RESUMO
In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.
Assuntos
Acanthamoeba castellanii , Amoeba , beta-Glucanas , Amoeba/metabolismo , Manose/metabolismo , Proteômica , beta-Glucanas/metabolismo , Glucanos/metabolismo , Histoplasma/metabolismoRESUMO
Fungal infections have increased in the last years, particularly associated to an increment in the number of immunocompromised individuals and the emergence of known or new resistant species, despite the difficulties in the often time-consuming diagnosis. The controversial efficacy of the currently available strategies for their clinical management, apart from their high toxicity and severe side effects, has renewed the interest in the research and development of new broad antifungal alternatives. These encompass vaccines and passive immunization strategies with monoclonal antibodies (mAbs), recognizing ubiquitous fungal targets, such as fungal cell wall ß-1,3-glucan polysaccharides, which could be used in early therapeutic intervention without the need for the diagnosis at species level. As additional alternatives, based on the Dectin-1 great affinity to ß-1,3-glucan, our group developed broad antibody-like Dectin1-Fc(IgG)(s) from distinct subclasses (IgG2a and IgG2b) and compared their antifungal in vitro and passive immunizations in vivo performances. Dectin1-Fc(IgG2a) and Dectin1-Fc(IgG2b) demonstrated high affinity to laminarin and the fungal cell wall by ELISA, flow cytometry, and microscopy. Both Dectin-1-Fc(IgG)(s) inhibited Histoplasma capsulatum and Cryptococcus neoformans growth in a dose-dependent fashion. For Candida albicans, such inhibitory effect was observed with concentrations as low as 0.098 and 0.049 µg/ml, respectively, which correlated with the impairment of the kinetics and lengths of germ tubes in comparison to controls. Previous opsonization with Dectin-1-Fc(IgG)(s) enhanced considerably the macrophage antifungal effector functions, increasing the fungi macrophages interactions and significantly reducing the intraphagosome fungal survival, as lower CFUs were observed. The administration of both Dectin1-Fc(IgG)(s) reduced the fungal burden and mortality in murine histoplasmosis and candidiasis models, in accordance with previous evaluations in aspergillosis model. These results altogether strongly suggested that therapeutic interventions with Dectin-1-Fc(IgG)(s) fusion proteins could directly impact the innate immunity and disease outcome in favor of the host, by direct neutralization, opsonization, phagocytosis, and fungal elimination, providing interesting information on the potential of these new strategies for the control of invasive fungal infections. LAY SUMMARY: Mycoses have increased worldwide, and new efficient therapeutics are needed. Passive immunizations targeting universally the fungal cell would allow early interventions without the species-level diagnosis. Lectins with affinity to carbohydrates could be used to engineer 'antibody-like' strategies.
Assuntos
Infecções Fúngicas Invasivas , Micoses , Animais , Antifúngicos/farmacologia , Modelos Animais de Doenças , Imunoglobulina G , Infecções Fúngicas Invasivas/veterinária , Lectinas Tipo C/metabolismo , CamundongosRESUMO
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.
Assuntos
Acanthamoeba castellanii/metabolismo , Fungos/patogenicidade , Lectina de Ligação a Manose/metabolismo , Acanthamoeba castellanii/química , Acanthamoeba castellanii/microbiologia , Acanthamoeba castellanii/ultraestrutura , Animais , Candida albicans/patogenicidade , Candida albicans/ultraestrutura , Concanavalina A/metabolismo , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Histoplasma/patogenicidade , Histoplasma/ultraestrutura , Interações Hospedeiro-Patógeno , Larva/microbiologia , Lepidópteros/microbiologia , Manose/química , Manose/metabolismo , Lectina de Ligação a Manose/química , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Saccharomyces cerevisiae/patogenicidade , Saccharomyces cerevisiae/ultraestrutura , Fatores de Tempo , Imagem com Lapso de Tempo , Virulência , Fatores de Virulência/metabolismoRESUMO
We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.
Assuntos
Acanthamoeba castellanii , Vesículas Extracelulares , Proteômica , Acanthamoeba castellanii/metabolismo , Acanthamoeba castellanii/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Humanos , Metabolismo dos Lipídeos/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteoma/metabolismo , Proteoma/genéticaRESUMO
Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of ß-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.
RESUMO
Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.
Assuntos
Acanthamoeba castellanii , Amoeba , Acanthamoeba castellanii/microbiologia , Amoeba/microbiologia , Animais , Antifúngicos , Parede Celular/metabolismo , Macrófagos/metabolismo , Manose/química , Camundongos , Trofozoítos/metabolismoRESUMO
The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans (Cn), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides, and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo. In this work, we characterized the ability of Hc to produce cellular-attached (C-gly-Hc) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly-Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly-Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly-Hc and E-gly-Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly-Hc and E-gly-Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly-Hc and E-gly-Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella. These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.
Assuntos
Criptococose , Cryptococcus neoformans , Basidiomycota , Genótipo , Histoplasma , Humanos , PolissacarídeosRESUMO
Extracellular vesicles (EVs) are membranous compartments of distinct cellular origin and biogenesis, displaying different sizes and include exosomes, microvesicles, and apoptotic bodies. The EVs have been described in almost every living organism, from simple unicellular to higher evolutionary scale multicellular organisms, such as mammals. Several functions have been attributed to these structures, including roles in energy acquisition, cell-to-cell communication, gene expression modulation and pathogenesis. In this review, we described several aspects of the recently characterized EVs of the protozoa Acanthamoeba castellanii, a free-living amoeba (FLA) of emerging epidemiological importance, and compare their features to other parasites' EVs. These A. castellanii EVs are comprised of small microvesicles and exosomes and carry a wide range of molecules involved in many biological processes like cell signaling, carbohydrate metabolism and proteolytic activity, such as kinases, glucanases, and proteases, respectively. Several biomedical applications of these EVs have been proposed lately, including their use in vaccination, biofuel production, and the pharmaceutical industry, such as platforms for drug delivery.
RESUMO
Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.
Assuntos
Acanthamoeba castellanii/metabolismo , Amebíase/parasitologia , Vesículas Extracelulares/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba castellanii/genética , Animais , Linhagem Celular , Vesículas Extracelulares/genética , Homeostase , Humanos , Transporte Proteico , Proteoma/genética , Proteômica , Proteínas de Protozoários/genética , Via SecretóriaRESUMO
O objetivo deste artigo foi refletir sobre a Sistematização da Assistência de Enfermagem na prática do enfermeiro e sua utilização enquanto instrumento de gestão do cuidado. Trata-se de uma reflexão, com apoio na literatura sobre a temática. A Sistematização da Assistência de Enfermagem é um instrumento metodológico da prática do enfermeiro, além de se constituir em dever legal para esse profissional. Apesar da sua importância para o processo de trabalho da enfermagem, há ainda dificuldades a respeito da sua implantação e implementação. Nesse sentido, surgem questionamentos acerca da pouca utilização da Sistematização da Assistência de Enfermagem como instrumento de trabalho do enfermeiro, apesar dos avanços tecnológicos verificados nas últimas décadas. Há a necessidade de um resgate de sua valorização enquanto instrumento de gestão, pautado no apoio dos enfermeiros, suas equipes e gestores das instituições para o enfrentamento das dificuldades de implantação e implementação deste instrumento gerencial...
The aim of this paper was to reflect on the Nursing Care Systematization (NCS) in nurses practice and its use as a care management tool. This is a theoretical reflection, supported in the literature on the subject. The NCS is a methodological tool of nursing practice, besides constituting as a legal duty for this professional. Despite its importance for the nursing work process, there are still difficulties regarding its deployment and implementation. In this sense, questions arise about the limited use of NCS as nursing work instrument, despite the technological advances observed in recent decades. There is the need for the rescue of the NCS valorization as an instrument of management, based on the support of nurses, their teams and managers of institutions to face the difficulties of deployment and implementation of this management tool...
El objetivo de este artículo fue reflexionar sobre la Sistematización de la Asistencia de Enfermería (SAE) en la práctica dei enfermero y su uso como instrumento de gestión del cuidado. Se trata de una reflexión teórica con apoyo en la literatura sobre la temática. La SAE es una herramienta metodológica de la práctica del enfermero, además de constituirse en deber legal para ese profesional. A pesar de su importancia para el proceso de trabajo de la enfermería, hay aún dificultades a respecto de su implementación y ejecución. En ese sentido, surgen cuestionamientos acerca del uso limitado de la SAE como instrumento de trabajo del enfermero, a pesar de los avances tecnológicos observados en las últimas décadas. Existe la necesidad de un rescate de la valorización de la SAE como instrumento de gestión, basado en el apoyo de los enfermeros, sus equipos y gestores de las instituciones para hacer frente a las dificultades de implementación y ejecución de esta herramienta de gestión...
Assuntos
Humanos , Cuidados de Enfermagem/métodos , Enfermagem , Padrões de Prática em Enfermagem , Supervisão de EnfermagemRESUMO
As micoses têm emergido nas últimas décadas dentre as principais causas de enfermidades humanas, particularmente em indivíduos sob risco constante de infecção. Devido à ausência de especificidade para um determinado hospedeiro, os fungos são capazes de causar doenças em múltiplas espécies animais. Estes microrganismos são capazes de interagir com hospedeiros ambientais, como a espécie de ameba de vida livre Acanthamoeba castellanii e macrófagos de diversas espécies, encontrando no interior destes, um ambiente propício para proteção, sobrevivência e disseminação. Nosso grupo descreveu a importância do receptor de manose (MR), identificado na superfície amebóide, como uma das principais vias de ligação fúngica. Na literatura, o receptor de manose também é descrito por sua participação na interação entre fungos e macrófagos. Assim, é possível estabelecer uma relação de similaridade entre amebas e macrófagos, não apenas por compartilharem propriedades de estrutura celular, motilidade, fisiologia e captura por emissão de pseudópodos e fagocitose, mas também pelo semelhante mecanismo de interação com microrganismos, apontando para possível evolução convergente dos receptores envolvidos. Diante disso, investigamos as proteínas de superfície, presentes em A. castellanii e macrófagos murinos, afim de encontrar possíveis candidatos à receptores de alta afinidade contra manoproteínas e mananas de parede celular fúngica. Inicialmente, após biotinilação das superfícies de ambas as células, processamos extratos contendo as proteínas de superfície de A. castellanii e macrófagos, que apresentaram um perfil proteico variando entre 10-250 KDa. Determinamos a ligação dessas proteínas aos fungos patogênicos Candida albicans, Cryptococcus neoformans e Histoplasma capsulatum. Confirmamos que os extratos de macrófagos apresentaram maior intensidade de ligação quando comparados aos extratos de amebas, apontando para a maior complexidade proteica de macrófagos em relação à superfície amebóide no mecanismo de interação com microrganismos. A partir disso, buscamos investigar as proteínas, presentes nesses extratos, que apresentavam afinidade por manose. Realizamos a purificação dos extratos protéicos de A. castellanii e dos macrófagos (RAW 264.7) por cromatografia de afinidade, em coluna de manose, além da confirmação por Western blot da presença desta lectina no extrato. Observamos afinidade dos extratos por ambos manose e mananas. Assim, direcionamos a nossa procura realizando o enriquecimento dos extratos de A. castellanii e macrófagos com manose. Analisamos os extratos enriquecidos por Western Blot, onde identificamos nos extratos de A. castellanii proteínas de, aproximadamente, 150 kDa, 75 kDa, 55 kDa e 35 KDa. Já nos extratos enriquecidos de macrófagos foram identificadas proteínas entre 50 e 170 KDa, além de bandas de menores pesos moleculares. Os extratos enriquecidos foram incubados com os fungos, para caracterizar as proteínas com capacidade de reconhecimento fúngico e os coprecipitados foram avaliados por espectometria de massas. Para determinar a importância desta lectina ligadora de manose na interação, realizamos ensaios de fagocitose, na presença e ausência de manose, caracterizando a importância deste receptor. Porém encontramos baixas porcentagens de inibição nos experimentos com macrófagos, concluindo que provavelmente, diferentemente das amebas, diversos outros receptores podem participar do mecanismo de interação, de forma compensatória, destacando a importância deste tipo de interação para cada espécie de hospedeiro e a possível diversidade. Os dados sugerem que, provavelmente, ocorre a evolução e o surgimento de outras vias de interação em hospedeiros superiores. Futuramente, após obtenção dos dados de caracterização de proteínas com alta afinidade por manose em ambos os organismos por espectrometria de massas, pretendemos realizar a construção, produção e caracterização de lectinas-Fc, utilizando sequências da lectinas identificadas na fusão com a porção Fc efetora de imunoglobulinas murinas. Como manoproteínas e mananas estão presentes universalmente na parede celular fúngica, estas proteínas quiméricas permitiriam o reconhecimento dos fungos por receptores Fc (FcR) presentes na superfície dos fagócitos, promovendo mecanismos de imunidade (inata e celular) e de ação antifúngica.