ABSTRACT
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.
Subject(s)
Acanthamoeba castellanii/metabolism , Fungi/pathogenicity , Mannose-Binding Lectin/metabolism , Acanthamoeba castellanii/chemistry , Acanthamoeba castellanii/microbiology , Acanthamoeba castellanii/ultrastructure , Animals , Candida albicans/pathogenicity , Candida albicans/ultrastructure , Concanavalin A/metabolism , Cryptococcus neoformans/pathogenicity , Cryptococcus neoformans/ultrastructure , Histoplasma/pathogenicity , Histoplasma/ultrastructure , Host-Pathogen Interactions , Larva/microbiology , Lepidoptera/microbiology , Mannose/chemistry , Mannose/metabolism , Mannose-Binding Lectin/chemistry , Mass Spectrometry , Microscopy, Electron, Scanning , Paracoccidioides/pathogenicity , Paracoccidioides/ultrastructure , Saccharomyces cerevisiae/pathogenicity , Saccharomyces cerevisiae/ultrastructure , Time Factors , Time-Lapse Imaging , Virulence , Virulence Factors/metabolismABSTRACT
INTRODUCTION: This study evaluated the frequency of intestinal parasites, emphasizing the identification and differentiation of Entamoeba spp. METHODS: Multiplex polymerase chain reaction (PCR), coproantigen tests and morphometric analysis were performed for Entamoeba spp. differentiation. RESULTS: The overall frequency of intestinal parasites was 65%. Entamoeba histolytica was detected by the coproantigen test, and the PCR showed that Entamoeba dispar predominated in the population. In contrast, morphometric analysis was important for identifying Entamoeba hartmanni. CONCLUSIONS: It is possible to identify the causative agent of amoebiasis and to differentiate this agent from other species by combining techniques.