RESUMO
The genus Paracoccidioides comprises the species complex causing paracoccidioidomycoses (PCM). These fungi are a serious public health problem due to the long-term drug therapy, follow-up treatment, and frequent sequelae generated by the infection, such as pulmonary fibrosis. In this sense, the objective of this work was to generate bioluminescent reporter strains of Paracoccidioides spp. harboring a thermostable, red-shifted luciferase gene under the control of different constitutive promoters. The strains were generated by the integration of a species-specific codon-optimized luciferase gene upon actin or enolase promoter's control. The insertion of the constructs in Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells were performed through Agrobacterium tumefaciens-mediated transformation. The results demonstrated the presence of several transformants harboring the luciferase gene. These transformants were further confirmed by the expression of luciferase and by the presence of the hygromycin resistance gene. Moreover, the luciferase activity could be detected in in vitro bioluminescence assays and in vivo models of infection. In general, this work presents the methodology for the construction of bioluminescent strains of Paracoccidioides spp., highlighting potential promoters and proposing an in vivo model, in which those strains could be used for the systemic study of PCM.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Actinas , Paracoccidioides/genética , Paracoccidioidomicose/microbiologia , Fosfopiruvato HidrataseRESUMO
Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis. We have previously characterized the <200-nt RNA sub-populations contained in fungal extracellular vesicles (EVs) from P. brasiliensis Pb18 and other pathogenic fungi. We have presently used the RNA-seq strategy to compare the <200- and >200-nt RNA fractions contained in EVs isolated from culture supernatants of P. brasiliensis Pb18, Pb3, and P. lutzii Pb01. Shared mRNA sequences were related to protein modification, translation, and DNA metabolism/biogenesis, while those related to transport and oxidation-reduction were exclusive to Pb01. The presence of functional full-length mRNAs was validated by in vitro translation. Among small non-coding (nc)RNA, 15 were common to all samples; small nucleolar (sno)RNAs were enriched in P. brasiliensis EVs, whereas for P. lutzii there were similar proportions of snoRNA, rRNA, and tRNA. Putative exonic sRNAs were highly abundant in Pb18 EVs. We also found sRNA sequences bearing incomplete microRNA structures mapping to exons. RNA-seq data suggest that extracellular fractions containing Pb18 EVs can modulate the transcriptome of murine monocyte-derived dendritic cells in a transwell system. Considering that sRNA classes are involved in transcription/translation modulation, our general results may indicate that differences in virulence among fungal isolates can be related to their distinct EV-RNA content.
Assuntos
Vesículas Extracelulares/genética , Paracoccidioides , Paracoccidioidomicose/microbiologia , RNA/genética , Animais , Células Cultivadas , Paracoccidioides/genética , Paracoccidioides/patogenicidade , VirulênciaRESUMO
Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in Cryptococcus neoformans. In fungal species, extracellular vesicles (EVs) participate in the export of polysaccharides, proteins and RNA. In the present work, we investigated if EV-mediated RNA export is functionally connected with GRASP in C. neoformans using a graspΔ mutant. Since GRASP-mediated unconventional secretion involves autophagosome formation in yeast, we included the atg7Δ mutant with defective autophagic mechanisms in our analysis. All fungal strains exported EVs but deletion of GRASP or ATG7 profoundly affected vesicular dimensions. The mRNA content of the graspΔ EVs differed substantially from that of the other two strains. The transcripts associated to the endoplasmic reticulum were highly abundant transcripts in graspΔ EVs. Among non-coding RNAs (ncRNAs), tRNA fragments were the most abundant in both mutant EVs but graspΔ EVs alone concentrated 22 exclusive sequences. In general, our results showed that the EV RNA content from atg7Δ and WT were more related than the RNA content of graspΔ, suggesting that GRASP, but not the autophagy regulator Atg7, is involved in the EV export of RNA. This is a previously unknown function for a key regulator of unconventional secretion in eukaryotic cells.
RESUMO
Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to ß-Galf (1,3) and α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1â6)-Manp polymer, (1â3)-glucan and (1â6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. The role of these newly identified components in the interaction with the host remains to be unraveled.
RESUMO
Extracellular vesicles (EVs) play an important role in the biology of various organisms, including fungi, in which they are required for the trafficking of molecules across the cell wall. Fungal EVs contain a complex combination of macromolecules, including proteins, lipids and glycans. In this work, we aimed to describe and characterize RNA in EV preparations from the human pathogens Cryptococcus neoformans, Paracoccidiodes brasiliensis and Candida albicans, and from the model yeast Saccharomyces cerevisiae. The EV RNA content consisted mostly of molecules less than 250 nt long and the reads obtained aligned with intergenic and intronic regions or specific positions within the mRNA. We identified 114 ncRNAs, among them, six small nucleolar (snoRNA), two small nuclear (snRNA), two ribosomal (rRNA) and one transfer (tRNA) common to all the species considered, together with 20 sequences with features consistent with miRNAs. We also observed some copurified mRNAs, as suggested by reads covering entire transcripts, including those involved in vesicle-mediated transport and metabolic pathways. We characterized for the first time RNA molecules present in EVs produced by fungi. Our results suggest that RNA-containing vesicles may be determinant for various biological processes, including cell communication and pathogenesis.
Assuntos
Espaço Extracelular/metabolismo , Transporte de RNA , RNA Fúngico/metabolismo , Vesículas Secretórias/metabolismo , Sequência de Bases , Éxons/genética , Fluorescência , Fungos/genética , Fungos/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Genoma Fúngico , Humanos , Íntrons/genética , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , Especificidade da EspécieRESUMO
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.
Assuntos
Proteínas Sanguíneas/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Paracoccidioides/metabolismo , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/microbiologia , Parede Celular/química , Parede Celular/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Paracoccidioides/química , Paracoccidioides/genética , Paracoccidioides/patogenicidade , Paracoccidioidomicose/genética , Ligação Proteica , VirulênciaRESUMO
The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.
Assuntos
Criptococose/microbiologia , Cryptococcus/metabolismo , Paracoccidioides/metabolismo , Paracoccidioidomicose/microbiologia , Polissacarídeos/metabolismo , Animais , Anticorpos Monoclonais/análise , Linhagem Celular , Criptococose/imunologia , Cryptococcus/química , Cryptococcus/genética , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Camundongos , Paracoccidioides/química , Paracoccidioides/genética , Paracoccidioidomicose/imunologia , Fagocitose , Polissacarídeos/químicaRESUMO
Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, one of the most important systemic fungal diseases in Latin America. This initiates in lung tissue and can subsequently disseminate to other tissues. Clinical manifestations range from localized forms to disseminated disease that can progress to lethality, probably depending on the relationships among the virulence of the fungus, the immune response and the ability to interact with the surface structures and invade epithelial cells and mononuclear cells of the host. It is generally regarded as a multifocal disease, with oral lesions as the prominent feature. The aim of this study was to evaluate P. brasiliensis yeast infection in normal oral keratinocytes (NOKs). The differential expression of mRNAs and proteins was also determined when the fungus was placed in contact with the cell in order to characterize differentially expressed genes and proteins during P. brasiliensis infection. After contact with NOKs, the fungus appeared to induce alterations in the cells, which showed cellular extensions and cavitations, probably resulting from changes in the actin cytoskeleton seen at 5 and 8 h after infection. Levels of protein expression were higher after reisolation of the fungus from infected NOK culture compared with culture of the fungus in medium. The analysis identified transcripts related to 19 proteins involved in different biological processes. Transcripts were found with multiple functions including induction of cytokines, protein metabolism, alternative carbon metabolism, zinc transport and the stress response during contact with NOKs. The proteins found suggested that the yeast was in a stress situation, as indicated by the presence of RDS1. Nevertheless, the yeast seemed to be proliferating and metabolically active, as shown by the presence of a proteasome, short-chain acetylator, glucosamine-6-phosphate isomerase and ADP/ATP carrier transcripts. Additionally, metabolic pathways may have been activated in order to eliminate toxic substances from the cell as a zinc transporter was detected, which is a potential target for the development of future drugs.