Campylobacter jejuni is the leading bacterial cause of gastrointestinal diarrheal disease in humans worldwide. This zoonotic pathogen has a complex epidemiology due to its presence in many different host organisms. The overall aim of this thesis was to explore the role of amoebae of the genus Acanthamoeba as an intermediate host and vector for survival and dissemination of C. jejuni. Earlier studies have shown that C. jejuni can enter, survive and replicate within Acanthamoebae spp. In this thesis, I have shown that C. jejuni actively invades Acanthamoeba polyphaga. Once inside, C. jejuni could survive within the amoebae by avoiding localization to degradative lysosomes. We also found that A. polyphaga could protect C. jejuni in acid environments with pH levels far below the range in which the bacterium normally survives. Furthermore, low pH triggered C. jejuni motility and invasion of A. polyphaga. In an applied study I found that A. polyphaga also could increase the survival of C. jejuni in milk and juice both at room temperature and at +4ºC, but not during heating to recommended pasteurization temperatures. In the last study we found that forty environmental C. jejuni isolates with low bacterial concentrations could be successfully enriched using the Acanthamoeba-Campylobacter coculture (ACC) method. Molecular genetic analysis using multilocus sequence typing (MLST) and sequencing of the flaA gene, showed no genetic changes during coculture. The results of this thesis have increased our knowledge on the mechanisms behind C. jejuni invasion and intracellular survival in amoebae of the genus Acanthamoeba. By protecting C. jejuni from acid environments, Acanthamoebae could serve as important reservoirs for C. jejuni e.g. during acid sanitation of chicken stables and possibly as vectors during passage through the stomach of host animals. Furthermore, Acanthamoeba spp. could serve as a vehicle and reservoir introducing and protecting C. jejuni in beverages such as milk and juice. Validation of the ACC method suggests that it is robust and could be used even in outbreak investigations where genetic fingerprints are compared between isolates. In conclusion, Acanthamoeba spp. are good candidates for being natural hosts and vectors of C. jejuni.

Free-living pathogenic amoebae Acanthamoeba spp. and Balamuthia mandrillaris are causative agents of important diseases of human: rarely occurring but highly fatal granulomatous amoebic encephalitis (both) and keratitis (Acanthamoeba). One of the reasons for the problematic therapy is differentiation into highly resistant cysts often found in affected tissues. In our study we have found that correct encystation in Acanthamoeba requires apart from others, the presence of functioning Golgi apparatus transporting the cyst wall material to the cell surface; glycogen phosphorylase degrading glycogen into glucose which seems to be further used for cellulose synthesis and two non-constitutive cellulose synthases. Acanthamoeba cellulose synthases seem not to be inhibited by known herbicides. In the cyst wall of acanthamoebae we detected cellulose, -mannan, and -1, 3-1, 4-linked glucan [lichenin or mixed-linkage glucan (MLG)]. Cellulose is present in the inner (endocyst) and the outer (exocyst) layers of the cyst wall, whereas-mannan and MLG are found in the endocyst. In a protozoan organism, MLG was detected for the first time. The MLG of Acanthamoeba has a similar composition to that found in barley with high amount of cellobiosyl and cellotriosyl followed by cellotetraosyl units. In contrast, with...

Amebic keratitis and granulomatous amebic encephalitis are diseases caused by free-living amoebas of the Acanthamoeba type. In the case of keratitis, the great problem is the relapse of this disease because of its resistance to medicines used for it, especially the cystic form of the organism. Plant essential oils have been used as potentially active agents against this protist. Therefore, the objective of this study was to determine the amebacide activity of plant essential oils of Lippia type against Acanthamoeba polyphaga trophozoites. To do this, 8 x 104 trophozoites were exposed to increased concentrations of essential oils of Lippia sidoides, Lippia gracilis, Lippia alba and Lippia pedunculosa during 24 hours, as well as of their majority compounds rotundifolona, carvone and carvacrol. Practically all the oil and compound concentrations presented amebacide activity. Based on these results, the researched oils in this study are potential candidates for a complementary healing for amebic keratitis and granulomatous amebic encephalitis. / A ceratite amebiana e a encefalite amebiana granulomatosa são doenças causadas por amebas de vida livre do gênero Acanthamoeba. No caso da ceratite, o grande problema é a recidiva da doença devido à resistência, especialmente da forma cística do organismo, aos medicamentos utilizados. Óleos essenciais de plantas têm sido utilizados como agentes potencialmente ativos contra este protista. Portanto, o objetivo deste estudo foi determinar a atividade amebicida de óleos essenciais de plantas do gênero Lippia frente à trofozoítos de Acanthamoeba polyphaga. Para isto 8 x 104 trofozoítos foram expostos durante 24 horas a concentrações crescentes dos óleos essenciais de Lippia sidoides, Lippia gracilis, Lippia alba e Lippia pedunculosa, bem como de seus compostos majoritários rotundifolona, carvona e carvacrol. Praticamente todas as concentrações dos óleos e compostos apresentaram atividade amebicida. Diante destes resultados, os óleos pesquisados neste estudo são candidatos em potencial para a terapêutica complementar na ceratite amebiana e encefalite amebiana granulomatosa.

This thesis examines unicellular microorganism of the genus Acanthamoeba, as well as the disease it causes - acanthamoeba keratitis. It is an eye disease, which occurs mainly in people using contact lenses. Since acanthamoeba attacks visual apparatus, several parts of the thesis are dedicated to the anatomy of said apparatus, especially to the eye ball. The symptoms, diagnostics, treatment as well as prevention of the disease are included as well. The thesis furthemore mentions individual types of contact lenses, their proper hygiene and care and most of all, the danger, that comes with improper handling - acanthamoeba keratitis. A survey, that took place among elementary school and high school students, found out, that almost none of the elementary school students wear contact lenses, whereas contact lenses are worn by approximately 15% of students at high school. Among other issues, the survey concludes, that only a small part of elementary school students prefers contact lenses to glasses, though glasses are needed by a vast majority of them. At high school level, wearing contact lenses becomes more common, for aesthetics or other reasons. Ophtalmologist is regularly visited by 35 % of interviewed students. An analysis of six different natural science and biology textbooks for the 8th grade of...

Les interactions entre les végétaux et les organismes telluriques sont déterminantes pour la décomposition des matières organiques et la nutrition minérale des plantes. L’objectif général de la thèse était de comprendre comment les interactions multi-trophiques dans la rhizosphere agissent sur la disponibilité en azote minéral et l’allocation en carbone dans la plante. Nous avons mis au point des dispositifs de culture de plante, permettant de contrôler l’environnement biotique des racines (inoculation par des espèces symbiotiques modèles : un protozoaire bactériophage et/ou une espèce mycorhizienne à arbuscules). Nous avons utilisé l’azote 15N et le carbone 13C pour tracer le cheminement de l’azote du sol vers la plante et le carbone assimilé par photosynthèse, de la plante vers le sol et les microorganismes du sol. L’allocation de C vers les racines et la rhizosphère est dépendante de la qualité de la litière foliaire enfouie. La structure de la communauté microbienne déterminée par l’analyse des profils d’acides gras (PLFA) est modifiée par la présence de protozoaires pour la litière à C/N élevé. Les mycorhizes à arbuscules et les protozoaires présentent une complémentarité pour l’acquisition du C et de N par la plante. Les protozoaires remobilisent l’azote de la biomasse microbienne par leur activité de prédation. Les hyphes fongiques transportent du C récent issu de la plante vers des sites riches en matière organique non accessibles aux racines. Ainsi, l’activité de la communauté microbienne est stimulée et la disponibilité en N augmentée lorsque des protozoaires sont présents. Les perspectives de ce travail sont de déterminer si (i) les interactions étudiées dans ce dispositif modèle peuvent être généralisées à d’autres interactions impliquant d’autres espèces de champignons mycorhiziens et de protozoaires (ii) la phénologie de la plante et la composition des communautés végétales influence la nature et l’intensité des réponses obtenues / Plants interact with multiple root symbionts for fostering uptake of growth-limiting nutrients. In turn, plants allocate a variety of organic resources in form of energy-rich rhizodeposits into the rhizosphere, stimulating activity, growth and modifying diversity of microorganisms. The aim of my study was to understand how multitrophic rhizosphere interactions feed back to plant N nutrition, assimilate partitioning and growth. Multitrophic interactions were assessed in a single-plant microcosm approach, with arbuscular mycorrhizal fungi (Glomus intraradices) and bacterial feeding protozoa (Acanthamoeba castellanii) as model root symbionts. Stable isotopes enabled tracing C (13C) and N (15N) allocation in the plant and into the rhizosphere. Plant species identity is a major factor affecting plant-protozoa interactions in terms of N uptake and roots and shoot morphology. Plants adjusted C allocation to roots and into the rhizosphere depending on litter quality and the presence of bacterial grazers for increasing plant growth. The effect of protozoa on the structure of microbial community supplied with both, plant C and litter N, varied with litter quality added to soil. AM-fungi and protozoa interact to complement each other for plant benefit in C and N acquisition. Protozoa re-mobilized N from fast growing rhizobacteria and by enhancing microbial activity. Hyphae of AM fungi acted as pipe system, translocating plant derived C and protozoan remobilized N from source to sink regions. Major perspectives of this work will be to investigate whether (i) multitrophic interactions in our model system can be generalized to other protozoa-mycorrhiza-plant interactions (ii) these interactions are depending on plant phenology and plant community composition

Les EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine). / EHEC O157:H7 are an emerging zoonotic food- and water-borne hazard highly pathogenic to humans and associated with diseases ranging from acute gastroenteritis to hemolytic uremic syndrome. From their commensal carriage by farm animals to human targets, EHEC pass through a crucial step of persistence in the open environment. Microbial endosymbiosis is one strategy used by pathogenic bacteria to survive in aquatic environments. Amoebae species are free-living protozoa found in diverse environmental habitats and known to host several water-borne pathogens. Thus amoebae could contribute to transmission of EHEC to humans. The first part of my PhD project was focused on interaction of the free-living amoebae Acanthamoeba castellanii with EHEC. The results showed that the presence of amoeba extends the persistence of EHEC that survived phagocytosis by amoebae. This demonstrates the real potential of amoebae to harbourd EHEC that may contribute to their transmission. However, absence of shiga toxins enhanced the intra-amoeba survival. Moreover, EHEC had a toxic and lethal effect on amoebae partially due to shiga toxins. The involvement of shiga toxins in the intracellular survival and mortality of amoebae suggests the value of using amoebae as a model of host/pathogen interactions to study the pathogenicity of EHEC. During their transmission cycle, EHEC encounter limitation inorganic phosphate (Pi) in the environment. Using jointly the PhoB-R two-component system (TCS) and the Pst (Pi specific transport) system, EHEC detect and respond to this Pi limitation by activating the Pho regulon. The interplay between the EHEC virulence, the Pho-Pst and/or the environmental Pi remains unknown. The second part of my project explored the role of Pho regulon (responding to Pi-limitation stress) in the virulence of EHEC. Transcriptomic analysis showed that EHEC has evolved a sophisticated response to Pi deficiency involving not only biochemical strategies that are likely critical to its survival, but also coordinating its virulence response. In these conditions, the regulator PhoB regulates directly the expression of LEE and Stx2 genes. This is confirmed by an increase in EspB secretion and Stx2 production and secretion in low Pi conditions. Moreover, the activation of Pho regulon increases biofilm formation and reduces motility in EHEC. This correlated with the induction of genes regulating curli production and repression of indole production pathway and the flagellum and PGA biosynthesis.

Les virus géants d'amibes( Acanthamoeba) sont des virus à ADN double brin . Ces virus géants ont été isolés depuis 2008 essentiellement à partir de prélèvements d'eaux et sols) collectés dans diverses régions géographiques à travers le monde, ou à partir de prélèvements humains (selle, liquide broncho-alvéolaire et sang). Ils sont repartis en 4 familles virales dont les plus représentées sont les familles Mimiviridae et Marseilleviridae avec pour membres fondateurs respectifs Mimivirus et Marseillevirus et comptent à ce jour respectivement 44 et 20 isolats. Les virus géants d'amibes sont ubiquitaires dans notre biosphère, et les êtres humains y sont potentiellement exposés. Au cours de cette Thèse, nous avons premièrement écrit une revue de la littérature décrivant les outils de mise en évidence des virus géants d'amibes chez l'homme incluant la sérologie, la culture, la PCR ou l'hybridation de sondes fluorescentes in situ. Deuxièmement, nous avons conçu et évalué 5 systèmes de PCR en temps réel détectant les membres des groupes de mimivirus d'amibes, leurs virophages et les marseillevirus. Nous avons participé à un 3ème travail décrivant les différentes procédures d'isolement sur amibes utilisées jusqu'à présent dans notre laboratoire . Enfin, dans un 4ème travail préliminaire, nous avons recherché par PCR la présence des mimivirus et marseillevirus dans 701 plasmas de patients infectés par HIV-1.Au total, nos travaux ont décrit les mises au point, performances et limites des tests de PCR pour l'étude des virus géants, et ont contribué aux outils et fourni des éléments pour l'étude de l'implication des virus géants d'amibes en pathologie humaine. / The giant viruses of amoebas( Acanthamoeba) are double stranded DNA viruses. These giant viruses have been isolated essentially from water and soil samples collected in various geographic regions around the world or from human samples (stool, blood and bronchoalveolar fluid). These giant viruses are divided into four viral families among which those comprising the largest number of representatives are the Mimiviridae and Marseilleviridae families, whose respective founders are Mimivirus and Marseillevirus and comprise 44 and 20 representative members, respectively. Giant viruses of amoeba are ubiquitous in our biosphere, which means that humans can be exposed to them. In this Thesis, we initially wrote a review of the literature describing the tools to detect the present of these giant viruses in humans, including serology, culture isolation, PCR and fluorescence in situ hybridization (FISH). Secondly, we designed and evaluated the performance of five real-time PCR systems targeting the members of the 3 groups of mimiviruses of amoeba, their virophages and the marseilleviruses. We were involved in a third work that described the different isolation procedures on amoebae used so far in our laboratory for giant viruses. Finally, in a fourth preliminary work, we looked by PCR for the presence of mimiviruses and marseilleviruses DNA in 701 plasma from patients infected with HIV-1. In summary, our work described the developed PCR assays for the study of giant viruses, and their performance and limitations, and it contributed to the tools and evidence for the study of the involvement of the giant amoeba virus in human pathology.

Amoebae of the genus Acanthamoeba spp. are free-living unicellular organisms found in disparate ecosystems all over the world. Due to their ability to invade human body, evade its defensive mechanisms and cause extensive tissue damage, Acanthamoeba infection can lead to serious, if rare, diseases, affecting most commonly the eye and the central nervous system. Specific therapy for Acanthamoeba infections is not available. A major reason for therapeutic failure in ameobiasis is the ability of the protist to differentiate into resistant stages. These are cysts, known to be formed under prolonged unfavorable conditions, both in the environment and the infected tissues, and the pseudocysts, less durable but rapidly formed under acute stress. The present thesis focuses on as yet unexplored mechanisms of resistance of cysts and pseudocysts. Moreover, further characteristics distinguishing cysts and pseudocysts as well as the processes involved in their formation are investigated. One of the issues addressed is a presence of protective carbohydrate compounds mannitol and trehalose that participate in defensive reactions against abiotic stress in many organisms. Although putative genes for enzymes of the trehalose and mannitol synthetic pathways are present in the genome of Acanthamoeba, only one of the...