spécifiques à leur mode de vie. Un jeu de MiSSPs de L. bicolor ont été localisées in planta. Trois d’entre eux ciblent spécifiquement des compartiments subcellulaires de la cellule végétale. Le motif répété DWRR présent dans la séquence de MiSSP8 est partagé par une famille de protéine fongique de champignons majoritairement saprotrophes. Ces résultats suggèrent que l’utilisation de SSPs comme moyen de communication est générique chez les champignons et démontrent aussi qu’au moins une petite protéine sécrétée requise pour la symbiose de L. bicolor a évoluée à partir de protéines de champignons saprotrophes / Roots of most trees form ectomycorrhizal (ECM) symbiosis with mutualistic soil-borne fungi, relying on a bi-directional exchange of nutrients between the two partners. Fungal colonization of cortical root cells form the Hartig net, a symbiotic interface. Functioning of this symbiotic interface is not well known. However, Laccaria bicolor genome sequencing sheds the light on upregulated small-secreted proteins, so-called MiSSPs (Mycorrhiza-Small Secreted Proteins). Several L. bicolor MiSSPs were demonstrated as symbiosis effectors. My PhD project aims to compare secretomes, in particular SSPs, of fungal with different lifestyles and pursue functional analysis of MiSSPs of L. bicolor. Based on the clustering analysis, we identified clusters of SSPs shared between saprotrophic and ECM fungi and clusters of SSPs specific to ECM-fungi. This study highlights that ECM fungi share SSPs with their saprotrophic ancestors but also possess lifestyle specific SSPs. In planta subcellular localization of a set of MiSSPs belonging to a core-regulon showed that three of them are able to target different plant subcellular compartments. Functional analysis of the symbiosis effector MiSSP8 does not lead to the identification of a putative interactor but the repetitive motif DWRR of MiSSP8 protein sequence is unique to fungi and is shared with SSPs from saprotrophic ancestors. Our results suggest the use of SSPs as mean of communication is common and generic and show at least one SSPs required for ectomycorhizal symbiosis of L. bicolor has evolved from SSPs found in saprotrophic fungi