Free-living amoebae (FLA) are protists of ubiquitous distribution characterised by their changing morphology and their crawling movements. They have no common phylogenetic origin but can be found in most protist evolutionary branches. Acanthamoeba is a common FLA that can be found worldwide and is capable of infecting humans. The main disease is a life altering infection of the cornea named Acanthamoeba keratitis. Additionally, Acanthamoeba has a close relationship to bacteria. Acanthamoeba feeds on bacteria. At the same time, some bacteria have adapted to survive inside Acanthamoeba and use it as transport or protection to increase survival. When conditions are adverse, Acanthamoeba is capable of differentiating into a protective cyst. This study had three objectives. First, isolate and identify new FLA and Acanthamoeba strains. Second, identify encystation factors of Acanthamoeba. Third, identify and characterise new potential antimicrobial proteins produced by Acanthamoeba. The isolation of environmental amoebae was performed, and several strains of Acanthamoeba were identified from previously known genotypes. Also, two new species of FLA were identified: Allovahlkampfia minuta and Leptomyxa valladaresi. The dynamics of encystment were studied in different strains of Acanthamoeba. RNAseq was used to study gene expression during differentiation and identify differentially expressed genes. We identified different encystment factors including at least two encystment related proteases. A new antimicrobial zymogram was developed that identified antimicrobial proteins being secreted by Acanthamoeba. A 33 kDa protease was found to be able to lyse bacteria. We created DNA constructs encoding the protease and a lysozyme from Acanthamoeba for heterologous expression. The genes were successfully cloned. However, bacteria were not able to produce the proteins most probably due to their antimicrobial characteristics. Further studies are required regarding encystment and antimicrobial factors identified. Such experiments should help elucidate critical factors of Acanthamoeba's biology that could help treat several infections.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Reproduction de : Thèse de doctorat : Microbiologie de l'eau : Poitiers : 2008. / Titre provenant de l'écran-titre. Bibliogr. 110 réf.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references (leaves 86-104). Also available online through Digital Dissertations.

Espécies de Acanthamoeba pertencem ao grupo das amebas de vida livre e constituem um agente etiológico da ceratite amebiana, doença que pode causar inflamação ocular grave e até mesmo cegueira. Plantas do gênero Pterocaulon (Asteraceae) são utilizadas na medicina popular como agente anti-séptico e antifúngico. Neste trabalho, foi investigada a composição química do óleo essencial de P. polystachyum e avaliada a sua atividade amebicida contra A. polyphaga. As folhas da planta fresca foram submetidas à hidrodestilação, fornecendo um rendimento de 0,15% (w/v) de óleo essencial. O óleo foi analisado por Cromatografia a Gás acoplada a Espectrômetro de Massas (CG/MS), sendo o acetato de E-sesquilavandulil o principal componente, representando 43,8% da amostra. Para avaliar a atividade amebicida do óleo, foram testadas as concentrações de 20, 10, 5, 2,5 e 1,25 mg/mL contra trofozoítos de duas cepas ATCC de A. polyphaga (uma de origem clínica e outra ambiental). O óleo essencial nas concentrações de 10 e 20 mg/mL foi letal para 100% dos trofozoítos das duas cepas tanto em 24 quanto em 48 horas. O mesmo resultado foi mostrado na concentração de 5 mg/mL de óleo essencial contra os trofozoítos da cepa de origem ambiental de A. polyphaga. O óleo essencial não induziu o encistamento de Acanthamoeba. O efeito citotóxico do óleo essencial também foi avaliado em células de mamíferos, sendo determinado pelo ensaio de Brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio (MTT). Os resultados da atividade amebicida encontrada no óleo estão em conformidade com relato anterior em que os compostos lipofílicos obtidos desta planta foram ativos contra trofozoítos de A. castellanii. A utilização do óleo como componente de soluções de limpeza de lentes de contato e de superfícies, não deve ser descartada, entretanto maiores estudos com seus componentes majoritários precisam ser feitos. / Acanthamoeba species are free-living amoebae that constitute an etiological agent of Acanthamoeba keratitis, an illness that may cause severe ocular inflammation and induce blindness. Plants of the genus Pterocaulon (Asteraceae) are used in folk medicine as antiseptic and antifungal agent. In this work, the activity of P. polystachyum essential oil against A. polyphaga was assessed. The leaves of the fresh plant submitted to hydrodistillation yielded 0.15% (w/v) of essential oil that was analyzed by GC/MS being E-sesquilavandulyl acetate the major component, representing 43.8% of the oil. For assessment of the amoebicidal activity concentrations of 20, 10, 5, 2.5 and 1.25 mg/mL were tested against two A. polyphaga ATCC strains (environmental and clinical strains). The essential oil at the concentrations of 10 and 20 mg/mL was lethal to 100% of the trophozoites of both strains in 24h and 48h. The same effect was observed when the essential oil at the concentration of 5 mg/mL was assayed against the environmental strain of A. polyphaga trophozoítes. Essential oil cytotoxic effect in mammalian cells was determined by MTT assay. The amoebicidal effect (activity) obtained in this work is in accordance with previous report in which the lipophilic compounds from this plant were active against A. castellanii. Essential oil utilization as a component of contact lenses cleaning and surface disinfectant solutions should not be discarded, however further studies with its major component should be performed.

Associações simbióticas, comensais e parasitárias são amplamente relatadas em insetos. Pelo fato de larvas de culicídeos e amebas de vida livre (AVL) habitarem meios aquáticos similares, objetivou-se verificar a prevalência de Acanthamoeba spp. em populações silvestres de Aedes aegypti. Esta AVL foi investigada em 60 pools contendo 10 larvas de A. aegypti, as quais foram coletadas através de ovitrampas instaladas em diversos bairros da cidade de Porto Alegre (RS, Brasil). Os isolados de Acanthamoeba spp. foram caracterizados morfologicamente e submetidos à técnica de Reação em Cadeia da Polimerase (PCR) para confirmação do gênero. Ademais, realizouse análise genotípica e testes presuntivos de patogenicidade para algumas cepas. Entre os pools, 54 (90%) foram positivos para AVL, dos quais 47 (87%) isolados eram pertencentes ao gênero Acanthamoeba. Os grupos genotípicos T4, T3 e T5 foram identificados, correspondendo a 14 (53,8%), 10 (38,5%) e dois (7,7%) isolados, respectivamente. De acordo com testes fisiológicos empregados para 14 cepas, 12 (85,7%) foram consideradas não patogênicas e duas (14,3%) foram consideradas com baixo potencial patogênico. Estes resultados servem como base para um maior conhecimento acerca da interação entre estes protozoários e mosquitos vetores, em seu habitat natural. Além disso, é o primeiro estudo dedicado ao isolamento de Acanthamoeba spp. a partir de culicídeos coletados do ambiente. / Symbiotic, commensal and parasitic associations are widely reported in insects. By the fact of mosquito larvae and free-living amoebae (FLA) occupy the similar aquatic sites, the aim of this study was to determine the prevalence of Acanthamoeba spp. in Aedes aegypti larvae collected in the environment. The amoebae were investigated in 60 pools, each containing 10 larvae of A. aegypti which were collected by using larvitraps installed in various districts of Porto Alegre (RS, Brazil). Acanthamoeba isolates were morphologically characterized and submitted to Polymerase Chain Reaction technique to confirm the genus. In addition, genotype analyses as well as presumptive tests for pathogenicity in some samples were performed. Among the pools, 54 (90%) were positive for FLA. From those isolates, 47 (87%) belong to the genus Acanthamoeba. The genotype groups T4, T3 and T5 have been identified corresponding to 14 (53.8%), 10 (38.5%) and two (7.7%) isolates respectively. The physiological tests performed in 14 strains showed that 12 (85.7%) were non pathogenic, while two (14.3%) were considered with low pathogenic potential. These results provide a basis for a better understanding between these protozoan and mosquitoes interaction in their natural habitat. Moreover, this study is the first to report isolation of Acanthamoeba spp. from mosquitoes collected in the environment.

O gênero Acanthamoeba compreende protozoários que pertencem ao grupo das amebas de vida livre e estão amplamente dispersos na natureza. São conhecidos por causarem duas graves doenças, a Encefalite Amebiana Granulomatosa, em pacientes imunodeprimidos e a Ceratite Amebiana, principalmente em usuários de lentes de contatos imunocompetentes. A própolis verde é uma substância resinosa e balsâmica, conhecida na medicina alternativa por apresentar diversas atividades biológicas. Neste trabalho foi avaliada a atividade amebicida de um extrato aquoso de própolis verde contra trofozoítos e cistos de A. castellanii . Nas concentrações de 10 e 20 mg/mL, o extrato foi capaz de inativar 100% dos trofozoítos em 24 e 48 horas. A dose de 5 mg/mL inativou 100% dos trofozoítos em 72 horas. Os cistos foram inativados na concentração de 40 mg/mL após 24 horas de exposição ao extrato. O efeito citotóxico do extrato foi avaliado sobre células VERO e HCE através do ensaio MTT. O extrato não causou efeito citotóxico significativo nas concentrações 0,312, 0,625, 1,25 e 2,5 mg/mL sobre as células HCE e nas concentrações 2,5 e 5 sobre as células VERO. O ensaio de adesão realizado mostrou que a ligação de Acanthamoeba às células HCE possui comportamento dose-dependente em relação ao extrato de própolis verde. Assim, o presente estudo demonstrou a atividade antiparasitária da própolis verde frente a ambas às formas de Acanthamoeba, mostrando-se uma substância promissora para o desenvolvimento de fármaco alternativo, bem como para utilização na composição de soluções de limpeza de lentes de contato ou superfícies. Entretanto, mais estudos são necessários para compreender seus mecanismos de ação. / The genus Acanthamoeba comprises protozoa belonging to the group of free-living amoebas and it is widely scattered in nature. These protozoa are known to cause two serious diseases, Granulomatous Amebic Encephalitis in immunocompromised patients and amebic keratitis, especially in immunocompetent contact lens wearers. Green Propolis is a resinous and balsamic substance, known in alternative medicine because of its several biological activities. In this study we evaluated the amoebicide activity of an aqueous extract of propolis against trophozoites and cysts of A. castellanii. At concentrations of 10 and 20 mg/mL, the extract was able to inactivate 100% of trophozoites within 24 and 48 hours. The dose of 5 mg/mL inactivated 100% of trophozoites within 72 hours. The cysts were inactivated at a concentration of 40 mg/mL after 24 hours of exposure to the extract. The cytotoxic effect of the extract was evaluated on VERO and HCE cells by MTT assay. The extract had no significant cytotoxic effect at concentrations of 0.312, 0.625, 1.25 and 2.5 mg/ml about HCE cells and at concentrations of 2.5 and 5 on VERO cells. The adhesion test performed showed that the attachment of Acanthamoeba to HCE cells have dose-dependent behavior in relation to the propolis extract. Thus, this study demonstrated the antiparasitic activity of propolis against both forms of Acanthamoeba and proved to be a promising substance for the development of an alternative drug, as well as its use in the formulation of cleaning solutions for contact lenses or surfaces. However, more studies are needed to understand their mechanisms of action.

Acanthamoeba é uma ameba de vida livre, capaz de causar raras e graves infecções oportunistas em olhos, pele e sistema nervoso de humanos e animais. É um protozoário ubiquitário e frequentemente isolado do ambiente. Pode infectar humanos através do uso de lentes de contato, cortes ou feridas na pele assim como ser inalada e conduzida aos pulmões. Possui duas formas em seu ciclo de vida: trofozoíto móvel e infectiva capaz de causar ceratite assim como uma fatal encefalite e de cisto resistente ao ambiente e ao ataque do sistema imune, facilitando a ocorrência da infecção. Existem vários fatores que contribuem na patogênese de Acanthamoeba, sendo a proteína de união à Manose (MBP) a única proteína de superfície descrita como principal fator de patogenicidade. Não existem trabalhos sobre o papel funcional ou patogênico de outras proteínas de superfície que poderiam ser relevantes na invasão ou infecção do hospedeiro por esta ameba. O objetivo geral deste estudo é identificar e analisar uma proteína de superfície de Acanthamoeba castellanii e avaliar seu potencial para utilização em diagnóstico da ceratite amebiana ou encefalite granulomatosa. Predições in silico deste estudo demostram que a calreticulina é uma proteína de superfície. A clonagem da sequência codificadora da calreticulina por recombinação homóloga in vivo foi realizada no vetor pGEX4T1 para permitir a expressão em Escherichia coli BL21 pLysE da proteína recombinante em fusão com a Glutariona-STransferase nas condições de 14°C por 16 horas tendo um rendimento final de 3,64 mg/L de proteína purificada. O potencial imunodiagnóstico foi avaliado através de ELISA usando soros de pacientes com encefalite e soros de rato com ceratite e encefalite testando como antígeno a proteína recombinante. A proteína foi reconhecida pelos soros de pacientes infectados e saudáveis, diferentemente dos soros de ratos, em que só foi reconhecida pelos ratos infectados. Estes resultados preliminares apontam que a proteína calreticulina de A. castellanii poderia ser um candidato para imunodiagnóstico das doenças causadas pelo protozoário em ratos; no caso dos resultados em pacientes humanos ainda são necessárias maiores investigações. / Acanthamoeba is a microscopic free-living amoeba able to cause rare and serious opportunistic infections in eyes, skin and nervous system in humans and animals. It’s one of the most common protist widely distributed and it has been isolated from the environment, including water and soil. The amoeba can infect contact-lenses wearers through skin lesions or via the nasal route. Acanthamoeba exists in two distinct forms: an active-infective trophozoite form during which Acanthamoeba reproduces being capable of cause Acanthamoeba keratitis and a fatal granulomatous amoebic encephalitis, and a dormant cyst form resistant to immune system defense making easier the recurrence of these infections. Several factors contribute with the pathogenesis of Acanthamoeba. The mannose bindingprotein (MBP) is the only surface protein as a main pathogenicity factor in Acanthamoeba; however, there are no evidence of any scientific work that describes the functional nor pathogenic role of another surface protein that could be relevant in the host-parasite invasion or infection by this amoeba. The general objective of this study is identify and analyze an Acanthamoeba castellanii surface protein and test its potential for use in diagnosis of amoebic keratitis or granulomatous encephalitis. In silico predictions showed that, A. castellanii calreticulin is a surface protein. In vivo homologous recombination cloning of the coding sequence was performed using the pGEX4T1 vector for expressing the GST fusion recombinant protein using an Escherichia coli BL21 pLysE strain at 14°C for 16 hours obtaining a final efficiency of 3,64 mg/L of purified protein. The immunodiagnostic potential was tested with specific antibody responses in serum from patients with encephalitis and infected rats with keratitis and encephalitis against recombinant calreticulin by ELISA. The protein was recognized by both infected and healthy patients serum, whereas the infected rat serum was the only recognized by the recombinant protein. These results would conclude that A. castellanii calreticulin probably could be an immunodiagnostic prospect of Acanthamoeba diseases in animals but in human further immunoassays are required.