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3.
PLoS Genet ; 15(12): e1008551, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31887136

RESUMO

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.


Assuntos
Aspergillus fumigatus/patogenicidade , Cálcio/efeitos adversos , Farmacorresistência Fúngica , Aspergilose Pulmonar/microbiologia , Fatores de Transcrição/genética , Animais , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Caspofungina , Parede Celular/metabolismo , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Mutação , Aspergilose Pulmonar/imunologia , Estresse Fisiológico , Virulência
4.
mSphere ; 4(6)2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694899

RESUMO

The release of extracellular vesicles (EVs) by fungi is a fundamental cellular process. EVs carry several biomolecules, including pigments, proteins, enzymes, lipids, nucleic acids, and carbohydrates, and are involved in physiological and pathological processes. EVs may play a pivotal role in the establishment of fungal infections, as they can interact with the host immune system to elicit multiple outcomes. It has been observed that, depending on the fungal pathogen, EVs can exacerbate or attenuate fungal infections. The study of the interaction between fungal EVs and the host immune system and understanding of the mechanisms that regulate those interactions might be useful for the development of new adjuvants as well as the improvement of protective immune responses against infectious or noninfectious diseases. In this review, we describe the immunomodulatory properties of EVs produced by pathogenic fungi and discuss their potential as adjuvants for prophylactic or therapeutic strategies.

5.
Nat Commun ; 10(1): 5273, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754185

RESUMO

Leishmania RNA virus (LRV) is an important virulence factor associated with the development of mucocutaneous Leishmaniasis, a severe form of the disease. LRV-mediated disease exacerbation relies on TLR3 activation, but downstream mechanisms remain largely unexplored. Here, we combine human and mouse data to demonstrate that LRV triggers TLR3 and TRIF to induce type I IFN production, which induces autophagy. This process results in ATG5-mediated degradation of NLRP3 and ASC, thereby limiting NLRP3 inflammasome activation in macrophages. Consistent with the known restricting role of NLRP3 for Leishmania replication, the signaling pathway triggered by LRV results in increased parasite survival and disease progression. In support of this data, we find that lesions in patients infected with LRV+ Leishmania are associated with reduced inflammasome activation and the development of mucocutaneous disease. Our findings reveal the mechanisms triggered by LRV that contribute to the development of the debilitating mucocutaneous form of Leishmaniasis.

6.
Biotechnol Biofuels ; 12: 269, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31754374

RESUMO

Background: ß-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. Results: In this study, BxlB-a highly secreted GH3 ß-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. Conclusions: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.

7.
Biomolecules ; 9(10)2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547546

RESUMO

Fungal diseases have been underestimated worldwide but constitute a substantial threat to several plant and animal species as well as to public health. The increase in the global population has entailed an increase in the demand for agriculture in recent decades. Accordingly, there has been worldwide pressure to find means to improve the quality and productivity of agricultural crops. Antifungal agents have been widely used as an alternative for managing fungal diseases affecting several crops. However, the unregulated use of antifungals can jeopardize public health. Application of fungicides in agriculture should be under strict regulation to ensure the toxicological safety of commercialized foods. This review discusses the use of antifungals in agriculture worldwide, the need to develop new antifungals, and improvement of regulations regarding antifungal use.

8.
Artigo em Inglês | MEDLINE | ID: mdl-31451502

RESUMO

The available antifungal therapeutic arsenal is limited. The search for alternative drugs with fewer side effects and new targets remains a major challenge. Decyl gallate (G14) is a derivative of gallic acid with a range of biological activities and broad-spectrum antifungal activity. Previously, our group demonstrated the promising anti-Paracoccidioides activity of G14. In this work, to evaluate the antifungal characteristics of G14 for Paracoccidioides lutzii, a chemical-genetic interaction analysis was conducted on a Saccharomyces cerevisiae model. N-glycosylation and/or the unfolded protein response pathway was identified as a high-confidence process for drug target prediction. The overactivation of unfolded protein response (UPR) signaling was confirmed using this model with IRE1/ATF6/PERK genes tagged with green fluorescent protein (GFP). In P. lutzii, this prediction was confirmed by the low activity of glycosylated enzymes [α-(1,3)-glucanase, N-acetyl-ß-d-glucosaminidase (NAGase), and α-(1,4)-amylase], by hyperexpression of genes involved with the UPR and glycosylated enzymes, and by the reduction in the amounts of glycosylated proteins and chitin. All of these components are involved in fungal cell wall integrity and are dependent on the N-glycosylation process. This loss of integrity was confirmed by the reduction in mitochondrial activity, impaired budding, enhancement of wall permeability, and a decrease in viability. These events led to a reduction of the ability of fungi to adhere on human lung epithelial cells (A549) in vitro Therefore, G14 may have an important role in balancing the inflammatory reaction caused by fungal infection, without interfering with the microbicidal activity of nitric oxide. This work provides new information on the activity of G14, a potential anti-Paracoccidioides compound.

9.
mSystems ; 4(4)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213522

RESUMO

Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.

10.
Biotechnol Biofuels ; 12: 146, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31223336

RESUMO

Background: Trichoderma reesei is the most important industrial producer of lignocellulolytic enzymes. These enzymes play an important role in biomass degradation leading to novel applications of this fungus in the biotechnology industry, specifically biofuel production. The secretory pathway of fungi is responsible for transporting proteins addressed to different cellular locations involving some cellular endomembrane systems. Although protein secretion is an extremely efficient process in T. reesei, the mechanisms underlying protein secretion have remained largely uncharacterized in this organism. Results: Here, we report for the first time the isolation and characterization of T. reesei extracellular vesicles (EVs). Using proteomic analysis under cellulose culture condition, we have confidently identified 188 vesicular proteins belonging to different functional categories. Also, we characterized EVs production using transmission electron microscopy in combination with light scattering analysis. Biochemical assays revealed that T. reesei extracellular vesicles have an enrichment of filter paper (FPase) and ß-glucosidase activities in purified vesicles from 24, 72 and 96, and 72 and 96 h, respectively. Furthermore, our results showed that there is a slight enrichment of small RNAs inside the vesicles after 96 h and 120 h, and presence of hsp proteins inside the vesicles purified from T. reesei grown in the presence of cellulose. Conclusions: This work points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus.

11.
Front Microbiol ; 10: 854, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31105662

RESUMO

Acquisition and subsequent metabolism of different carbon and nitrogen sources have been shown to play an important role in virulence attributes of the fungal pathogen Aspergillus fumigatus, such as the secretion of host tissue-damaging proteases and fungal cell wall integrity. We examined the relationship between the metabolic processes of carbon catabolite repression (CCR), nitrogen catabolite repression (NCR) and virulence in a variety of A. fumigatus clinical isolates. A considerable amount of heterogeneity with respect to the degree of CCR and NCR was observed and a positive correlation between NCR and virulence in a neutropenic mouse model of pulmonary aspergillosis (PA) was found. Isolate Afs35 was selected for further analysis and compared to the reference strain A1163, with both strains presenting the same degree of virulence in a neutropenic mouse model of PA. Afs35 metabolome analysis in physiological-relevant carbon sources indicated an accumulation of intracellular sugars that also serve as cell wall polysaccharide precursors. Genome analysis showed an accumulation of missense substitutions in the regulator of protease secretion and in genes encoding enzymes required for cell wall sugar metabolism. Based on these results, the virulence of strains Afs35 and A1163 was assessed in a triamcinolone murine model of PA and found to be significantly different, confirming the known importance of using different mouse models to assess strain-specific pathogenicity. These results highlight the importance of nitrogen metabolism for virulence and provide a detailed example of the heterogeneity that exists between A. fumigatus isolates with consequences for virulence in a strain-specific and host-dependent manner.

12.
mSphere ; 4(2)2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-31019001

RESUMO

The thermodimorphic pathogenic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiologic causes of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Galectin-3 (Gal-3), an animal ß-galactoside-binding protein, modulates important roles during microbial infections, such as triggering a Th2-polarized immune response in PCM. Herein, we demonstrate that Gal-3 also plays other important roles in P. brasiliensis infection. We verified that Gal-3 levels are upregulated in human and mice infections and established that Gal-3 inhibited P. brasiliensis growth by inhibiting budding. Furthermore, Gal-3 affected disruption and internalization of extracellular vesicles (EVs) from P. brasiliensis by macrophages. Our results suggest important protective roles for Gal-3 in P. brasiliensis infection, indicating that increased Gal-3 production during P. brasiliensis infection may affect fungal growth and EV stability, thus promoting beneficial effects that could influence the course of PCM. The finding that Gal-3 has effects against P. brasiliensis together with previously reported effects against Cryptococcus neoformans suggests that molecule has a general antifungal role in innate defenses against fungal pathogens.IMPORTANCE Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Although the immune mechanisms to control PCM are still not fully understood, several events of the host innate and adaptive immunity are crucial to determine the progress of the infection. Mammalian ß-galactoside-binding protein galectin-3 (Gal-3) plays significant roles during microbial infections and has been studied for its immunomodulatory roles, but it can also have direct antimicrobial effects. We asked whether this protein plays a role in Paracoccidioides brasiliensis We report herein that Gal-3 indeed has direct effects on the fungal pathogen, inhibiting fungal growth and reducing extracellular vesicle stability. Our results suggest a direct role for Gal-3 in P. brasiliensis infection, with beneficial effects for the mammalian host.


Assuntos
Galectina 3/genética , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/imunologia , Animais , Antifúngicos , Modelos Animais de Doenças , Vesículas Extracelulares , Galectina 3/imunologia , Humanos , Imunidade Inata , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Viabilidade Microbiana , Regulação para Cima
13.
Front Microbiol ; 10: 214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809213

RESUMO

In the past few years, fungal diseases caused estimated over 1.6 million deaths annually and over one billion people suffer from severe fungal diseases (Brown et al., 2012; Anonymous, 2017b). Public health surveillance of fungal diseases is generally not compulsory, suggesting that most estimates are conservative (Casadevall, 2017; Anonymous, 2017a). Fungal disease can also damage plants and crops, causing major losses in agricultural activities and food production (Savary et al., 2012). Animal pathogenic fungi are threatening bats, amphibians and reptiles with extinction (Casadevall, 2017). It is estimated that fungi are the highest threat for animal-host and plant-host species, representing the major cause (approximately 65%) of pathogen-driven host loss (Fisher et al., 2012). In this complex scenario, it is now clear that the global warming and accompanying climate changes have resulted in increased incidence of many fungal diseases (Garcia-Solache and Casadevall, 2010). On the basis of all these factors, concerns on the occurrence of a pandemic of fungal origin in a near future have been raised (Casadevall, 2017). In this context, to stop forgetting and underestimating fungal diseases is mandatory.

14.
Front Immunol ; 9: 2343, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30356863

RESUMO

The release of biomolecules critically affects all pathogens and their establishment of diseases. For the export of several biomolecules in diverse species, the use of extracellular vesicles (EVs) is considered to represent an alternative transport mechanism, but no study to date has investigated EVs from dermatophytes. Here, we describe biologically active EVs from the dermatophyte Trichophyton interdigitale, a causative agent of mycoses worldwide. EV preparations from T. interdigitale were examined using nanoparticle-tracking analysis, which revealed vesicular structures 20-380 nm in diameter. These vesicles induced the production of proinflammatory mediators by bone marrow-derived macrophages (BMDMs) and keratinocytes in a dose-dependent manner, and an addition of the EVs to BMDMs also stimulated the transcription of the M1-polarization marker iNOS (inducible nitric oxide synthase) and diminished the expression of the M2 markers arginase-1 and Ym-1. The observed M1 macrophages' polarization triggered by EVs was abolished in cells obtained from knockout Toll-like receptor-2 mice. Also, the EVs-induced productions of pro-inflammatory mediators were blocked too. Furthermore, the EVs from T. interdigitale enhanced the fungicidal activity of BMDMs. These results suggest that EVs from T. interdigitale can modulate the innate immune response of the host and influence the interaction between T. interdigitale and host immune cells. Our findings thus open new areas of investigation into the host-parasite relationship in dermatophytosis.


Assuntos
Vesículas Extracelulares/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Tinha/imunologia , Tinha/microbiologia , Trichophyton/imunologia , Trichophyton/metabolismo , Animais , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Imunomodulação , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Fagocitose/imunologia
15.
Sci Rep ; 8(1): 12314, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120327

RESUMO

Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H2O2 at elevated velocities (rate constants in the 107 M-1s-1 range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakAHOG1 MAP kinase and the Yap1YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.


Assuntos
Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Peróxido de Hidrogênio/metabolismo , Peroxirredoxinas/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Estimativa de Kaplan-Meier , Cinética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peroxidase , Fator de Necrose Tumoral alfa/metabolismo , Virulência
16.
Biotechnol Biofuels ; 11: 84, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29619080

RESUMO

Background: Trichoderma reesei is a saprophytic fungus implicated in the degradation of polysaccharides present in the cell wall of plants. T. reesei has been recognized as the most important industrial fungus that secretes and produces cellulase enzymes that are employed in the production of second generation bioethanol. A few of the molecular mechanisms involved in the process of biomass deconstruction by T. reesei; in particular, the effect of sugar transporters and induction of xylanases and cellulases expression are yet to be known. Results: In our study, we characterized a novel sugar transporter, which was previously identified by our group through in silico analysis of RNA-seq data. The novel T. reesei 69957-sugar transport system (Tr69957) is capable of transporting xylose, mannose, and cellobiose using a T. reesei 69957-sugar transport system in Saccharomyces cerevisiae. The deletion of Tr69957 in T. reesei affected the fungal growth and biomass accumulation, and the sugar uptake in the presence of mannose, cellobiose, and xylose. Molecular docking studies revealed that Tr69957 shows reduced protein-ligand binding energy for interactions towards disaccharides in comparison with monosaccharides. Furthermore, the deletion of Tr69957 affected the gene expression of cellobiohydrolases (cel7a and cel6a), ß-glucosidases (cel3a and cel1a), and xylanases (xyn1 and xyn2) in the cultures of parental and mutant strains in the presence of cellobiose and sugarcane bagasse (SCB). Conclusion: The transporter Tr69957 of T. reesei can transport cellobiose, xylose, and mannose, and can affect the expression of a few genes encoding enzymes, such as cellulases and xylanases, in the presence of SCB. We showed for the first time that a filamentous fungus (T. reesei) contains a potential mannose transporter that may be involved in the degradation of cellulose.

17.
Sci Rep ; 8: 12314, 2018.
Artigo | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib15579

RESUMO

Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H2O2 at elevated velocities (rate constants in the 107?M-1s-1 range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakAHOG1 MAP kinase and the Yap1YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.

18.
Nat Commun ; 8(1): 1968, 2017 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-29213074

RESUMO

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian ß-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host.


Assuntos
Antifúngicos/imunologia , Antifúngicos/farmacologia , Criptococose/tratamento farmacológico , Criptococose/imunologia , Cryptococcus neoformans/efeitos dos fármacos , Galectina 3/imunologia , Galectina 3/farmacologia , Imunidade Adaptativa , Animais , Cápsulas Bacterianas/efeitos dos fármacos , Encéfalo/imunologia , Criptococose/microbiologia , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Galectina 3/sangue , Galectina 3/genética , Expressão Gênica , Humanos , Pulmão/imunologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia
19.
PLoS One ; 12(8): e0184010, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28846733

RESUMO

Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-ß-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72-96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.


Assuntos
Proteínas Fúngicas/metabolismo , Paracoccidioides/metabolismo , Paracoccidioides/crescimento & desenvolvimento , Aglutininas do Germe de Trigo/metabolismo
20.
mBio ; 8(4)2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28720727

RESUMO

Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (AsPCN1, AsPCN2, and AsPCN3) and characterized them with regard to P. brasiliensis biology and pathogenicity. AsPCN1, AsPCN2, and AsPCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with AsPCN1, AsPCN2, and AsPCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis.IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis.


Assuntos
Proteínas Fúngicas/metabolismo , Inativação Gênica , Lectinas/metabolismo , Paracoccidioides/patogenicidade , Fatores de Virulência/metabolismo , Animais , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Lectinas/genética , Masculino , Camundongos Endogâmicos BALB C , Micélio/citologia , Micélio/crescimento & desenvolvimento , Paracoccidioides/citologia , Paracoccidioides/genética , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/patologia , Análise de Sobrevida , Virulência , Fatores de Virulência/genética
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