RESUMO
The genus Paracoccidioides includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which comprises four phylogenetic species. A key feature distinguishing planktonic growth from biofilm is the presence of a 3D extracellular matrix (ECM). Therefore, in this study, we analyzed biofilm formation in different species of Paracoccidioides yeast phase, characterized the structural elements of the matrix of P. brasiliensis (Pb18), P. lutzii (Pl01 and 8334) and P. restrepiensis (339 and 192) and evaluated the expression of glucan genes, according to the stage of biofilm evolution for P. brasiliensis. The strains were cultivated in planktonic and biofilm form for 24-144 h. The fungi biomass and metabolic activity were determined by crystal violet and tetrazolium salt reduction (XTT) tests and colony-forming unit (CFU) by plating. The biofilm structure was designed using scanning electron microscopy and confocal laser scanning microscopy techniques. The extracellular matrix of P. brasiliensis and P. lutzii biofilms was extracted by sonication, and polysaccharides, proteins, and extracellular DNA (eDNA) were quantified. The RNA was extracted with the Trizol® reagent and quantified; then, the cDNA was synthesized to analyze the enolase expression, 14-3-3, FKS1, AGS1, GEL3, and KRE6 genes by real-time PCR. All strains of Paracoccidioides studied form a biofilm with more significant metabolic activity and biomass values in 144 h. The extracellular matrix of P. brasiliensis and P. lutzii had a higher content of polysaccharides in their composition, followed by proteins and eDNA in smaller quantities. The P. brasiliensis biofilm kinetics of formation showed greater expression of genes related to glucan's synthesis and its delivery to the external environment in addition adhesins during the biofilm's adhesion, initiation, and maturation. The GEL3 and enolase genes increased in expression within 24 h and during the biofilm maturation period, there was an increase in 14-3-3, AGS1, and FKS1. Furthermore, at 144 h, there was a decrease in KRE6 expression and an increase in GEL3. This study highlights the potential for biofilm formation for three species of Paracoccidioides and the main components of the extracellular matrix that can contribute to a better understanding of biofilm organization.
RESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Paracoccidioides/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases , Biofilmes , Adesinas Bacterianas/metabolismo , Fosfopiruvato Hidratase/genéticaRESUMO
Considering the toxicity of conventional therapeutic approaches and the importance of precise mechanistic targets, it is important to explore signaling pathways implicated in fungal pathobiology. Moreover, treatment of paracoccidioidomycosis, a systemic mycosis caused by a dimorphic fungus, requires prolonged therapeutic regimens. Among the numerous factors underpinning the establishment of Paracoccidioides spp. infection, the capacity to transition from the mycelial to the yeast form is of pivotal importance. The Drk1 protein of Paracoccidioides brasiliensis likely plays a decisive role in this morphological shift and subsequent virulence. We identified peptides with affinity for the PbDrk1 protein using the phage-display method and assessed the effects of these peptides on P. brasiliensis. The peptides were found to inhibit the phase transition of P. brasiliensis. Furthermore, a substantial proportion of these peptides prevented adhesion to pneumocytes. Although these peptides may not possess inherent antifungal properties, they can augment the effects of certain antifungal agents. Notably, the cell wall architecture of P. brasiliensis appears to be modulated by peptide intervention, resulting in a reduced abundance of glycosylated proteins and lipids. These peptides were also evaluated for their efficacy in a Galleria mellonella model and shown to contribute to enhanced larval survival rates. The role of PbDrk1, which is notably absent in mammals, should be further investigated to improve the understanding of its functional role in P. brasiliensis, which may be helpful for designing novel therapeutic modalities.
RESUMO
Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3'hydroxychalcone (3'chalc) -loaded nanoemulsion (NE) (NE3'chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3'chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3'hidroxychalcone (3'chalc) range from 0.97- 7.8 µg/mL and NE3'chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3'chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3'chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3'chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.
Assuntos
Mycobacterium tuberculosis , Paracoccidioides , Biofilmes , Humanos , Testes de Sensibilidade MicrobianaRESUMO
P. brasiliensis is a thermally dimorphic fungus belonging to Paracoccidioides complex, causative of a systemic, endemic mycosis limited to Latin American countries. Signal transduction pathways related to important aspects as surviving, proliferation according to the biological niches are linked to the fungal pathogenicity in many species, but its elucidation in P. brasiliensis remains poorly explored. As Drk1, a hybrid histidine kinase, plays regulators functions in other dimorphic fungi species, mainly in dimorphism and virulence, here we investigated its importance in P. brasilensis. We, therefore generated the respective recombinant protein, anti-PbDrk1 polyclonal antibody and a silenced strain. The Drk1 protein shows a random distribution including cell wall location that change its pattern during osmotic stress condition; moreover the P. brasiliensis treatment with anti-PbDrk1 antibody, which does not modify the fungus's viability, resulted in decreased virulence in G. mellonella model and reduced interaction with pneumocytes. Down-regulating PbDRK1 yielded phenotypic alterations such as yeast cells with more elongated morphology, virulence attenuation in G. mellonella infection model, lower amount of chitin content, increased resistance to osmotic and cell wall stresses, and also caspofungin, and finally increased sensitivity to itraconazole. These observations highlight the importance of PbDrk1 to P. brasiliensis virulence, stress adaptation, morphology, and cell wall organization, and therefore it an interesting target that could help develop new antifungals.
RESUMO
Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins, acting as essential regulators of diverse constitutive metabolic processes. The Hsp60 of the dimorphic fungal Histoplasma capsulatum is the major surface adhesin to mammalian macrophages and studies of antibody-mediated protection against H. capsulatum have provided insight into the complexity involving Hsp60. However, nothing is known about the role of Hsp60 regarding biofilms, a mechanism of virulence exhibited by H. capsulatum. Considering this, the present study aimed to investigate the influence of the Hsp60 on biofilm features of H. capsulatum. Also, the non-conventional model Galleria mellonella was used to verify the effect of this protein during in vivo interaction. The use of invertebrate models such as G. mellonella is highly proposed for the evaluation of pathogenesis, immune response, virulence mechanisms, and antimicrobial compounds. For that purpose, we used a monoclonal antibody (7B6) against Hsp60 and characterized the biofilm of two H. capsulatum strains by metabolic activity, biomass content, and images from scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). We also evaluated the survival rate of G. mellonella infected with both strains under blockage of Hsp60. The results showed that mAb 7B6 was effective to reduce the metabolic activity and biomass of both H. capsulatum strains. Furthermore, the biofilms of cells treated with the antibody were thinner as well as presented a lower amount of cells and extracellular polymeric matrix compared to its non-treated controls. The blockage of Hsp60 before fungal infection of G. mellonella larvae also resulted in a significant increase of the larvae survival compared to controls. Our results highlight for the first time the importance of the Hsp60 protein to the establishment of the H. capsulatum biofilms and the G. mellonella larvae infection. Interestingly, the results with Hsp60 mAb 7B6 in this invertebrate model suggest a pattern of fungus-host interaction different from those previously found in a murine model, which can be due to the different features between insect and mammalian immune cells such as the absence of Fc receptors in hemocytes. However further studies are needed to support this hypothesis.
Assuntos
Chaperonina 60 , Histoplasma , Animais , Anticorpos Monoclonais , Biofilmes , Chaperonina 60/genética , Macrófagos , CamundongosRESUMO
In recent years, research has focused on the immunoreactive components of the Sporothrix schenckii cell wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an emergent worldwide mycosis. In a previous study, we identified a 47-kDa enolase as an immunodominant antigen in mice vaccinated with an adjuvanted mixture of S. schenckii cell wall proteins. Here, we sought to assess the protective potential of a Sporothrix spp. recombinant enolase (rSsEno) formulated with or without the adjuvant Montanide Pet-GelA (PGA) against the S. brasiliensis infection in mice. Mice that were immunized with rSsEno plus PGA showed increased antibody titters against rSsEno and increased median survival time when challenged with S. brasiliensis as compared with mice that had not been immunized or that were immunized with rSsEno alone. Immunization with rSsEno plus PGA induced a predominantly T-helper 1 cytokine pattern after in vitro stimulation of splenic cells with rSsEno: elevated levels of IFN-γ and IL-2, as well as of other cytokines involved in host defense against sporotrichosis, such as TNF-alpha, IL-6, and IL-4. Furthermore, we show for the first time the presence of enolase in the cell wall of both S. schenckii and S. brasiliensis. As a whole, our results suggest that enolase could be used as a potential antigenic target for vaccinal purposes against sporotrichosis.
Assuntos
Anticorpos Antifúngicos/imunologia , Proteínas Fúngicas/imunologia , Imunidade Celular/imunologia , Fosfopiruvato Hidratase/imunologia , Sporothrix/enzimologia , Sporothrix/imunologia , Esporotricose/prevenção & controle , Sequência de Aminoácidos , Animais , Citocinas/metabolismo , Proteínas Fúngicas/administração & dosagem , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosfopiruvato Hidratase/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Homologia de Sequência , Esporotricose/imunologia , Esporotricose/microbiologiaRESUMO
The genus Paracoccidioides consist of dimorphic fungi geographically limited to the subtropical regions of Latin America, which are responsible for causing deep systemic mycosis in humans. However, the molecular mechanisms by which Paracoccidioides spp. causes the disease remain poorly understood. Paracoccidioides spp. harbor genes that encode proteins involved in host cell interaction and mitochondrial function, which together are required for pathogenicity and mediate virulence. Previously, we identified TufM (previously known as EF-Tu) in Paracoccidioides brasiliensis (PbTufM) and suggested that it may be involved in the pathogenicity of this fungus. In this study, we examined the effects of downregulating PbTUFM using a silenced strain with a 55% reduction in PbTUFM expression obtained by antisense-RNA (aRNA) technology. Silencing PbTUFM yielded phenotypic differences, such as altered translation elongation, respiratory defects, increased sensitivity of yeast cells to reactive oxygen stress, survival after macrophage phagocytosis, and reduced interaction with pneumocytes. These results were associated with reduced virulence in Galleria mellonella and murine infection models, emphasizing the importance of PbTufM in the full virulence of P. brasiliensis and its potential as a target for antifungal agents against paracoccidioidomycosis.
Assuntos
Comunicação Celular , Interações Hospedeiro-Patógeno , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Virulência/metabolismo , Virulência , Animais , Regulação para Baixo , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/metabolismo , Paracoccidioidomicose/metabolismo , FagocitoseRESUMO
The Paracoccidioides brasiliensis strain downregulated the expression of adhesin Pb14-3-3 (Pb14-3-3 aRNA) was evaluated in a murine model of paracoccidioidomycosis (PCM). Pb14-3-3 aRNA displays attenuated virulence and triggered the formation of fewer granulomas by lowering the fungal burden in the lungs. Additionally, the Pb14-3-3 aRNA showed more elongated yeast cells and less ability to induce pneumocytes apoptosis in vitro. Our results show that 14-3-3 is an important virulence factor in P. brasiliensis-induced pulmonary infection.
Assuntos
Proteínas 14-3-3/genética , Proteínas Fúngicas/genética , Paracoccidioides/genética , Paracoccidioides/patogenicidade , Fatores de Virulência/genética , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/patologia , Animais , Apoptose/genética , Modelos Animais de Doenças , Expressão Gênica , Pulmão/citologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioidomicose/microbiologiaRESUMO
Paracoccidioidomycosis (PCM) is a fungal disease restricted to Latin countries, and its etiologic agents derive from the Paracoccidioides genus. Attenuation or loss of virulence in Paracoccidioides spp. following successive subculturing has been described. However, virulence can be recovered by passage in mammalian host. In this study, the recovery of adhesion of P. brasiliensis through passage in mice was compared to that in the insect Galleria mellonella. Analysis of in vitro fungal-host cell interaction, gene expression of adhesins, and analysis of the survival curves revealed that Galleria mellonella is useful for the reactivation of P. brasiliensis adhesion.
Assuntos
Adesinas Bacterianas/metabolismo , Mariposas/microbiologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/mortalidade , Fatores de Virulência/metabolismo , Adesinas Bacterianas/genética , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/genética , Paracoccidioidomicose/patologia , Taxa de Sobrevida , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Apoptosis is considered an escape mechanism from the host immune system for the fungus Paracoccidioides spp, and it serves as a vehicle for entry into macrophages without stimulating microbicidal activities. Recently, gp43 of P. brasiliensis was demonstrated to be involved in this process. Therefore, as a new therapeutic alternative, it is very important to study compounds that could reduce the modulation of the induction of apoptosis caused by this fungus. Decyl gallate (G14) is a known antifungal compound, and we decided to investigate its anti-apoptotic properties. Our results demonstrate that G14 was effective against apoptosis induced by gp43, as observed in epithelial cells, and led to a reduction in DNA damage, Bak down-regulation and Bcl-2 up-regulation. Together, these data show that G14 presents promising anti-apoptotic activity.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Glicoproteínas/fisiologia , Paracoccidioides/fisiologia , Células A549 , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/patologia , Antígenos de Fungos/metabolismo , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes bcl-2/genética , Humanos , Paracoccidioidomicose/fisiopatologia , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
Paracoccidioides spp., which are temperature-dependent dimorphic fungi, are responsible for the most prevalent human systemic mycosis in Latin America, the paracoccidioidomycosis. The aim of this study was to characterise the involvement of elongation factor Tu (EF-Tu) in Paracoccidioides brasiliensis-host interaction. Adhesive properties were examined using recombinant PbEF-Tu proteins and the respective polyclonal anti-rPbEF-Tu antibody. Immunogold analysis demonstrated the surface location of EF-Tu in P. brasiliensis. Moreover, PbEF-Tu was found to bind to fibronectin and plasminogen by enzyme-linked immunosorbent assay, and it was determined that the binding to plasminogen is at least partly dependent on lysine residues and ionic interactions. To verify the participation of EF-Tu in the interaction of P. brasiliensis with pneumocytes, we blocked the respective protein with an anti-rPbEF-Tu antibody and evaluated the consequences on the interaction index by flow cytometry. During the interaction, we observed a decrease of 2- and 3-fold at 8 and 24 h, respectively, suggesting the contribution of EF-Tu in fungal adhesion/invasion.
Assuntos
Interações Hospedeiro-Patógeno , Paracoccidioides/enzimologia , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Virulência/metabolismo , Células Epiteliais Alveolares/microbiologia , Adesão Celular , Linhagem Celular , Fibronectinas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Paracoccidioides/fisiologia , Plasminogênio/metabolismo , Ligação ProteicaRESUMO
The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by Ë55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis.
Assuntos
Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Paracoccidioides/genética , Paracoccidioides/patogenicidade , Agrobacterium tumefaciens/genética , Animais , Adesão Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/genética , Larva/microbiologia , Mariposas/microbiologia , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/ultraestrutura , RNA Antissenso/genética , Transformação Genética , Virulência/genéticaRESUMO
Understanding the possible methodologies for the rapid and inexpensive identification of fungal infections is essential for disease diagnosis, but there are some limitations. To help with this problem, serological methods that detect antigens or antibodies are widely used and are useful for the diagnosis of paracoccidioidomycosis (PCM) through the detection of gp43, which is the main antigen employed for the immunodiagnosis of this disease caused by Paracoccidioides brasiliensis. However, the use of gp43 has become restricted because it was recently found that this marker is not identified in the infections caused by Paracoccidioides lutzii. Therefore, it is necessary to identify new antigens in both species or antigens specific for P. lutzii to decrease the morbidity and/or mortality associated with PCM. This review provides a discussion of new diagnostic challenges after the recent discoveries regarding the taxonomy of the Paracoccidioides genus.
Assuntos
Paracoccidioides/classificação , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/diagnóstico , Paracoccidioidomicose/microbiologia , Testes Sorológicos/métodos , Anticorpos Antifúngicos/imunologia , Antígenos de Fungos/imunologia , HumanosRESUMO
BACKGROUND: 14-3-3 proteins comprise a family of eukaryotic multifunctional proteins involved in several cellular processes. The Pb14-3-3 of Paracoccidioides brasiliensis seems to play an important role in the Paracoccidioides-host interaction. Paracoccidioides brasiliensis is an etiological agent of paracoccidioidomycosis, which is a systemic mycosis that is endemic in Latin America. In the initial steps of the infection, Paracoccidioides spp. synthetizes adhesins that allow it to adhere and invade host cells. Therefore, the aim of this work was to perform a functional analysis of Pb14-3-3 using Saccharomyces cerevisiae as a model. RESULTS: The functional analysis of Pb14-3-3 was performed in S. cerevisiae, and it was found that Pb14-3-3 partially complemented S. cerevisiae proteins Bmh1p and Bmh2p, which are recognized as two yeast 14-3-3 homologues. When we evaluated the adhesion profile of S. cerevisiae transformants, Pb14-3-3 acted as an adhesin in S. cerevisiae; however, Bmh1p did not show this function. The influence of Pb14-3-3 in S. cerevisiae ergosterol pathway was also evaluated and our results showed that Pb14-3-3 up-regulates genes involved in ergosterol biosynthesis. CONCLUSIONS: Our data showed that Pb14-3-3 was able to partially complement Bmh1p and Bmh2p proteins in S. cerevisiae; however, we suggest that Pb14-3-3 has a differential role as an adhesin. In addition, Pb-14-3-3 may be involved in Paracoccidioides spp. ergosterol biosynthesis which makes it an interest as a therapeutic target.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Fúngicas/metabolismo , Paracoccidioides/genética , Saccharomyces cerevisiae/genética , Proteínas 14-3-3/genética , Clonagem Molecular , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Paracoccidioides/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.
Assuntos
Proteínas 14-3-3/fisiologia , Antígenos de Fungos/fisiologia , Apoptose , Células Epiteliais/microbiologia , Proteínas Fúngicas/fisiologia , Glicoproteínas/fisiologia , Paracoccidioides/fisiologia , Linhagem Celular/microbiologia , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.
Assuntos
Humanos , Apoptose , Antígenos de Fungos/fisiologia , /fisiologia , Células Epiteliais/microbiologia , Proteínas Fúngicas/fisiologia , Glicoproteínas/fisiologia , Paracoccidioides/fisiologia , Linhagem Celular/microbiologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Paracoccidioides/fisiologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Proteínas 14-3-3/análise , Proteínas 14-3-3/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Parede Celular/química , Parede Celular/metabolismo , Clonagem Molecular , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Paracoccidioides/citologia , Paracoccidioides/genética , Paracoccidioidomicose/patologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Paracoccidioidomycosis is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis. Understanding the interactions between P. brasiliensis and the host tissue depends on the study of the different steps of the process of colonization, especially adhesion, in which the pathogen recognizes ligands on the surface of host cells. This study aimed to verify the role of enolase in the host cell-fungus interaction and the ability of enolase to bind to extracellular matrix components, to determine its subcellular localization, and to study the P. brasiliensis enolase amino acid sequence. The data revealed that fibronectin is the major ligand of enolase. Evaluation of the location of enolase at an ultrastructural level revealed that it is distributed in various cellular compartments, but at a high level in the cell wall. The analysis of the amino acid sequence revealed an internal plasminogen-binding motif ((254)FYKADEKKY(262)), which is conserved in most organisms and described as an important interaction site of the enolase with the host cell surface. This suggests that enolase performs additional functions related to the glycolytic pathway and also plays a role of adhesion in P. brasiliensis. Therefore, this study increases the knowledge about the characteristics of enolase and its influence on the binding process of P. brasiliensis.