RESUMO
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
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
Natural and human-made disasters have long played a role in shaping the environment and microbial communities, also affecting non-microbial life on Earth. Disaster microbiology is a new concept based on the notion that a disaster changes the environment causing adaptation or alteration of microbial populations -growth, death, transportation to a new area, development traits, or resistance- that can have downstream effects on the affected ecosystem. Such downstream effects include blooms of microbial populations and the ability to colonize a new niche or host, cause disease, or survive in former extreme conditions. Throughout history, fungal populations have been affected by disasters. There are prehistoric archeological records of fungal blooms after asteroid impacts and fungi implicated in the fall of the dinosaurs. In recent times, drought and dust storms have caused disturbance of soil fungi, and hurricanes have induced the growth of molds on wet surfaces, resulting in an increased incidence of fungal disease. Probably, the anticipated increase in extreme heat would force fungi adaptation to survive at high temperatures, like those in the human body, and thus be able to infect mammals. This may lead to a drastic rise of new fungal diseases in humans.
Los desastres naturales o los causados por el hombre impactan la formación de ecosistemas y comunidades microbianas, y también afectan las formas de vida no microbianas. Este concepto es conocido como "microbiología de desastres", una subespecialización de la microbiología, basada en los cambios ambientales generados por un desastre y las posibles adaptaciones o alteraciones de las poblaciones microbianas crecimiento, muerte, trasporte a una nueva región, o adquisición de resistencia o de nuevas características que influirán en el moldeamiento del ecosistema transformado. Algunos de los efectos de estas adaptaciones pueden ser: el surgimiento de poblaciones microbianas, la habilidad de colonizar nuevos nichos u huéspedes, la generación de nuevas enfermedades, o el crecimiento de microorganismos en condiciones que antes eran "extremas" para ellos. A lo largo de la historia, varias poblaciones de hongos han sido afectadas por desastres. Existen registros arqueológicos prehistóricos que evidencian la presencia y el crecimiento de hongos luego del impacto de asteroides, y otros de hongos relacionados con la extinción de los dinosaurios. Actualmente, las sequías y las tormentas de polvo causan perturbaciones en las comunidades de hongos del suelo, y los huracanes inducen el crecimiento de hongos filamentosos en superficies húmedas, lo que aumenta la cantidad de enfermedades por hongos. Además, con el aumento de las temperaturas extremas es posible que los hongos puedan adaptarse para sobrevivir a temperaturas más altas, equivalentes a las temperaturas corporales, y nuevas especies puedan infectar mamíferos. Esto puede llevar a un aumento drástico de las infecciones fúngicas en humanos.
Assuntos
Desastres , Microbiota , Humanos , Animais , Micologia , Secas , MamíferosRESUMO
Natural and human-made disasters have long played a role in shaping the environment and microbial communities, also affecting non-microbial life on Earth. Disaster microbiology is a new concept based on the notion that a disaster changes the environment causing adaptation or alteration of microbial populations-growth, death, transportation to a new area, development traits, or resistance-that can have downstream effects on the affected ecosystem. Such downstream effects include blooms of microbial populations and the ability to colonize a new niche or host, cause disease, or survive in former extreme conditions. Throughout history, fungal populations have been affected by disasters. There are prehistoric archeological records of fungal blooms after asteroid impacts and fungi implicated in the fall of the dinosaurs. In recent times, drought and dust storms have caused disturbance of soil fungi, and hurricanes have induced the growth of molds on wet surfaces, resulting in an increased incidence of fungal disease. Probably, the anticipated increase in extreme heat would force fungi adaptation to survive at high temperatures, like those in the human body, and thus be able to infect mammals. This may lead to a drastic rise of new fungal diseases in humans.
Los desastres naturales o los causados por el hombre impactan la formación de ecosistemas y comunidades microbianas, y también afectan las formas de vida no microbianas. Este concepto es conocido como "microbiología de desastres", una subespecialización de la microbiología, basada en los cambios ambientales generados por un desastre y las posibles adaptaciones o alteraciones de las poblaciones microbianas -crecimiento, muerte, trasporte a una nueva región, o adquisición de resistencia o de nuevas características- que influirán en el moldeamiento del ecosistema transformado. Algunos de los efectos de estas adaptaciones pueden ser: el surgimiento de poblaciones microbianas, la habilidad de colonizar nuevos nichos u huéspedes, la generación de nuevas enfermedades, o el crecimiento de microorganismos en condiciones que antes eran "extremas" para ellos. A lo largo de la historia, varias poblaciones de hongos han sido afectadas por desastres. Existen registros arqueológicos prehistóricos que evidencian la presencia y el crecimiento de hongos luego del impacto de asteroides, y otros de hongos relacionados con la extinción de los dinosaurios. Actualmente, las sequías y las tormentas de polvo causan perturbaciones en las comunidades de hongos del suelo, y los huracanes inducen el crecimiento de hongos filamentosos en superficies húmedas, lo que aumenta la cantidad de enfermedades por hongos. Además, con el aumento de las temperaturas extremas es posible que los hongos puedan adaptarse para sobrevivir a temperaturas más altas, equivalentes a las temperaturas corporales, y nuevas especies puedan infectar mamíferos. Esto puede llevar a un aumento drástico de las infecciones fúngicas en humanos.
Assuntos
Micologia , Desastres Naturais , Mudança Climática , Fungos , Candida aurisRESUMO
Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.
RESUMO
Fungal infections are associated with high mortality rates in humans. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungus-host interaction process. We hypothesized that fungal EVs work as an additional element in the communication routes regulating fungal responses in intraspecies interaction systems. In this respect, the aim of this study was to address the gene regulation profiles prompted by fungal EVs in intraspecies recipient cells. Our data demonstrated the intraspecies uptake of EVs in pathogenic fungi, such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in the yeast-to-hypha transition, while in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and triggering phenotypic changes. The EVs treatment induced cellular proliferation and boosted the yeast to hyphal transition in C. albicans, while they enhanced stress responsiveness in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intraspecies communication. Thus, EVs regulate fungal behavior, acting as potent message effectors, and understanding their effects and mechanism(s) of action could be exploited in antifungal therapies. IMPORTANCE Here, we report a study about extracellular vesicles (EVs) as communication mediators in fungi. Our results demonstrated the role of EVs from Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis regulating the expression of target genes and phenotypic features. We asked whether fungal EVs play a role as message effectors. We show that fungal EVs are involved in fungal interaction systems as potent message effectors, and understanding their effects and mechanisms of action could be exploited in antifungal therapies.
Assuntos
Vesículas Extracelulares , Micoses , Humanos , Antifúngicos/farmacologia , Aspergillus fumigatus/genética , Candida albicans , Comunicação CelularRESUMO
Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Cápsulas Bacterianas/química , Cápsulas Bacterianas/imunologia , Cryptococcus neoformans/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos , Células CHO , Linhagem Celular , Cricetulus , Criptococose/imunologia , Epitopos/química , Epitopos/imunologia , Camundongos , Proteínas Recombinantes de Fusão , Relação Estrutura-AtividadeRESUMO
Several studies have shown the immunomodulatory effects of extracellular vesicles (EVs) released by pathogenic fungi. Herein, we discuss the data regarding the immunomodulatory properties of fungal EVs, but also of EVs produced by infected leukocytes. This characterizes a two-way path, in which both host and fungal EVs could coexist and play crucial roles in disease progression or protection in fungal infections. We suggest that EVs can dictate the progress of fungal diseases, and their potential as therapeutic targets.
Assuntos
Vesículas Extracelulares , Micoses , Fungos , Humanos , LeucócitosRESUMO
Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant Δcap67 in inflammasome activation in an in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1ß secretion and LDH release via pyroptosis) more strongly than conditioned media from Δcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501's conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by C. neoformans. These results were confirmed by in vivo experiments, where exposure to conditioned media led to higher fungal burdens in Acanthamoeba castellanii culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and in vivo and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.
Assuntos
Cryptococcus neoformans/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Polissacarídeos/química , Animais , Caspase 1/metabolismo , Criptococose , Meios de Cultivo Condicionados , Células Dendríticas/metabolismo , Imunofluorescência , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Polissacarídeos/metabolismo , Fatores de Virulência/metabolismoRESUMO
Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host's antifungal immunity. In this work we show the generation of and promising results with an mAb against Heat Shock Protein (HSP)90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM.