Isolation and characterization of extracellular vesicles in Candida albicans

Authors

  • Virginia Pérez-Doñate Grupo de Investigación GMCA. Departamento de Microbiología. Facultad de Farmacia. Universitat de Valencia. / Servicio de Microbiología. Hospital Universitario de la Ribera. Alzira. Valencia.
  • Facundo Pérez-Giménez Unidad de Investigación de Radiofarmacia y Estabilidad de Medicamentos. Departamento de Química Física. Facultad de Farmacia. Universitat de València.
  • Lucas del Castillo Agudo Grupo de Investigación GMCA. Departamento de Microbiología. Facultad de Farmacia. Universitat de Valencia.
  • Juan Alberto Castillo-Garit Unidad de Toxicología Experimental. Universidad de Ciencias Médicas de Villa Clara. Santa Clara. Cuba. / Unidad de Investigación de Diseño de Fármacos y Conectividad Molecular. Departamento de Química Física. Facultad de Farmacia. Universitat de València.
  • Mar Soria-Merino Servicio de Radiofarmacia. Hospital Clínico Universitario de Valencia.
  • Eulogio Valentín Gómez Grupo de Investigación GMCA. Departamento de Microbiología. Facultad de Farmacia. Universitat de Valencia.

DOI:

https://doi.org/10.46583/nereis_2020.12.611

Keywords:

exosomes, extracellular vesicles, Candida albicans, tetraspanins

Abstract

Background: The occurrence of systemic infections due to C. albicans has increased especially in critically ill patients. In fungal infections, secretory mechanisms are key events for disease establishment. Recent findings demonstrate that fungal organisms release many molecular components to the extracellular space in extracellular vesicles.
Aims: We develop a method to obtain exosomes from yeast cultures of the Candida albicans.
Methods: Yeast strains used in this work were C. albicans SC5314, C. parapsilosis (ATCC 22019) and C. krusei (ATCC 6258). Yeasts were grown at 37.º in liquid YPD medium. The cell cultures were centrifuged and the supernatant filtered through sterile nitrocellulose. Filtrates were concentrated and centrifuged using an ultracentrifuge. The sediment was analyzed by electron microscopy of transmission.
Results: The transmission of electron microscopy and nanoparticle tracking analysis confirmed the presence of extracellular vesicles (exosomes) of sizes between 100 and 200 nm and the absence of cellular contaminants. This was ratified by the characterization of proteins performed through the western blot technique, where the absence of cell contamination in the preparations was assessed.
Conclusions: The method proves to be highly effective due to the homogeneity and purity of the obtained microvesicles. The protocol developed in this paper proves to be effective for obtaining exosomes of other Candida species, which will allow future studies to determine its protein composition and the role that these vesicles can play.

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Published

2020-07-16