Lipidomic Analysis of Extracellular Vesicles from the Pathogenic Phase of Paracoccidioides brasiliensis

Background: Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery.Methodology/Principal Findings: Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. the prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells.Conclusions/Significance: the extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institutes of Health (NIH)Universidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USAUniversidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 06/05095-6FAPESP: 07/04757-8FAPESP: 07/59768-4CNPq: 301666/2010-5National Institutes of Health (NIH): 5G12RR008124-16A1National Institutes of Health (NIH): 5G12RR008124-16A1S1National Institutes of Health (NIH): G12MD007592Web of Scienc

Vesicle and Vesicle-Free Extracellular Proteome of Paracoccidioides brasiliensis: Comparative Analysis with Other Pathogenic Fungi

Microorganisms release effector molecules that modulate the host machinery enabling survival, replication, and dissemination of a pathogen. Here we characterized the extracellular proteome of Paracoccidioides brasiliensis at its pathogenic yeast phase. Cell-free culture supernatants from the Pb18 isolate, cultivated in defined medium, were separated into vesicle and vesicle-free fractions, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. in vesicle and vesicle-free preparations we identified, respectively, 205 and 260 proteins with two or more peptides, including 120 overlapping identifications. Almost 70% of the sequences were predicted as secretory, mostly using nonconventional secretory pathways, and many have previously been localized to fungal cell walls. A total of 72 proteins were considered as commonly transported by extracellular vesicles, considering that orthologues have been reported in at least two other fungal species. These sequences were mostly related to translation, carbohydrate and protein metabolism, oxidation/reduction, transport, response to stress, and signaling. This unique proteomic analysis of extracellular vesicles and vesicle-free released proteins in a pathogenic fungus provides full comparison with other fungal extracellular vesicle proteomes and broadens the current view on fungal secretomes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)NIHBBRC/Biology/UTEP (NIH)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, UNIFESP, BR-04023062 São Paulo, BrazilUniv Texas El Paso, Border Biomed Res Ctr, Dept Biol Sci, El Paso, TX 79968 USANatl Ctr Res Energy & Mat, Natl Lab Biosci LNBio, BR-13083970 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, UNIFESP, BR-04023062 São Paulo, BrazilNIH: 5G12RR008124-16A1NIH: 5G12RR008124-16A1S1BBRC/Biology/UTEP (NIH): 5G12RR008124-16A1BBRC/Biology/UTEP (NIH): 5G12RR008124-16A1S1Web of Scienc

Neutral glycolipid analysis of Pb3 and Pb18 extracellular vesicles.

<p>Glycolipid-enriched fractions from total lipids were permethylated and analyzed by ESI-MS/MS. (A) Full-scan spectrum by ESI-MS in the positive-ion mode of Pb3 extracellular vesicle glycolipids. (B) Proposed fragmentation and structure of the major glycolipid identified in Pb3 and Pb18 extracellular vesicles. The positions of unsaturation, hydroxyl and methyl groups of the ceramide were based on the structure proposed by Toledo et al. (1999). (C) MS/MS spectrum of the major glycolipid species at <i>m/z</i> 876.8. The number at the top right corner indicates ion intensity. <i>m/z</i>, mass to charge ratio. The same glycolipid species have been identified in Pb3 and Pb18, however since the analysis is qualitative we cannot assure that are not quantitative differences.</p

Phospholipid fatty acid analysis of Pb3 and Pb18 extracellular vesicles and whole yeast cells.

<p>Fatty acids from phospholipid-enriched fractions resulting from silica gel-60 column separation were released with NH₄OH, methylated with methanolic HCl, and analyzed by GC-MS. Signal intensity was normalized by the C16:0 peak area.</p