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Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody's target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba's encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.

Free-living amoeba (FLA) group includes the potentially pathogenic genera Acanthamoeba, Naegleria, Balamuthia, Sappinia, and Vermamoeba, causative agents of human infections (encephalitis, keratitis, and disseminated diseases). In Brazil, the first report on pathogenic FLA was published in the 70s and showed meningoencephalitis caused by Naegleria spp. FLA studies are emerging, but no literature review is available to investigate this trend in Brazil critically. Thus, the present work aims to integrate and discuss these data. Scopus, PubMed, and Web of Science were searched, retrieving studies from 1974 to 2020. The screening process resulted in 178 papers, which were clustered into core and auxiliary classes and sorted into five categories: wet-bench studies, dry-bench studies, clinical reports, environmental identifications, and literature reviews. The papers dating from the last ten years account for 75% (134/178) of the total publications, indicating the FLA topic has gained Brazilian interest. Moreover, 81% (144/178) address Acanthamoeba-related matter, revealing this genus as the most prevalent in all categories. Brazil’s Southeast, South, and Midwest geographic regions accounted for 96% (171/178) of the publications studied in the present work. To the best of our knowledge, this review is the pioneer in summarising the FLA research history in Brazil.

Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.

This work explored hybrid films of silver nanowires (AgNWs) with indium-zinc oxide (IZO) for developing high-performance and low-cost electrocorticography (ECoG) electrodes. The transparent hybrid films achieved a sheet resistance of 6 Ω/sq enabling electrodes with 500 μm diameter to reach an impedance of 20 kΩ at 1 kHz and a charge storage capacity of 3.2 mC/cm2, an improvement in properties over IZO electrodes, whose performance is on par with the classical tin doped indium oxide (ITO). Characterization of light-induced artifacts was performed showing that light intensities 2 elicit minimal electrical potential variation, which falls within the magnitude of baseline noise. The validation of electrodes in vivo was achieved by recording electrical neural activity from the surface of rat cortex under anaesthesia. Moreover, the presence of the hybrid films did not cause distortion of light during fluorescence microscopy. This study highlighted the capabilities of the hybrid structure of AgNWs with IZO, that can be fabricated with industrially-established processes at low cost, to be used for transparent ECoG electrodes, offering a new way to record neural electrical activity on a large and fast scale with direct visualization of neurons.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais INCT – Instituto nacional de ciência e tecnologia (Antigo Instituto do Milênio) The zoonotic potential of giardiasis, as proposed by WHO since the late 70's, has been largely confirmed in this century. The genetic assemblages A and B of Giardia duodenalis are frequently isolated from human and canine hosts. Most of the assemblage A strains are not infective to adult mice, which can limit the range of studies regarding to biology of G. duodenalis, including virulence factors and the interaction with host immune system. This study aimed to determine the infectivity in mice of an assemblage A Giardia duodenalis strain (BHFC1) isolated from a dog and to classify the strain in sub-assemblages (AI, AII, AIII) through the phylogenetic analysis of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. In addition, the proteomic profile of soluble and insoluble protein fractions of trophozoites was analyzed by 2D-electrophoresis. Accordingly, trophozoites of BHFC1 were highly infective to Swiss mice. The phylogenetic analysis of tpi and gdh revealed that BHFC1 clustered to sub-assemblage AI. The proteomic map of soluble and insoluble protein fractions led to the identification of 187 proteins of G. duodenalis, 27 of them corresponding to hypothetical proteins. Considering both soluble and soluble fractions, the vast majority of the identified proteins (n = 82) were classified as metabolic proteins, mainly associated with carbon and lipid metabolism, including 53 proteins with catalytic activity. Some of the identified proteins correspond to antigens while others can be correlated with virulence. Besides a significant complementation to the proteomic data of G. duodenalis, these data provide an important source of information for future studies on various aspects of the biology of this parasite, such as virulence factors and host and pathogen interactions.

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred. INTRODUÇÃO: A produção de anticorpos policlonais anti-Cryptosporidium e sua utilização na imunofluorescência para determinar a presença de Cryptosporidium em água são descritas no presente trabalho. MÉTODOS: Dois coelhos foram imunizados com antígeno solúvel e particulado provenientes de oocistos purificados de Cryptosporidium. O soro produzido foi preparado para a extração de imunoglobulinas G, que foram purificadas e conjugadas com isotiocianato de fluoresceína (FITC). Lâminas contendo quantidades conhecidas de oocistos foram preparadas para determinar a sensibilidade da técnica. Para testar a especificidade foram preparadas lâminas contendo cistos de Giardia duodenalis. RESULTADOS: O conjugado foi usado com sucesso em amostras de água contaminadas experimentalmente com oocistos de Cryptosporidium, sendo capaz de detectar até cinco oocistos/spots que corresponde a uma contaminação de 250 oocistos/mL. CONCLUSÕES: As três imunizações realizadas nos coelhos foram suficientes para produção de anticorpos contra Cryptosporidium; a reação de imunofluorescência direta padronizada permitiu a detecção de cinco oocistos em 20% das amostras; não houve reação cruzada com cistos de Giardia duodenalis.

Free-living amoeba of the genus Acanthamoeba can eventually act as parasites, causing infections in humans. Some physiological characteristics of Acanthamoeba have been related to the grade of pathogenicity, allowing inferences about the pathogenic potential. The main goal of this study was to characterize isolates of Acanthamoeba obtained in Brazil and evaluate properties associated with their pathogenicity. A total of 39 isolates obtained from keratitis cases (n = 16) and environmental sources (n = 23) were classified into morphological groups and genotyped by sequencing the 18S rDNA fragments ASA.S1 and GTSA.B1. Samples were also tested regarding their thermo-tolerance, osmo-tolerance, and cytopathogenicity in MDCK cells. Isolates were identified and classified as follows: group I (T17, T18); group II (T1, T3, T4, T11); and group III (T5, T15), with the predominance of genotype T4 (22/39). Clinical isolates were genotyped as T3 (1/16), T4 (14/16) and T5 (1/16). The majority of isolates (38/39) were able to grow at 37 °C, but tolerance to 40 °C was more frequent among environmental samples. The tolerance to 1 M mannitol was infrequent (4/39), with three of these corresponding to clinical samples. The variable ability to cause cytopathic effects was observed among isolates of distinct genotypes and origins. This study identified, for the first time, T1, T15, and T18 in Brazil. It also indicated a weak association between the clinical origin of the isolates and tolerance to high temperatures, high osmolarity, and cytopathogenicity, demonstrating that some in vitro parameters do not necessarily reflect a higher propensity of Acanthamoeba to cause a disease.

Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N -acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N -acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.

Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Amoebae of the genus Acanthamoeba are free-living protozoa that can cause granulomatous encephalitis and keratitis in humans. In this study, four clinical and three household dust isolates obtained in Vitoria, Espirito Santo, Brazil were characterized by their morphological, genotypic, and physiological properties. All isolates belonged to group II according to Pussard and Pons’ cyst morphology. Analysis of their 18S rDNA sequence identified one isolate from household dust as genotype T11 and the others six samples as genotype T4. Five T4 isolates presented a highly variable region (DF3) in 18S rDNA identical to those previously described. Physiological assays carried out with trophozoites in co-culture with bacteria or in axenic conditions showed all samples tolerated temperatures up to 37 °C, regardless of culture method. One keratitis isolate grew at 42 °C in co-culture with bacteria. Most isolates in co-culture survived at 1.0 M, except a T11 isolate, which tolerated up to 0.5 M. The isolates did not grow at 42 °C and did not tolerate 0.5 M and 1.0 M under axenic condition. This is the first report of 18S rRNA gene genotyping applied to Acanthamoeba isolated from keratitis patients in Brazil. The results also indicated that osmo-tolerance is dependent on the culture system.

Free Living Amoeba (FLA) has gained increasing visibility due to human health concerns. They are unicellular heterotrophic protozoans, widely spread in nature. Water is the main source of amoebic contaminations that may result in encephalitis, keratitis, and skin infections. Pathogenic species belong to the Naegleria, Acanthamoeba, Sappinia, Balamuthia, and Hartmannella genera. To date, there is a lack of effective treatment and accurate diagnosis for amoebic infections, which has motivated the environmental characterization of FLA as a human health risk assessment strategy. However, FLA epidemiology in Brazil is scarce, contrasting with the country´s freshwater abundance. To address this gap, the present research aimed at the FLA characterization along the Monjolinho River, São Carlos, Brazil. The water abiotic evaluation as well as the morphological, and molecular investigation of FLA encompasses the central methodology. Dissolved oxygen and electrical conductivity were measured as the most informative features to inspect anthropogenic driving forces acting upon the hydric ecosystem. Eutrophic sites, downstream of the urban areas, were revealed as hotspots to a diversity of FLA. Non-nutrient agar (NNA) plates seeding confirmed the amoeba growth by light and electron microscopy characterization. Based on these results, relevant taxonomic details (e.g.: flagella, cyst walls, and pseudopodia branching patterns) were described to all FLA life cycle stages. Axenization attempts revealed the persistence of bacteria contamination, suggestive of the presence of amoebic resistant microorganisms, enhancing its threat to humans. Molecular characterization enabled us to detect the potentially pathogenic genera Naegleria (N. australiensis and N. phillipinensis), Acanthamoeba (A. genotype T4 and A. hatchetti), and Hartmanella (V. vermiforms); besides the non-pathogenic Vannela sp, Stenamoeba, Filamoeba, and Naegleria (N. canariensisi, N.gruberi, and N. dobsoni). In this work, the knowledge of FLA distribution allowed the recognition of Brazilian freshwaters as suitable ecological niches to FLA spread, as well as the development of the methodology necessary for the future identification and characterization of FLA in the environment. Although conducted to a local extend, this research suggests that other FLA genus can be equally isolated in Brazilian freshwater systems that can finally booster later expansion to other water streams. Amebas de vida livre (AVL) tem ganhado visibilidade devido à preocupação que provocam à saúde humana. São protozoários unicelulalres heterotróficos amplamente distribuídos na natureza, importantes para a saúde humana. A água é a principal fonte de contaminação por amebas, causando encefalites, ceratites, e infecções de pele. Espécies patogênicas pertencem aos gêneros Naegleria, Acanthamoeba, Sappinia, Balamuthia, e Hartmanella. Atualmente, os tratamentos e diagnósticos são pouco efetivos, o que impulsiona a caracterização ambiental de AVL como estratégia para monitoramento do risco de contaminação ao homem. Entretanto a epidemiologia de AVL no Brasil é pouco explorada, contrastando com a abundância do país em fontes de água doce. Para abordar esta lacuna, a presente pesquisa teve como objetivo a identificação de AVL no Rio Monjolinho, São Carlos, Brasil. A avaliação dos parâmetros abióticos da água, assim como investigação morfológica e molecular de AVL compuseram a metodologia central deste trabalho. Oxigênio dissolvido e condutividade elétrica foram os parâmetros limnologicos mais informativos para caracterizar as ações antropogênicas atuando sobre o ecossistema aquático. Sítios eutrofizados, à jusante da área urbana, revelaram ser foco para uma diversidade de AVL. As caracterizações por microscopia de luz e microscopia eletrônica confirmaram o crescimento de amebas em placas de ágar não nutritivo. Baseado nestes resultados, relevantes detalhes taxonômicos típicos dos estágios trofozoito, cisto, e flagelar (ex: flagelos, parede dos cistos e padrão de ramificação dos pseudopodes) foram evidenciados. Ensaios de axenização indicaram persistente contaminação bacteriana, possível consequência das AVLs atuando como reservatório para microrganismos resistentes à AVL, aumentando sua ameaça ao homem. A caracterização molecular permitiu a detecção de gêneros potencialmente patogênicos: Naegleria (N. australiensis and N. phillipinensis), Acanthamoeba (A. genotype T4 e A. hatchetti) e Hartmanella (V. vermiformis); além de não patogênicos Vannela, Stenamoeba, Filamoeba e Naegleria (N. canariensisi, N.gruberi, e N. dobsoni). No presente trabalho, o conhecimento da distribuição de AVLs permitiu o reconhecimento de corpos de água doce no Brasil como nichos ecológicos apropriados para a dispersão de AVL, assim como o desenvolvimento da metodologia necessária para a identificação e caracterização de AVL no ambiente em trabalhos futuros. Embora realizada em uma escala local, esta pesquisa sugere que outros gêneros de AVL podem ser igualmente isolados em sistemas de água doce do Brasil e, consequentemente impulsionar a expansão das investigações de AVL para outros sistemas aquáticos.

As amebas de vida livre do gênero Acanthamoeba são protozoários ubíquos que eventualmente agem como parasitas, causando infecções graves como a encefalite amebiana granulomatosa e a ceratite amebiana. Um dos aspectos importantes da fisiopatologia das acantamebíases é a capacidade de encistamento do protozoário nos tecidos infectados. Os cistos de Acanthamoeba são formados por paredes duplas resistentes, resultando em menor eficácia de medicamentos e consequente demora no tratamento das infecções e ainda em falha terapêutica. O processo de encistamento ainda não é amplamente compreendido e as ciências ômicas podem fornecer importantes contribuições na avaliação do fenótipo de organismo. Portanto, esse trabalho teve como objetivo determinar o perfil metabólico de Acanthamoeba castellanii durante o processo de diferenciação celular de trofozoítos para cistos. Uma cepa de A. castellanii em cultivo axênico foi induzida ao encistamento em salina de Neff, estabelecendo-se a cinética de formação de pré-cistos e cistos. Culturas em tempo zero (somente trofozoítos), 24 horas (encistamento iniciado) e 72 horas (somente cistos) foram submetidas à extração de metabólitos para avaliação por cromatografia gasosa acoplada a espectrometria de massas (GC/MS). Foram identificados dezoito metabólitos com significância estatística, incluindo aminoácidos, lipídios, carboidratos, compostos nitrogenados e poliaminas. De forma geral, houve redução da concentração dos metabólitos com o avanço do encistamento, com exceção da arbutina, celobiose e lactose. Os resultados indicam uma diminuição do metabolismo de Acanthamoeba durante o encistamento, além de alteração de sua fonte energética e de carbono. Sugere-se ainda o envolvimento dos transportadores ABC no encistamento, como possíveis exportadores do produto da degradação de organelas e macromoléculas no processo. The free-living amoebae of the genus Acanthamoeba are ubiquitous protozoa that can eventually act as parasites, causing serious infections such as granulomatous amoebic encephalitis and amoebic keratitis. A relevant aspect of the pathophysiology of acanthamoebiasis is the ability of the protozoa to encyst on infected tissues. Acanthamoeba cysts present resistant double walls, resulting in lower efficacy of drugs, delay in the treatment and therapeutic failure. The encystment process is not entirely understood and the omics sciences can contribute to evaluate the phenotype of organisms. Thus, the aim of this work was to determine the metabolic profile of Acanthamoeba castellanii during the process of cell differentiation of trophozoites into cysts, since the omics sciences can contribute to the evaluation of gene expression and phenotype of organisms. Encystment was induced in a strain of A. castellanii in axenic culture with Neff saline, establishing the kinetics of pre-cysts and cysts formation. Time zero (throphozoites only), twenty-four hours (transformation initiated) and seventy-two hours (cysts only) cultures were submitted to extraction of metabolites for evaluation by gas chromatography coupled to mass spectrometry (GC/MS). Eighteen metabolites with statistical significance were identified, including amino acids, lipids, carbohydrates, nitrogen compounds and polyamines. In general, there was a reduction of the concentration of metabolites with the advance of the encystment, with the exception of arbutine, cellobiose and lactose. The results indicated a decrease of Acanthamoeba metabolism during encystment, as well as an alteration of its energy and carbon source. The involvement of ABC transporters in encystment was also suggested. They could act as exporters of degradation products from organelles and macromolecules during the process.

As leishmanioses compõem um grupo de doenças causadas por parasitos do gênero Leishmania e possuem grande importância em saúde pública. O controle dessas doenças ainda é um desafio, devido a crescente resistência às drogas utilizadas no tratamento. Nesse contexto, o estudo de modelos resistentes a drogas é essencial para se explorar o potencial metabólico dos parasitos e melhor compreender os mecanismos envolvidos na aquisição de resistência a fármacos. Neste estudo, foi gerada uma linhagem mutante de Leishmania braziliensis resistente ao antibiótico tunicamicina, fármaco que atua na inibição da glicosilação proteica. Para a geração dos mutantes foi utilizado o método de aumento gradual de concentração de droga (stepwise). Com o intuito de avaliar as adaptações metabólicas induzidas durante o processo de aquisição de resistência à tunicamicina, foram realizadas análises genômicas, ensaios de microscopia, infectividade, citometria e análise do metaboloma da linhagem resistente. As promastigotas de Leishmania braziliensis resistentes à tunicamicina (até 80 vezes o valor da DL50), apresentaram amplificação gênica e um concomitante aumento de virulência. Modificações morfológicas foram observadas na linhagem resistente como, desorganização das cisternas do complexo de Golgi indicando possíveis alterações no processo de glicosilação proteica. Alterações no metabolismo de várias fontes de carbono, relacionados à produção de substrato para a síntese de dolicol, molécula que desempenha papel importante na glicosilação de proteínas, foram detectadas como resposta ao bloqueio do processo induzido pela tunicamicina. Os dados gerados permitiram a concepção de um modelo de estudo dos mecanismos de resistência a drogas, bem como a influencia da glicosilação proteica nas relações parasito-hospedeiro em Leishmania. Leishmaniasis is a group of diseases caused by parasites of the Leishmania type and is of great importance in public health. Disease control is still a challenge due to its resistance to the drugs used in treatment. In this context, the study of potential resistance is essential for the exploration of the potential metabolism of parasitism and improve the structures of the drugs of the stress of drugs In this study, a mutant strain of Leishmania braziliensis resistant to the antibiotic tunicamycin, a drug that inhibits the glycosylation of protein, was generated by the gradual increase in drug concentration (stepwise). In order to evaluate the metabolic adaptations caused during the process of developing resistance to tunicamycin. In order to evaluate the metabolic changes during the acquisition of resistance to tunicamycin, genomic analyzes, microscopy, infectivity, cytometry and resistance metabolism analysis were performed. Promastigotes of Leishmania braziliensis resistant to tunicamycin (up to 80 times the value of LD50), gene amplification and a concomitant increase in virulence. Morphological changes were observed in the lineage such, disorganization of cisterns of the Golgi complex, changes in the process of protein glycosylation. Changes in the metabolism of various carbon sources, related to the production of substrate for a synthesis of fatty acid, molecules that play an important role in the glycosylation of proteins, were detected as responses to the induction blocking process of tunicamycin. The results suggests that main mechanisms of drug resistance, as well as the influence of protein glycosylation on host parasitic infections in Leishmania.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As amebas de vida livre do gênero Acanthamoeba são protozoários ubíquos isolados dos mais variados ambientes. Eventualmente, são encontradas causando sérias doenças nos seres humanos. A infecção mais comum causada por esse protozoário é a ceratite amebiana (CA), caracterizada por uma grave inflamação na córnea que pode levar à cegueira. O conhecimento sobre as proteínas parasitárias envolvidas no processo fisiopatológico pode ser útil para desenvolver estratégias de diagnóstico e terapêuticas para CA. Assim, realizou-se de um estudo proteômico comparativo para identificar proteínas diferencialmente expressas por trofozoítos de Acanthamoeba, provenientes de cultura de longo prazo e de re-isolados obtidos de lesão da córnea induzida em modelo animal de CA. Para tal, uma cepa de Acanthamoeba castellani isolada de um paciente com CA, mantida em cultura axênica em meio PYG desde 2006, foi utilizada. Ratos Winstar (45 dias) foram utilizados para induzir CA, após a inoculação intraestromal de 104 trofozoítos. Com o objetivo de definir qual o método de obtenção de proteínas mais adequado para a análise proteômica, trofozoítos da cepa ALX de longo prazo foram submetidos aos métodos de extração proteica por sonicação e “freeze/thaw” na presença de detergente CHAPS. O método de sonicação mostrou-se mais adequado, resultando em mapas bidimensionais de maior resolução. Para a confecção dos géis bidimensionais das culturas avaliadas, as proteínas totais de ALXltc, ALXrec1, ALXrec2 e ALXrec3 foram focalizadas em strips. Posteriormente, as proteínas já focalizadas foram separadas na segunda dimensão em eletroforese de poliacrilamida. A detecção das proteínas diferencialmente expressas foi realizada através do software ImageMaster 2D Platinum. Sessenta e dois spots foram selecionados para a espectrometria de massas, sendo 42 identificados na base de dados específica de Acanthamoeba. Destas, oito correspondiam a proteínas hipotéticas com função desconhecida. Análises de função molecular revelaram que a maioria das demais proteínas estão associadas à processos biológicos relacionados à oxirredução e função molecular de ligação à outra proteína. Por intermédio da representação em um heatmap da análise de expressão diferencial entre a cultura de longo prazo e culturas recuperadas, foi possível inferir que Acanthamoeba modula a expressão de várias proteínas associadas principalmente ao metabolismo energético, à atividade proteolítica, ao controle da expressão gênica, pela degradação de proteínas ou metilação do DNA, à resposta ao extresse e reparo do DNA, para adaptação ao novo ambiente e resistência aos mecanismos imunes do hospedeiro, promovendo lesões teciduais. The free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa isolated from the most varied environments. Eventually, they are found causing serious illness in humans. The most frequently infection caused by this protozoan is amoebic keratitis (KA), characterized by severe inflammation in the cornea that can lead to blindness. Knowledge about the parasitic proteins involved in the pathophysiological process may be useful to develop diagnostic and therapeutic strategies for CA. Thus, a comparative proteomic study was performed to identify proteins differentially expressed by Acanthamoeba trophozoites from long-term culture and from re-isolates obtained from KA-induced in a animal model. For this purpose, a strain of Acanthamoeba castellanii isolated from a patient with KA maintained in axenic culture in PYG medium since 2006 was used. Winstar rats where used to induce KA after intraestromal inoculation of 104 trophozoites. In order to define the most suitable method of obtaining proteins for the proteomic analysis, trophozoites of the longo-term culture ALX were submitted to the methods of protein extraction by sonication and freeze/thaw in the presence of detergent CHAPS. The sonication method was more adequate, resulting in two-dimensional maps of higher resolution. For the preparation of the two-dimensional gels of the evaluated cultures, total proteins of ALXltc, ALXrec1, ALXrec2 and ALXrec3 were focused. Subsequently, the already focused proteins were separated in the second dimension in polyacrylamide electrophoresis. Detection of differentially expressed proteins was performed using ImageMaster 2D Platinum software. Sixty-two spots were selected for mass spectrometry, of which 42 were identified in the Acanthamoeba specific database. Of these, eight hypothetical proteins with unknown function could be detected. Molecular function analyzes revealed that most proteins are associated with biological processes related to oxidation and molecular function of binding to the other protein. Through the representation in a heatmap of the analysis of differential expression between long-term culture and re-isolated cultures, it was possible to infer that Acanthamoeba modulates the expression of several proteins associated mainly to energy metabolism, proteolytic activity, control of gene expression, protein degradation or DNA methylation, response to DNA stress and repair, adaptation to the new environment, resistance to host immune mechanisms, and promotion of tissue damage.

A amebíase é a protozoose mais grave que atinge o ser humano e a segunda maior causa de morte entre as doenças parasitárias, superada apenas pela malária. Considera-se que apenas a Entamoeba histolytica é capaz de penetrar nos tecidos e produzir amebíase invasiva. Contudo, alguns estudos têm demonstrado que a Entamoeba dispar é capaz de produzir lesões em modelos experimentais. Com o objetivo de determinar se as bactérias enteropatogênicas contribuem para aumentar a expressão gênica dos fatores de virulência amebianos, bem como de induzir lesões mais intensas, propusemos avaliar a influência da bactéria Salmonella enterica sorovar Typhimurium sobre a virulência da Entamoeba dispar e da Entamoeba histolytica em trofozoítos isolados da cultura in vitro e avaliar patologicamente a colite amebiana, em ratos infectados com trofozoítos amebianos e co-infectados com S. Typhimurium. As cepas MCR, ADO e VEJ de E. dispar e, EGGp e EGGa de E. histolytica foram cultivadas separadamente e, associadas ou não à bactéria S. Typhimurium. Em seguida foi realizada a extração do RNA dessas culturas para produção de cDNA dos fatores de virulência de E. histolytica e E.dispar e quantificada a expressão dos fatores de virulência. Para a indução de colite amebiana, 8 ratos wistar (Rattus norvegicus) por grupo, previamente submetidos a uma intubação intragástrica da S. Typhimurium, foram inoculados pela via intracecal com 5x105 trofozoítos. Como grupos controles, 40 ratos foram somente inoculados com trofozoítos amebianos das cepas de Entamoeba estudadas. Outros 16 ratos constituíram os grupos controles somente infectados com S. Typhimurium e controle não infectado. Todos os ratos foram sacrificados 7 dias após a infecção amebiana para a coleta do ceco, confecção de lâminas histológicas para análise histopatológica e morfométrica e, para a realização de reações imuno-histoquímicas anti IL-1á, IL4, COX-2 e MUC-2. Nosso estudo permitiu observar que a co-cultura de E. dispar com a enterobactéria S. Typhimurium foi capaz de aumentar a expressão dos fatores de virulência amebianos amebaporo A, cisteína proteinase-5 e lectina ligante de Galactose e N-Acetilgalactosamina. Ao contrário do observado por outros autores, as cepas de E. dispar por nós estudados foram capazes de expressar o fator de virulência amebiano cisteína proteinase-5. A co-cultura de E. histolytica com a enterobactéria S. Typhimurium foi capaz de aumentar a expressão do fator de virulência amebiano amebaporo A. Também verificamos que na maioria dos grupos analisados, a associação in vivo da E. dispar ou da E. histolytica com a S. Typhimurium foi capaz de aumentar a intensidade da colite amebiana ulcerativa, também acompanhada pelo aumento da expressão de mediadores pró-inflamatórios e da redução da expressão de IL-4. Além disso, na maioria dos grupos analisados, a associação in vivo da E. dispar ou da E. histolytica com a S. Typhimurium foi capaz de aumentar a expressão da mucina MUC2, em virtude da maior intensidade da agressão à mucosa colônica. Diante dos resultados obtidos, concluímos que a interação da S. Typhimurium com a E. dispar e a E. histolytica foi capaz aumentar a virulência destas espécies de amebas levando à colite ulcerativa amebiana de maior intensidade. Amebiasis is the most serious protozoa that hits the human intestine and is the second-leading cause of death among parasitic diseases, surpassed only by malaria. It is considered that only Entamoeba histolytica is able to penetrate tissues and produce invasive amebiasis. However, some studies have shown that Entamoeba dispar is capable of producing lesions in experimental models. In order to determine if the enteropathogenic bacteria contribute to increase the gene expression of amebic virulence factors, as well as to induce more intense lesions, we propose to evaluate the influence of Salmonella Enterica sorovar Typhimurium on the virulence of Entamoeba dispar and Entamoeba histolytica in trophozoites isolated from the in vitro culture and pathologically evaluate amoebic colitis in rats infected with amoebic trophozoites and co-infected with S. typhimurium. E. dispar, MCR, ADO and VEJ strains from E. dispar and, EGGp and EGGa from E. histolytica were cultured separately and associated or not with the S. Typhimurium bacterium. Then, the RNA extraction from these cultures was carried out to produce cDNA from the virulence factors of E. histolytica and E.dispar and quantified the expression of the virulence factors. For the induction of amoebic colitis, 8 wistar rats (Rattus norvegicus) per group, previously submitted to an intragastric intubation of S. Typhimurium, were inoculated by the intracecal route with 5x105 trophozoites. As control groups, 40 rats were only inoculated with amebian trophozoites from the Entamoeba strains studied. Another 16 rats were the control groups infected only with S. typhimurium and uninfected control. All rats were sacrificed 7 days after amebic infection to collect cecum, histological slides for histopathological and morphometric analysis, and for the immunohistochemical anti-IL1á, IL4, COX-2 and MUC-2 immunohistochemical reactions . Our study showed that the co-culture of E. dispar with the S. typhimurium enterobacterium was able to increase the expression of amebaporo A, cysteine proteinase-5 and Galactose-binding lectin and N-acetylgalactosamine virulence factors. Contrary to what was observed by other authors, E. dispar strains studied by us were able to express the amoebic virulence factor cysteine proteinase-5. The co-culture of E. histolytica with the S. typhimurium enterobacterium was able to increase the expression of the amebian virulence factor amebaporo A. We also found that in the majority of the groups analyzed, the in vivo association of E. dispar or E. histolytica with S. typhimurium was able to increase the intensity of ulcerative amebic colitis, also accompanied by increased expression of proinflammatory mediators and reduced IL-4 expression. In addition, in most of the analyzed groups, the in vivo association of E. dispar or E. histolytica with S. typhimurium was able to increase MUC-2 mucin expression, due to the greater intensity of aggression to the colonic mucosa. In view of the results obtained, we conclude that the interaction of S. Typhimurium with E. dispar and E. histolytica was able to increase the virulence of these species of amoebae leading to ulcerative amebic colitis of greater intensity.

As amebas do gênero Acanthamoeba são organismos de vida livre presentes nos mais variados ambientes, e eventualmente causam graves infecções no ser humano, como a encefalite amebiana granulomatosa e a ceratite por Acanthamoeba. Diferentes espécies de Acanthamoeba foram descritas e atualmente são classificadas em mais de 20 diferentes genótipos, com base em sequências do 18S rDNA. Um fator relacionado à patogenia e virulência de Acanthamoeba é a capacidade de secretar proteases, que parecem estar envolvidas na patogênese da infecção. Alguns estudos têm avaliado o perfil quantitativo e qualitativo desses produtos de secreção, buscando correlações com a virulência entre diferentes linhagens. O objetivo desse trabalho foi realizar uma análise comparativa do perfil de proteases secretadas por amostras clínicas e ambientais de Acanthamoeba de diferentes genótipos, e avaliar se ocorre alteração desse perfil após a interação com células de cultivo MDCK. Seis isolados de Acanthamoeba de origem clínica e ambiental, sendo três pertencentes ao genótipo T4 e as demais de genótipo T1, T2, e T11, foram cultivados em meio PYG a 32o C. Os isolados foram avaliados por curva de crescimento e efeito citopático sobre células MDCK. Para os ensaios de zimografia e azocaseína, utilizou-se meio condicionado a 32º C, por 24 horas. O meio condicionado também foi obtido de culturas interagidas por três dias consecutivos com células MDCK. O crescimento dos isolados foi variado e todos produziram efeito citopático. Os perfis de zimografia variaram entre os isolados, que apresentaram proteases de massa molecular entre 170 e 58 kDa, a maioria identificada pelo uso de inibidores como serinoproteases. O isolado de genótipo T1, obtido de poeira ambiental, apresentou maior produção de proteases que os demais e uma provável cisteínoprotease de alto peso molecular, ainda não descrita para esse genótipo (170 kDa). Após a interação com células MDCK, houve alteração do perfil de zimografia em todos os isolados, com a supressão de algumas proteases e o surgimento de outras, de baixa massa molecular (

Neste trabalho, aspectos da patogenicidade e virulência da cepa ICB-ADO polixênica de Entamoeba dispar foram abordados, visando um melhor conhecimento dos mecanismos de agressão que essa cepa apresenta. Considerada uma espécie não-patogênica, E. dispar tem sido isolada de pacientes brasileiros com colite não disentérica e demonstrado capacidade de ocasionar lesões em animais de experimentação, quando associada à flora da microbiota intestinal de seus hospedeiros. Estudos in vivo utilizando hamsters como modelo experimental, verificaram diferenças na cinética das lesões hepáticas ocasionadas pela cepa ICB-ADO polixênica de E. dispar, diferindo substancialmente do observado para as cepas ICB-EGG e HM1-IMSS de E. histolytica. Apesar de ocasionar lesões extensas após sete dias de inoculação, a cepa ICB-ADO demonstra uma capacidade de lesar mais lentamente os tecidos. Estudos in vitro demonstraram ser a cepa ICB-ADO de E. dispar capaz de ocasionar a destruição e a queda da medida da resistência transepitelial de células MDCK em monocamadas, embora de forma mais lenta e gradual que cepas de E. histolytica. A cepa ICB-ADO polixênica apresentou também uma maior atividade de proteases quando comparada com a cepa HM1-IMSS de E. histolytica, devendo a relação bactérias/amebas ser melhor estudada, objetivando conhecer mais profundamente as alterações que elas causam nos trofozoítos quando em associação.