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Comparative Study
. 2012 Mar 2;11(3):1676-85.
doi: 10.1021/pr200872s. Epub 2012 Feb 14.

Vesicle and Vesicle-Free Extracellular Proteome of Paracoccidioides Brasiliensis: Comparative Analysis With Other Pathogenic Fungi

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Free PMC article
Comparative Study

Vesicle and Vesicle-Free Extracellular Proteome of Paracoccidioides Brasiliensis: Comparative Analysis With Other Pathogenic Fungi

Milene C Vallejo et al. J Proteome Res. .
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Abstract

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.

Figures

Figure 1
Figure 1
A, Venn diagrams showing the number of proteins detected in vesicle and vesicle-free (ves-free) fractions of the Pb18 extracellular proteome. B, Proportion of proteins in vesicle (ves) and ves-free preparations classified as secretory or not (no) when using the FSD: SP (containing signal peptide identified by SignaIP 3.0), SP3 (bearing signal peptide predicted by SigPred, SigCleave or RPSP), SL (sub-cellular localization predicted by PSort II and/or Target 1.1b), and NS (non-classical, predicted by SecretomeP 1.0f).
Figure 2
Figure 2
Analysis of orthologous proteins found in fungal extracellular vesicles. The Venn diagram shows the number of proteins detected in extracellular vesicle preparations from P. brasiliensis (this work) and orthologues described in vesicles from H. capsulatum, C. neoformans and S. cerevisiae. Overlapping groups are shown in gray scale.
Figure 3
Figure 3
Functional categorization of fungal extracellular vesicle proteins commonly found in P. brasiliensis and at least two other species (total of 72, Table 1); * p ≤ 0.05. The figure shows enriched functional clusters in comparison with the genome, represented by bars of fold-increase.
Figure 4
Figure 4
Phosphatase activity (A405) at different Pb18 extracellular vesicles concentrations expressed as equivalents of sterol contents. Background values were subtracted. * p ≤ 0.05.

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