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
The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-3 secretion systems encoded in SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively). Recently, we reported that S. Typhimurium requires T3SS-1 and T3SS-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.
Assuntos
Proteínas de Bactérias/metabolismo , Dictyostelium/metabolismo , Proteoma/metabolismo , Salmonella typhimurium/metabolismo , Vacúolos/metabolismo , Amoeba/metabolismo , Animais , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Mutação , Proteômica , Proteínas de Protozoários/metabolismo , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genéticaRESUMO
The unusual life cycle of Dictyostelium discoideum, in which an extra-cellular stressor such as starvation induces the development of a multicellular fruiting body consisting of stalk cells and spores from a culture of identical amoebae, provides an excellent model for investigating the molecular control of differentiation and the transition from single- to multi-cellular life, a key transition in development. We utilized serial analysis of gene expression (SAGE), a molecular method that is unbiased by dependence on previously identified genes, to obtain a transcriptome from a high-density culture of amoebae, in order to examine the transition to multi-cellular development. The SAGE method provides relative expression levels, which allows us to rank order the expressed genes. We found that a large number of ribosomal proteins were expressed at high levels, while various components of the proteosome were expressed at low levels. The only identifiable transmembrane signaling system components expressed in amoebae are related to quorum sensing, and their expression levels were relatively low. The most highly expressed gene in the amoeba transcriptome, dutA untranslated RNA, is a molecule with unknown function that may serve as an inhibitor of translation. These results suggest that high-density amoebae have not initiated development, and they also suggest a mechanism by which the transition into the development program is controlled.
Assuntos
Amoeba/genética , Amoeba/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Perfilação da Expressão Gênica , Actinas/genética , Actinas/metabolismo , Amoeba/citologia , Amoeba/crescimento & desenvolvimento , Animais , Transporte Biológico/genética , Adesão Celular/genética , Citoesqueleto/genética , Dictyostelium/citologia , Dictyostelium/crescimento & desenvolvimento , Dineínas/genética , Endossomos/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Genes de Protozoários , Membranas Intracelulares/metabolismo , Estágios do Ciclo de Vida/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Percepção de Quorum/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.
Assuntos
Animais , Amoeba/metabolismo , Amoeba/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , /metabolismo , Proteínas de Membrana/metabolismo , Sintaxina 1/metabolismo , Microscopia Eletrônica de VarreduraRESUMO
Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.(AU)
Assuntos
Animais , Amoeba/metabolismo , Amoeba/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Membrana/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Microscopia Eletrônica de VarreduraRESUMO
Acanthamoeba castellanii were incubated in vivo with 1(-14)C linoleic and 1(-14)C-alpha-linolenic acids. The incorporation of the acids into lipid fractions was studied. Labeling was found mainly in triglycerides and phospholipids. Homogenized cells and subcellular fractions separated by centrifugation were incubated with 1-14C-linoleic. 1-14C -alpha-linoleic and 1-14C eicosa-8,11-dienoic acids in the presence of NADH, ATP, and CoA. Different metabolic routes were demonstrated. omega3 and omega6 desaturases of the vegetal type, as well as a delta6 desaturation of alpha-linolenic acid of the animal type were present. The supernatant of 100000 x g contained both types of desaturating enzymes, whereas the corresponding particulated fraction was inactive. The ultrastructure of Acanthamoeba showed the endoplasmic reticulum with a poorly developed membrane component. The metabolic pathways found with Acanthamoeba were compared to Ochromonas danica incubated with linoleic acid in the light and in darkness. Desaturases typical of "vegetal" and "animal" pathways were found in both organisms. In both of them, alpha-linolenic and arachidonic acids could be synthetized. However, alpha-linolenic acid, typical of vegetal synthesis, was only stored in Ochromonas due to the presence of a photosynthetic machinery.