Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process.
Ustilago maydis is a pathogenic fungus that produces the corn smut. It is a biotrophic parasite that depends on living plant tissues for its proliferation and development. Polygalacturonases are secreted by pathogens to solubilize the plant cell-wall and are required for pathogen virulence. In this paper, we report the isolation of a U. maydis polygalacturonase gene (Pgu1) and the functional and structural characterization of the encoded enzyme. The U. maydis Pgu1 gene is expressed when the fungus is grown in liquid culture media containing different carbon sources. In plant tissue, the expression increased as a function of incubation time. Pgu1 gene expression was detected during plant infection around 10 days post-infection with U. maydis FB-D12 strain in combination with teliospore formation. Synthesis and secretion of active recombinant PGU1 were achieved using Pichia pastoris, the purified enzyme had a optimum temperature of 34 °C, optimum pH of 4.5, a Km of 57.84 g/L for polygalacturonic acid, and a Vmax of 28.9 µg/min mg. Structural models of PGU1 based on homologous enzymes yielded a typical right-handed β-helix fold of pectinolytic enzymes classified in the glycosyl hydrolases family 28, and the U. maydis PGU1 is related with endo rather than exo polygalacturonases.
Nanotechnology opens new perspectives in many science and technology fields through new materials, such as metal nanoparticles. The biomedical field is one of these areas where nanoparticles offer promising application in the diagnosis and therapy of disease, generating biosensors for disease detection, bioimaging, and drug delivery. However, the controversy about whether nanoparticles are inert or exhibit different degrees of toxicity related to their physicochemical properties remains a subject of study and debate. This work shows how gold nanoparticles and nanorods were simultaneously functionalized with 4-aminothiophenol (4-ATP) and methoxypolyethylene glycol thiol (mPEG-SH). Then, folic acid (FA) gold nanoparticle bioconjugates caused diverse biological effects on HeLa and SH-SY5Y cell cultures after 24 h of incubation, when they were evaluated in the range of gold concentrations from 0.17 to 350 μM. We found notable changes in cell metabolic activity, viability, and biomass. 16 nm nanoparticles produced the most enhanced damage for functionalized (AuNPs) and bioconjugated (AuNPs-FA) nanoparticles, which we expected to be more biocompatible when coated with FA. Besides, epifluorescence images showed damage to F-actin microfilaments, adhesion and cell attachment loss, morphology changes (cells became round and detached), presence of blebs, and rupture of cell membrane. These results suggested that both AuNPs and AuNPs-FA have significant biological impact on HeLa and SH-SY5Y cell cultures, whereas gold nanorods showed significant changes only in the metabolic activity of SH-SY5Y cells when they were exposed to gold nanorods of 23 and 27 nm in length with a diameter of 5 nm, and crystal violet did not show evidence of toxicity.
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