Isolation and characterization of extracellular vesicles in Candida albicans
Keywords:exosomes, extracellular vesicles, Candida albicans, tetraspanins
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.
Beck-Sague C, Jarvis WR. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. The Journal of infectious diseases. 1993;167(5):1247-51.
Hsueh PR, Graybill JR, Playford EG, Watcharananan SP, Oh MD, Ja’alam K, et al. Consensus statement on the management of invasive candidiasis in Intensive Care Units in the Asia-Pacific Region. International journal of antimicrobial agents. 2009;34(3):205-9.
Mean M, Marchetti O, Calandra T. Bench-to-bedside review: Candida infections in the intensive care unit. Critical care (London, England). 2008;12(1):204.
Ajenjo HM, Aquevedo SA, Guzman DA, Poggi MH, Calvo AM, Castillo VC, et al. [Epidemiologial profile of invasive candidiasis in intensive care units at a university hospital]. Revista chilena de infectologia : organo oficial de la Sociedad Chilena de Infectologia. 2011;28(2):118-22.
Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clinical microbiology reviews. 2007;20(1):133-63.
Nanjappa S, Kynaston K, Moreland S, Carraway S, Sandin RL, Jani D, et al. Candidemia in Cancer Patients: A Retrospective Analysis; 2001-20142015.
Bennett JE. 257 - Introduction to Mycoses. In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (Eighth Edition). Philadelphia: Content Repository Only; 2015. p. 2874-8.
Corry DB, Ampel NM, Christian L, Locksley RM, Galgiani JN. Cytokine Production by Peripheral Blood Mononuclear Cells in Human Coccidioidomycosis. The Journal of infectious diseases. 1996;174(2):440-3.
Vazquez JA, Beckley A, Sobel JD, Zervos MJ. Comparison of restriction enzyme analysis and pulsed-field gradient gel electrophoresis as typing systems for Candida albicans. Journal of clinical microbiology. 1991;29(5):962-7.
Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms2013.
Batanghari JW, Deepe GS, Jr., Di Cera E, Goldman WE. Histoplasma acquisition of calcium and expression of CBP1 during intracellular parasitism. Molecular microbiology. 1998;27(3):531-9.
Kmetzsch L, Staats CC, Rodrigues ML, Schrank A, Vainstein MH. Calcium signaling components in the human pathogen: Cryptococcus neoformans. Communicative & integrative biology. 2011;4(2):186-7.
Rodrigues ML, Nimrichter L, Oliveira DL, Nosanchuk JD, Casadevall A. Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism for the Delivery of Virulence-Associated Macromolecules? Lipid insights. 2008;2:27-40.
Albuquerque PC, Nakayasu ES, Rodrigues ML, Frases S, Casadevall A, Zancope-Oliveira RM, et al. Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cellular microbiology. 2008;10(8):1695-710.
Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, et al. Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema. PloS one. 2011;6(7):e21480.
Vallejo MC, Matsuo AL, Ganiko L, Medeiros LC, Miranda K, Silva LS, et al. The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic alpha-Galactosyl epitopes. Eukaryotic cell. 2011;10(3):343-51.
Thery C. Exosomes: secreted vesicles and intercellular communications. F1000 biology reports. 2011;3:15.
Oliveira DL, Nakayasu ES, Joffe LS, Guimaraes AJ, Sobreira TJ, Nosanchuk JD, et al. Biogenesis of extracellular vesicles in yeast: Many questions with few answers. Communicative & integrative biology. 2010;3(6):533-5.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 1976;72(1):248-54.
Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970;227:680.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America. 1979;76(9):4350-4.
Burnette WN. “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981;112(2):195-203.
Kumar D, Gupta D, Shankar S, Srivastava RK. Biomolecular characterization of exosomes released from cancer stem cells: Possible implications for biomarker and treatment of cancer. Oncotarget. 2015;6(5):3280-91.
Daaboul GG, Gagni P, Benussi L, Bettotti P, Ciani M, Cretich M, et al. Digital Detection of Exosomes by Interferometric Imaging. Scientific reports. 2016;6:37246.
Deregibus MC, Figliolini F, D’Antico S, Manzini PM, Pasquino C, De Lena M, et al. Charge-based precipitation of extracellular vesicles. International journal of molecular medicine. 2016;38(5):1359-66.
Yuan Z, Kolluri KK, Gowers KH, Janes SM. TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy. Journal of extracellular vesicles. 2017;6(1):1265291.