Improved proteomic approach for the discovery of potential vaccine targets in Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, is a devastating parasitic infection affecting millions of people. Although many efforts have been made for the development of immunotherapies, there is no available vaccine against this deadly infection. One major hurdle for the rational approach to develop a T. cruzi vaccine is the limited information about the proteins produced by different phylogenetic lineages, strains, and stages of the parasite. Here, we have adapted a 1D nanoHPLC system to perform online 2D LC-MS/MS, using the autosampler to inject the eluting salt solutions in the first dimension separation. The application of this methodology for the proteomic analysis of the infective trypomastigote stage of T. cruzi led to the identification of 1448 nonredundant proteins. Furthermore, about 14% of the identified sequences comprise surface proteins, most of them glycosylphosphatidylinositol (GPI)-anchored and related to parasite pathogenesis. Immunoinformatic analysis revealed thousands of potential peptides with predicted high-binding affinity for major histocompatibility complex (MHC) class I and II molecules. The high diversity of proteins expressed on the trypomastigote surface may have many implications for host-cell invasion and immunoevasion mechanisms triggered by the parasite. Finally, we performed a rational approach to filter potential T-cell epitopes that could be further tested and validated for development of a Chagas disease vaccine.

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.

The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic α-Galactosyl epitopes.

Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after ß-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.

Paracoccin, an N-acetyl-glucosamine-binding lectin of Paracoccidioides brasiliensis, is involved in fungal growth.

Paracoccin is an N-acetyl-glucosamine-binding lectin from Paracoccidioides brasiliensis, which can be obtained in small amounts either from culture supernatants or yeast cell extracts. In the present work, immunoelectron microscopy with mouse anti-paracoccin IgG localized the antigen to the cell wall of P. brasiliensis yeast forms. Paracoccin interacted with chitin, and colocalized with beta-1,4-homopolymer of GlcNAc to the budding sites of P. brasiliensis yeast cell. In order to evaluate the role of paracoccin on fungal growth, yeast cells were cultivated in the presence of anti-paracoccin antibodies. A significant reduction of both colony forming units and individual yeast cells was observed as well as morphological alterations such as smaller colonies and cells more loosely aggregated than in control cultures without the antibody. A role of paracoccin on the cell wall organization was reinforced by alterations in the labeling pattern of chitin when yeasts were treated with anti-paracoccin antibodies. Binding of specific antibodies to paracoccin may disrupt the paracoccin/chitin interactions, resulting in the inhibition of P. brasiliensis growth.

Lectin KM+-induced neutrophil haptotaxis involves binding to laminin.

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 microM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis.

Virulence of Paracoccidioides brasiliensis and gp43 expression in isolates bearing known PbGP43 genotype.

Paracoccidioides brasiliensis is the dimorphic fungus responsible for human paracoccidioidomycosis (PCM). We previously observed that P. brasiliensis isolates bearing highly polymorphic PbGP43 of genotype A (Pb2, Pb3 and Pb4) were phylogenetically distant from the others. The PbGP43 gene encodes an immune dominant diagnostic antigen (gp43), and its polymorphism reflects broader genetic diversity in the species. In the present study, we observed that isolates with PbGP43 of genotype A showed low virulence when inoculated in B10.A mice by the intraperitoneal, intratracheal and intravenous routes. In vitro studies detected sharp and prolonged down-regulation of PbGP43 in Pb3 (and not in Pb18 or Pb339) as a result of heat shock at 42 degrees C and temperature shift to prompt mycelium to yeast transition, which was, however, not disturbed. Differences in transcriptional regulation are possibly a consequence of mutations in the PbGP43 promoter region, which we here show to be more polymorphic in genotype A isolates. As opposed to Pb3's rapid adaptation to in vitro culture conditions after isolation from the lung, Pb12, the most aggressive isolate tested here, showed slow growth and phase transition in vitro. Interestingly, animals that were highly infected by Pb12 produced small amounts of anti-gp43 antibodies. That was apparently due to down-regulation in PbGP43 expression. We present the first evidence of transcriptional regulation of gp43 expression, but our results suggest that gene expression is also regulated at the protein and/or secretion levels.

Lack of iGb3 and Isoglobo-Series Glycosphingolipids in Pig Organs Used for Xenotransplantation: Implications for Natural Killer T-Cell Biology.

α-1,3-Terminated galactose residues on glycoproteins and glycosphingolipids are recognized by natural anti-α-1,3-galactose antibodies in human serum and cause hyperacute rejection in pig-to-human xenotransplantation. Genetic depletion of α-1,3-galactosyltransferase-1 in pigs abolishes the hyperacute rejection reaction. However, the isoglobotriosylceramide (iGb3) synthase in pigs may produce additional α-1,3-terminated galactose residues on glycosphingolipids. In both α-1,3-galactosyltranserase-1 knockout mice and pigs, cytotoxic anti-α-1,3-galactose antibodies could be induced; thus, a paradox exists that anti-α-1,3-galactose antibodies are present in animals with functional iGb3 synthases. Furthermore, iGb3 has been found to be an endogenous antigen for natural killer T (NKT) cells, an innate type of lymphocyte that may initiate the adaptive immune responses. It has been reasoned that iGb3 may trigger the activation of NKT cells and cause the rejection of α-1,3-galactosyltransferase-1-deficient organs through the potent stimulatory effects of NKT cells on adaptive immune cells (see ref.([20])). In this study, we examined the expression of iGb3 and the isoglobo-series glycosphingolipids in pig organs, including the heart, liver, pancreas, and kidney, by ion-trap mass spectrometry, which has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 5 µg/mL of the total iGb3/Gb3 mixture is present (see ref.([35])). We did not detect iGb3 or other isoglobo-series glycosphingolipids in any of these organs, although they were readily detected in mouse and human thymus and dendritic cells. The lack of iGb3 and isoglobo-series glycosphingolipids in pig organs indicates that iGb3 is unlikely to be a relevant immune epitope in xenotransplantation.

Identification of human plasma proteins associated with the cell wall of the pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermodimorphic species that cause paracoccidioidomycosis. The cell wall is the outermost fungal organelle to form an interface with the host. A number of host effector compounds, including immunologically active molecules, circulate in the plasma. In the present work, we extracted cell-wall-associated proteins from the yeast pathogenic phase of P. brasiliensis, isolate Pb3, grown in the presence of human plasma and analyzed bound plasma proteins by liquid chromatography-tandem mass spectrometry. Transport, complement activation/regulation, and coagulation pathway were the most abundant functional groups identified. Proteins related to iron/copper acquisition, immunoglobulins, and protease inhibitors were also detected. Several human plasma proteins described here have not been previously reported as interacting with fungal components, specifically, clusterin, hemopexin, transthyretin, ceruloplasmin, alpha-1-antitrypsin, apolipoprotein A-I, and apolipoprotein B-100. Additionally, we observed increased phagocytosis by J774.16 macrophages of Pb3 grown in plasma, suggesting that plasma proteins interacting with P. brasiliensis cell wall might be interfering in the fungal relationship with the host.

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.

The PbMDJ1 gene belongs to a conserved MDJ1/LON locus in thermodimorphic pathogenic fungi and encodes a heat shock protein that localizes to both the mitochondria and cell wall of Paracoccidioides brasiliensis.

J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones, besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1, a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The anti-recombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline beta-mercaptoethanol extract) component, compatible with the predicted size of the protein devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro, possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood preferentially close to the plasma membrane, and gold particles were detected in the thin space between them, toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and Aspergillus, where the genes are adjacent and have a common 5' region. This is the first time a DnaJ member has been observed on the cell surface, where its function is speculative.

The novel lectin KM+ detects a specific subset of mannosyl-glycoconjugates in the rat cerebellum.

KM+ is a D(+)mannose-specific lectin with a carbohydrate structure-affinity relationship different from those of most mannose-binding lectins. KM+ elicits carbohydrate-dependent biological effects in several mammalian cell types, but it has not yet been employed as a probe for the detection of its specific ligands. We show here for the first time the screening and partial identification of cerebellar mannosyl-glycoconjugates recognized by KM+, by means of lectin-histochemistry and lectin-blotting. Biotinylated KM+ stained most cellular structures in the adult rat cerebellum, particularly Purkinje cells bodies and the surface of granule cells, but not cellular processes. Capillaries in the choroid plexus were also strongly decorated, while blood vessels in the cerebellar parenchyma remained unstained. D(+)mannose, but not D(+)galactose, abolished the staining of all cerebellar structures. Higher inhibitory potencies were found for mannosyl-glycans such as mannotriose (man-alpha1,3-[man-alpha1,6]-man) and the biantennary heptasaccharide carried by the enzyme horseradish peroxidase. After separation of cerebellar proteins by SDS-PAGE, KM+ recognized three major unidentified mannosyl-glycoproteins of 132, 83 and 49 kDa. KM+ also detected high-Mw bands corresponding to the light and heavy chains of Type-I laminin, but not a 160-kDa cleavage product of laminin. We conclude that KM+ binds preferentially to a specific subset of mannose-containing glycoproteins in cerebellar tissue, thus being much more restricted than other mannose-specific lectins. KM+ can be used as a novel probe to screen the central nervous system for this specific subset of complex mannosyl-glycoconjugates.