La kératite à Acanthamoeba (KA) est une kératite infectieuse rare et grave,potentiellement cécitante. L'infection est causée par Acanthamoeba spp., unprotozoaire ubiquitaire présent dans le sol, l'air et l'eau. Jusqu'à 85% des cas de KAsont associés au port de lentilles cornéennes, et plus rarement suite à untraumatisme.Actuellement, aucune molécule n’a d’autorisation de mise sur le marché danscette indication dans l'Union européenne et aux États-Unis. Ces dernières années,des combinaisons d'agents anti-amibiens tels que les biguanides et les diamidinesont été utilisées comme traitement de référence. Cependant, les schémasthérapeutiques et les concentrations d'agents actifs reposent sur des donnéesempiriques. Récemment, le voriconazole, antifongique triazolé, a été utilisé avecsuccès pour traiter des KA humaines. Malgré cela, la communauté ophtalmologiquese heurte le plus souvent dans les formes sévères à de grandes difficultés de prise encharge et se retrouve parfois en situation d’impasse thérapeutique. La pertefonctionnelle et anatomique de l’oeil est encore possible.A partir d’un modèle de KA chez le rat, plusieurs molécules et voiesd’administration ont été testées. Dans une première partie, en lien avec projeteuropéen ODAK (Orphan drug for Acanthamoeba Keratitis), nos travaux ont suggéréqu’une concentration de collyre PHMB supérieure ou égale à 0,04% devait êtrepréférée. Dans une deuxième partie, nous avons pu montrer la supériorité duvoriconazole en collyre par rapport à la voie orale. Enfin, l’étude de lapharmacocinétique du voriconazole et du posaconazole après injections directesintracornéennes, démontre leur faible utilité en clinique humaine du fait de lafréquence nécessaire de réinjection, bien que des analyses complémentairesconcernant le posaconazole en collyre pour confirmer son intérêt soient nécessaires.L'ensemble de ces travaux pourrait permettre d’adapter les protocolesthérapeutiques de la KA. / Acanthamoeba keratitis (AK) is a rare and severe form of infectious keratitis,which is potentially sight-threatening. The infection is caused by Acanthamoeba spp.a common protozoan present in soil, air and water. Up to 85% of AK cases areassociated with contact lens wearing, more rarely after corneal injury.Currently, there are no agents approved for the treatment of AK in theEuropean Union or in the United States of America. In recent years, combinations ofunlicensed anti-amoebic agents such as biguanides and diamidines have been usedas the reference treatment. Treatment regimens and concentrations of active agentsare based on empirical data. Recently, voriconazole, a mono-triazole, wassuccessfully used to treat cases of human AK. Despite this, the ophthalmologicalcommunity is most often faced with severe forms of the disease with severemanagement difficulties and sometimes with a situation of therapeutic impasse. Thefunctional and anatomical loss of an eye can occur.Several agents and routes of administration have been tested in a rat model ofAK. First, as part of the European ODAK project (Orphan drug for AcanthamoebaKeratitis), our work suggested that a concentration of PHMB eye drops greater thanor equal to 0.04% should be preferred. Second, we were able to show the superiorityof voriconazole in eye drops compared to the oral route. Finally, our study on thepharmacokinetics of voriconazole and posaconazole after intrastromal injections,demonstrates their low utility in human because of the need for frequent reinjection.Nevertheless, additional analyses are necessary to confirm the interest ofposaconazole eye drops. All of this work could make it possible to adapt thetherapeutic protocols of AK.

Les espèces bactériennes opportunistes responsables d'infections nosocomiales chez l'Homme se rencontrent dans les environnements terrestres et aquatiques. Elles sont très souvent caractérisées par une résistance naturelle aux antibiotiques leur conférant un phénotype appelé Multi-Drug Resistant (MDR). L'efflux d'antibiotiques via des pompes, est un des mécanismes à l'origine de cette multi-résistance. Alors que le rôle de ces pompes chez des bactéries isolées en milieu clinique est connu, aucune donnée n'est disponible concernant leur rôle chez les bactéries associées avec d'autres organismes eucaryotes du sol tels que les amibes. Pourtant des données de la littérature indiquent que les amibes, jusqu'alors principalement connues pour leur rôle prédateur de bactéries sont susceptibles d'héberger des bactéries " résistantes " aux amibes (ARB). Parmi ces ARB, des pathogènes opportunistes ont été identifiés dont certains sont connus pour être porteurs de pompes à efflux. Les pompes à efflux de ces bactéries pourraient donc intervenir dans l'adaptation aux amibes du sol. Afin de vérifier cette hypothèse, nous avons, dans un premier temps, isolé et identifié la flore amibienne et les ARB de différents sols. Parmi les ARB identifiées, Stenotrophomonas maltophilia, Pseudomonas aeruginosa et Burkholderia cepacia sont caractérisées par des propriétés d'antibiorésistance contrastées et de virulence élevées. Des études d'interaction ont montré que S. maltophilia se multipliait dans des amibes axéniques et que des pompes à efflux Sme étaient surexprimées. Par ailleurs des molécules sécrétées par l'amibe stimulent la croissance bactérienne et des études préliminaires de profilage métabolique ont montré la présence de différents métabolites secondaires produits par l'amibe au cours de l'interaction avec S. maltophilia pouvant jouer un rôle dans l'expression des pompes à efflux / The opportunistic bacterial species, responsible for nosocomial infections in humans, occurs in terrestrial and aquatic environments. They are often characterized by natural resistance to antibiotics giving them a phenotype called Multi-Drug Resistant (MDR). The efflux of antibiotics via pumps, is one of the mechanisms behind this multi-resistance. While the role of these pumps in bacteria isolated from hospital is known, no data are available regarding their role in bacteria associated with other soil eukaryotic organisms such as amoebae. Nevertheless, data from the literature indicate that amoebae, mainly known to be predators of bacteria, are likely to harbour "amoeba resistant bacteria” (ARB). Among these ARB, opportunistic pathogens have been identified, some of which are known to be carriers of efflux pumps. The efflux pumps of these bacteria could thus interfere in the adaptation to soil amoebae. In order to verify this hypothesis, we first isolated and identified the amoebal population and the ARB of different soils. Among the identified ARB, Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Burkholderia cepacia are characterized by high contrast antibiotic resistance and high virulence. Interaction studies showed that S. maltophilia could multiplied in axenic amoebae and Sme efflux pumps were overexpressed. Furthermore, molecules secreted by the amoeba stimulate bacterial growth and preliminary studies of metabolic profile have shown that production of various secondary metabolites by the amoeba during the interaction with S. maltophilia could play a role in the efflux pumps expression

Exogenous and endogenous cannabinoids have been reported to modulate functional activities of macrophages. It is recognized that macrophages express primarily the CB2 cannabinoid receptor, but recent studies indicate that its expression is differential in relation to activation state with maximal levels occurring when cells are in "responsive" and "primed" states. The functional activities of macrophages when in these states of activation are the most susceptible to the action of cannabinoids, at least in terms of a functional linkage to the CB2. To assess the effect of cannabinoid treatment on macrophage chemotaxis and test the hypothesis that cannabinoids inhibit the chemotactic response of macrophages and microglia to endogenous and exogenous, pathogen-derived stimuli, primary murine peritoneal macrophages and neonatal rat microglia were used. Chemotaxis assays and scanning electron microscopy studies demonstrated that cannabinoids inhibit chemotaxis, a signature activity attributed to "responsive" macrophage-like cells, to the endogenous chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted) and to Acanthamoeba conditioned medium containing secreted proteases. The partial agonist delta-9-tetrahydrocannabinol (THC), administered in vitro, inhibited the chemotactic response of peritoneal macrophages to the chemokine RANTES and to Acanthamoeba conditioned medium. In vivo treatment with THC also resulted in inhibition of the in vitro chemotactic response of murine peritoneal macrophages to RANTES and amoebic conditioned medium. Pharmacological studies employing cannabinoid receptor agonists and antagonists demonstrated the involvement of CB2 in cannabinoid-mediated inhibition of peritoneal macrophage chemotaxis to RANTES and Acanthamoeba conditioned medium, implying that signaling through cannabinoid receptors may desensitize chemokine receptors. Treatment with cannabinoids had no apparent effect on chemokine receptor mRNA levels, but did enhance CCR5 protein phosphorylation. Macrophage migration to Acanthamoeba conditioned medium may involve activation and signaling through protease activated receptors (PARs), as pathogen-derived proteases have been shown to activate PARs and initiate cellular migration; however, further studies are required to demonstrate PAR activation by amoebic conditioned medium and to assess the effects of cannabinoids on PAR signaling. Acanthamoeba are opportunistic pathogens that cause Granulomatis amoebic encephalitis, an infection of the CNS that is often fatal. THC treatment has been shown to increase mortality to Acanthamoeba infections and is characterized by an absence of granuloma formation. We hypothesize that inhibitory effect of THC on macrophage migration may be a key factor in cannabinoid-mediated immunosuppression. To assess the effect of cannabinoids on microglial migration to Acanthamoeba conditioned medium, chemotaxis assays were performed using primary rat microglia treated with cannabinoids. These studies demonstrated that cannabinoids inhibit microglial chemotaxis to amoebic conditioned medium. Furthermore, the studies demonstrate that cannabinoids, acting through cannabinoid receptors, may cross-talk with a diverse array G-protein coupled receptors so as to modulate responsiveness of macrophage and macrophage-like cells.

Acanthamoeba culbertsoni (A. culbertsoni) is an amphizoic amoeba that is the causative agent of Granulomatous Amoebic Encephalitis (GAE), an often fatal central nervous system infection that is seen most frequently in severely immunocompromised patients and is characterized by hemorrhagic and necrotic lesions of the brain as well as varying degrees of granuloma formation. A.culbertsoni isolates have also been identified in a few cases of Amoebic Keratitis, a painful, sight-threatening corneal infection that disproportionately affects contact lens users irrespective of immune status. Common features of both infections include amoebic interaction with host extracellular matrix (ECM) components as requisites for both attachment to, and subsequent invasion of, host tissues to facilitate disease establishment. Previous studies have demonstrated that pathogenic species of Acanthamoeba , such as A.culbertsoni, bind to the ECM proteins Laminin-1 and Collagen I to a greater extent than non-pathogenic species. It has also been documented in the literature that secreted Acanthamoeba proteases have the ability to degrade components of the extracellular matrix. The role of amoebic proteases in mediating the attachment and invasion processes is not entirely understood. Initial experiments conducted in the present study revealed secretion of approximately 150 and 55-kDa serine proteases during attachment as well as invasion of the ECM by A. culbertsoni. However, inhibition of these serine proteases using phenylmethylsulfonyl fluoride (PMSF) did not diminish the ability of amoebae to attach or invade. It was demonstrated that secretion of the observed proteases occurred in a constitutive rather than substrate-induced manner and that amoebae secrete these proteases under a number of different conditions. Additionally, a 140-kDa membrane-associated serine protease was identified which may prove to play a role in focal proteolytic degradation. Collectively, our results suggest that attachment to extracellular matrix components is mediated through non-protease-dependent mechanisms. We also suggest that ECM invasion by A.culbertsoni is predominately a mechanical process that may be supplemented or enhanced by focal proteolytic degradation of extracellular matrix components by membrane-associated proteases.

Cryptococcus gattii é um dos principais agentes de infecção oportunista em todo o mundo. Esse fungo está presente no meio ambiente e, por isso, pode infectar diversos hospedeiros, inclusive seres humanos, nematóides e células ameboides. Acanthamoeba spp. são protozoários de vida livre que fagocitam diversos organismos, especialmente bactérias e fungos. Apesar de macrófagos e amebas serem evolutivamente distantes, eles compartilham diversas etapas comuns no processo de fagocitose e eliminação do patógeno. Além disso, existem teorias de que amebas e macrófagos possuem um ancestral comum. Para averiguar se essas duas células fagocíticas apresentam estratégias antifúngicas similares, nós analisamos o mecanismo de imunidade nutricional. Essa estratégia imunológica reduz a disponibilidade de nutrientes essenciais para o patógeno, inclusive metais de transição como o zinco. Neste trabalho, nós analisamos se há modulação da homeostase de zinco em Acanthamoeba castellanii durante sua interação com C. gattii. Testes de fagocitose e taxa de replicação intracelular (IPR) realizados através da interação de amebas com a linhagem selvagem (WT) e mutante do gene ZIP1 de C. gattii. O mutante utilizado (zip1Δ) é caracterizado pela sua incapacidade de crescer sem a presença de zinco. Nós observamos que a linhagem mutante foi mais fagocitada por células de A. castellanii comparado com WT. Também, o teste de IPR mostrou que a atividade antifúngica das células hospedeiras apresentou-se mais efetiva contra as células mutantes. Entretanto, a sobrevivência de zip1Δ foi maior quando zinco extracelular (10 M) foi adicionado ao meio de interação. Esses resultados sugerem que as células criptocócicas internalizadas podem estar sofrendo uma privação da disponibilidade de zinco no interior do fagossomo. Para analisar alterações nos transportadores de zinco das células hospedeiras durante sua interação com C. gattii, análises de PCR quantitativo em tempo real (RT-qPCR) foram realizadas para os transportadores de zinco das famílias ZIP e ZnT. Uma intensa modulação de alguns genes foi observado após 3 e 24 horas pós-infecção. Além disso, análises de citometria de fluxo mostraram que os níveis de zinco livre das amebas estavam reduzidos devido a presença do fungo. Esses resultados sugerem que amebas podem modular a disponibilidade de zinco, afim de prejudicar o patógeno. / Cryptococcus gattii is one of the most important agents of opportunistic infections worldwide. They are found in the environment, where it can interact with different host types, including humans, nematodes and amoebic cells. Acanthamoeba spp. are free-living protozoa that basically feed on bacteria and yeast through phagocytosis. Macrophages and amoebae, although evolutionarily distant, share conserved mechanisms related to steps of phagocytosis and microbial killing. In addition, it has been hypothesized that amoeba and macrophage have a common ancestor. To investigate if there are similar antifungal strategies between both cellular types, we analyzed the nutritional immunity mechanism. It is a process defined as a reduction of essential nutrients availability to the pathogen, such as zinc. In this context, we investigate if amoeba cells are able to modulate zinc homeostasis during the interaction with C. gattii. Phagocytosis and intracellular replications (IPR) analysis performed through the interaction between amoebae and wild-type (WT) and mutant for the ZIP1 gene (zip1Δ) strains of C. gattii. The mutant is unable to grow in absence of zinc. We found that zip1Δ strain is more readily engulfed by A. castellanii cells compared to WT. In addition, IPR analysis showed that the antifungal activity of such host cells was more effective against the mutant cells. However, the mutant strain survival was increased when additional extracellular zinc (10 M) was added to the interaction medium. This data suggests that engulfed cryptococcal cells might have been experiencing a deprivation of zinc inside the phagosome. To further evaluate alterations of zinc transporters in host cells due to cryptococcal infection, RT-qPCR analysis was performed for the ZIP and ZnT zinc transporter families. An intense modulation of some genes was found after 3 and 24 hours’ post-infection. Furthermore, flow cytometry analysis showed that free zinc levels from amoebae are reduced by the cryptococcal presence. These results indicate that amoebae are able to modulate zinc availability to harm the pathogen.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 117-136.

Foram coletadas amostras de água de piscinas térmicas e não térmicas na cidade de Porto Alegre, RS, Brasil entre os meses de maio de 2006 e março de 2007, com o objetivo de determinar a presença do gênero Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e trofozoítos e na amplificação por PCR com oligonucleotídeos gênero-específico. O potencial patogênico foi avaliado usando testes de osmotolerância e termotolerância. Das 65 amostras analisadas, 13 (20%) foram positivas para amebas de vida livre e identificados morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 9 possuíam características compatíveis com o grupo morfológico II e 4 com o grupo III. Todos os isolados identificados morfologicamente quando submetidos à Reação de PCR, confirmaram pertencer ao gênero Acanthamoeba e 38% (5/13) dos isolados foram considerados potencialmente patogênicos a partir dos testes de osmotolerância e termotolerância. Neste estudo, o método molecular de RAPD ("Random Amplified Polymorphic-DNA") foi utilizado para investigar a relação genética entre os 13 isolados de piscinas e dois isolados de referência da ATCC. De 10 oligonucleotídeos decaméricos testados, quatro foram selecionados por gerarem produtos de amplificação passíveis de análise. A similaridade entre os isolados foi calculada utilizando-se o coeficiente de Jaccard e o dendrograma construído pelo método da média das distâncias entre grupos ("Average Linkage"). Quatro grupos distintos (G1-G4) de isolados foram formados de acordo com a similaridade genética entre eles. Sugeriu-se que os isolados do G1 por agruparem-se aos isolados de referência de A. castellanii (ATCC 30010 e 50492) possam pertencer a esta espécie. Os dados fenotípicos, tais como, morfologia e testes de tolerância foram relacionados aos dados genotípicos de RAPD e permitiram a caracterização dos isolados. Os resultados deste primeiro estudo de isolamento e caracterização de Acanthamoeba de água de piscinas na cidade de Porto Alegre-RS, Brasil confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana. / Water samples were collected from both heated and unheated swimming pools in the city of Porto Alegre, RS, Brazil between May 2006 and March 2007, to determine the presence of Acanthamoeba in the water of swimming pools as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Escherichia coli. The identification of the isolates was based on the trophozoites and cysts morphology and on the amplification through PCR with genus-specific oligonucleotides. The potential pathogenic was assessed by osmotolerance and temperature tolerance assays. From the 65 samples analyzed, 13 (20%) were positive for free-living amoebae, and the isolates morphologically identified as belonging to the genus Acanthamoeba. Out of these, 9 presented characteristics compatible with morphological group II, and 4 with group III. All the morphologically identified isolates, when submitted to PCR, were confirmed as belonging to the genus Acanthamoeba, and 38% (5/13) of the isolates were considered potentially pathogenic according to osmotolerance and temperature tolerance assays. In this study, the molecular RAPD method (Random Amplified Polymorphic-DNA) was used to investigate the genetic relationship among 13 isolates from swimming pools and two strains from the ATCC reference. From the ten decameric oligonucleotides tested, four were selected for generating products of amplification possible to be analyzed. The similarity between isolates was calculated using the Jaccard coefficient and the dendrogram constructed by using the method of the average distances between groups ("Average Linkage"). Four distinct groups (G1-G4) of isolates were separated according to genetic similarity between them. It was suggested that the isolates from G1 once group up with the reference isolates of A. castellanii may belong to this species. The phenotypic data such as morphology and tolerance tests were related to RAPD genotypic data and led to the characterization of isolates. The results of this study about isolation and characterization of Acanthamoeba in swimming pools water at Porto Alegre, RS, Brazil confirm the presence of potentially pathogenic isolates which can present risks to human health.

Cryptococcus gattii é um dos principais agentes de infecção oportunista em todo o mundo. Esse fungo está presente no meio ambiente e, por isso, pode infectar diversos hospedeiros, inclusive seres humanos, nematóides e células ameboides. Acanthamoeba spp. são protozoários de vida livre que fagocitam diversos organismos, especialmente bactérias e fungos. Apesar de macrófagos e amebas serem evolutivamente distantes, eles compartilham diversas etapas comuns no processo de fagocitose e eliminação do patógeno. Além disso, existem teorias de que amebas e macrófagos possuem um ancestral comum. Para averiguar se essas duas células fagocíticas apresentam estratégias antifúngicas similares, nós analisamos o mecanismo de imunidade nutricional. Essa estratégia imunológica reduz a disponibilidade de nutrientes essenciais para o patógeno, inclusive metais de transição como o zinco. Neste trabalho, nós analisamos se há modulação da homeostase de zinco em Acanthamoeba castellanii durante sua interação com C. gattii. Testes de fagocitose e taxa de replicação intracelular (IPR) realizados através da interação de amebas com a linhagem selvagem (WT) e mutante do gene ZIP1 de C. gattii. O mutante utilizado (zip1Δ) é caracterizado pela sua incapacidade de crescer sem a presença de zinco. Nós observamos que a linhagem mutante foi mais fagocitada por células de A. castellanii comparado com WT. Também, o teste de IPR mostrou que a atividade antifúngica das células hospedeiras apresentou-se mais efetiva contra as células mutantes. Entretanto, a sobrevivência de zip1Δ foi maior quando zinco extracelular (10 M) foi adicionado ao meio de interação. Esses resultados sugerem que as células criptocócicas internalizadas podem estar sofrendo uma privação da disponibilidade de zinco no interior do fagossomo. Para analisar alterações nos transportadores de zinco das células hospedeiras durante sua interação com C. gattii, análises de PCR quantitativo em tempo real (RT-qPCR) foram realizadas para os transportadores de zinco das famílias ZIP e ZnT. Uma intensa modulação de alguns genes foi observado após 3 e 24 horas pós-infecção. Além disso, análises de citometria de fluxo mostraram que os níveis de zinco livre das amebas estavam reduzidos devido a presença do fungo. Esses resultados sugerem que amebas podem modular a disponibilidade de zinco, afim de prejudicar o patógeno. / Cryptococcus gattii is one of the most important agents of opportunistic infections worldwide. They are found in the environment, where it can interact with different host types, including humans, nematodes and amoebic cells. Acanthamoeba spp. are free-living protozoa that basically feed on bacteria and yeast through phagocytosis. Macrophages and amoebae, although evolutionarily distant, share conserved mechanisms related to steps of phagocytosis and microbial killing. In addition, it has been hypothesized that amoeba and macrophage have a common ancestor. To investigate if there are similar antifungal strategies between both cellular types, we analyzed the nutritional immunity mechanism. It is a process defined as a reduction of essential nutrients availability to the pathogen, such as zinc. In this context, we investigate if amoeba cells are able to modulate zinc homeostasis during the interaction with C. gattii. Phagocytosis and intracellular replications (IPR) analysis performed through the interaction between amoebae and wild-type (WT) and mutant for the ZIP1 gene (zip1Δ) strains of C. gattii. The mutant is unable to grow in absence of zinc. We found that zip1Δ strain is more readily engulfed by A. castellanii cells compared to WT. In addition, IPR analysis showed that the antifungal activity of such host cells was more effective against the mutant cells. However, the mutant strain survival was increased when additional extracellular zinc (10 M) was added to the interaction medium. This data suggests that engulfed cryptococcal cells might have been experiencing a deprivation of zinc inside the phagosome. To further evaluate alterations of zinc transporters in host cells due to cryptococcal infection, RT-qPCR analysis was performed for the ZIP and ZnT zinc transporter families. An intense modulation of some genes was found after 3 and 24 hours’ post-infection. Furthermore, flow cytometry analysis showed that free zinc levels from amoebae are reduced by the cryptococcal presence. These results indicate that amoebae are able to modulate zinc availability to harm the pathogen.

Foram coletadas amostras de água de piscinas térmicas e não térmicas na cidade de Porto Alegre, RS, Brasil entre os meses de maio de 2006 e março de 2007, com o objetivo de determinar a presença do gênero Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e trofozoítos e na amplificação por PCR com oligonucleotídeos gênero-específico. O potencial patogênico foi avaliado usando testes de osmotolerância e termotolerância. Das 65 amostras analisadas, 13 (20%) foram positivas para amebas de vida livre e identificados morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 9 possuíam características compatíveis com o grupo morfológico II e 4 com o grupo III. Todos os isolados identificados morfologicamente quando submetidos à Reação de PCR, confirmaram pertencer ao gênero Acanthamoeba e 38% (5/13) dos isolados foram considerados potencialmente patogênicos a partir dos testes de osmotolerância e termotolerância. Neste estudo, o método molecular de RAPD ("Random Amplified Polymorphic-DNA") foi utilizado para investigar a relação genética entre os 13 isolados de piscinas e dois isolados de referência da ATCC. De 10 oligonucleotídeos decaméricos testados, quatro foram selecionados por gerarem produtos de amplificação passíveis de análise. A similaridade entre os isolados foi calculada utilizando-se o coeficiente de Jaccard e o dendrograma construído pelo método da média das distâncias entre grupos ("Average Linkage"). Quatro grupos distintos (G1-G4) de isolados foram formados de acordo com a similaridade genética entre eles. Sugeriu-se que os isolados do G1 por agruparem-se aos isolados de referência de A. castellanii (ATCC 30010 e 50492) possam pertencer a esta espécie. Os dados fenotípicos, tais como, morfologia e testes de tolerância foram relacionados aos dados genotípicos de RAPD e permitiram a caracterização dos isolados. Os resultados deste primeiro estudo de isolamento e caracterização de Acanthamoeba de água de piscinas na cidade de Porto Alegre-RS, Brasil confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana. / Water samples were collected from both heated and unheated swimming pools in the city of Porto Alegre, RS, Brazil between May 2006 and March 2007, to determine the presence of Acanthamoeba in the water of swimming pools as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Escherichia coli. The identification of the isolates was based on the trophozoites and cysts morphology and on the amplification through PCR with genus-specific oligonucleotides. The potential pathogenic was assessed by osmotolerance and temperature tolerance assays. From the 65 samples analyzed, 13 (20%) were positive for free-living amoebae, and the isolates morphologically identified as belonging to the genus Acanthamoeba. Out of these, 9 presented characteristics compatible with morphological group II, and 4 with group III. All the morphologically identified isolates, when submitted to PCR, were confirmed as belonging to the genus Acanthamoeba, and 38% (5/13) of the isolates were considered potentially pathogenic according to osmotolerance and temperature tolerance assays. In this study, the molecular RAPD method (Random Amplified Polymorphic-DNA) was used to investigate the genetic relationship among 13 isolates from swimming pools and two strains from the ATCC reference. From the ten decameric oligonucleotides tested, four were selected for generating products of amplification possible to be analyzed. The similarity between isolates was calculated using the Jaccard coefficient and the dendrogram constructed by using the method of the average distances between groups ("Average Linkage"). Four distinct groups (G1-G4) of isolates were separated according to genetic similarity between them. It was suggested that the isolates from G1 once group up with the reference isolates of A. castellanii may belong to this species. The phenotypic data such as morphology and tolerance tests were related to RAPD genotypic data and led to the characterization of isolates. The results of this study about isolation and characterization of Acanthamoeba in swimming pools water at Porto Alegre, RS, Brazil confirm the presence of potentially pathogenic isolates which can present risks to human health.

Cryptococcus gattii é um dos principais agentes de infecção oportunista em todo o mundo. Esse fungo está presente no meio ambiente e, por isso, pode infectar diversos hospedeiros, inclusive seres humanos, nematóides e células ameboides. Acanthamoeba spp. são protozoários de vida livre que fagocitam diversos organismos, especialmente bactérias e fungos. Apesar de macrófagos e amebas serem evolutivamente distantes, eles compartilham diversas etapas comuns no processo de fagocitose e eliminação do patógeno. Além disso, existem teorias de que amebas e macrófagos possuem um ancestral comum. Para averiguar se essas duas células fagocíticas apresentam estratégias antifúngicas similares, nós analisamos o mecanismo de imunidade nutricional. Essa estratégia imunológica reduz a disponibilidade de nutrientes essenciais para o patógeno, inclusive metais de transição como o zinco. Neste trabalho, nós analisamos se há modulação da homeostase de zinco em Acanthamoeba castellanii durante sua interação com C. gattii. Testes de fagocitose e taxa de replicação intracelular (IPR) realizados através da interação de amebas com a linhagem selvagem (WT) e mutante do gene ZIP1 de C. gattii. O mutante utilizado (zip1Δ) é caracterizado pela sua incapacidade de crescer sem a presença de zinco. Nós observamos que a linhagem mutante foi mais fagocitada por células de A. castellanii comparado com WT. Também, o teste de IPR mostrou que a atividade antifúngica das células hospedeiras apresentou-se mais efetiva contra as células mutantes. Entretanto, a sobrevivência de zip1Δ foi maior quando zinco extracelular (10 M) foi adicionado ao meio de interação. Esses resultados sugerem que as células criptocócicas internalizadas podem estar sofrendo uma privação da disponibilidade de zinco no interior do fagossomo. Para analisar alterações nos transportadores de zinco das células hospedeiras durante sua interação com C. gattii, análises de PCR quantitativo em tempo real (RT-qPCR) foram realizadas para os transportadores de zinco das famílias ZIP e ZnT. Uma intensa modulação de alguns genes foi observado após 3 e 24 horas pós-infecção. Além disso, análises de citometria de fluxo mostraram que os níveis de zinco livre das amebas estavam reduzidos devido a presença do fungo. Esses resultados sugerem que amebas podem modular a disponibilidade de zinco, afim de prejudicar o patógeno. / Cryptococcus gattii is one of the most important agents of opportunistic infections worldwide. They are found in the environment, where it can interact with different host types, including humans, nematodes and amoebic cells. Acanthamoeba spp. are free-living protozoa that basically feed on bacteria and yeast through phagocytosis. Macrophages and amoebae, although evolutionarily distant, share conserved mechanisms related to steps of phagocytosis and microbial killing. In addition, it has been hypothesized that amoeba and macrophage have a common ancestor. To investigate if there are similar antifungal strategies between both cellular types, we analyzed the nutritional immunity mechanism. It is a process defined as a reduction of essential nutrients availability to the pathogen, such as zinc. In this context, we investigate if amoeba cells are able to modulate zinc homeostasis during the interaction with C. gattii. Phagocytosis and intracellular replications (IPR) analysis performed through the interaction between amoebae and wild-type (WT) and mutant for the ZIP1 gene (zip1Δ) strains of C. gattii. The mutant is unable to grow in absence of zinc. We found that zip1Δ strain is more readily engulfed by A. castellanii cells compared to WT. In addition, IPR analysis showed that the antifungal activity of such host cells was more effective against the mutant cells. However, the mutant strain survival was increased when additional extracellular zinc (10 M) was added to the interaction medium. This data suggests that engulfed cryptococcal cells might have been experiencing a deprivation of zinc inside the phagosome. To further evaluate alterations of zinc transporters in host cells due to cryptococcal infection, RT-qPCR analysis was performed for the ZIP and ZnT zinc transporter families. An intense modulation of some genes was found after 3 and 24 hours’ post-infection. Furthermore, flow cytometry analysis showed that free zinc levels from amoebae are reduced by the cryptococcal presence. These results indicate that amoebae are able to modulate zinc availability to harm the pathogen.