Ethanol modulates the effector functions of human monocyte-derived macrophages in response to Paracoccidioides brasiliensis yeast cells.

We aimed to investigate the effects of ethanol and its metabolites (ß-hydroxybutyrate and sodium acetate) in the effector functions of macrophages in response to Paracoccidioides brasiliensis yeast cells and to determine their influence in the development of the adaptive response. Purified peripheral blood monocytes were differentiated into macrophages and were treated with ethanol, ß-hydroxybutyrate, and sodium acetate, and stimulated with P. brasiliensis yeast cells and evaluated for their phenotypic characteristics, functional activity, and capability to induce T cells activation/differentiation. We found that the ethanol treatment diminished the expression of HLA-AB, HLA-DR, CD80, and CD86, modulating the expression of dectin-1, as well as Syk phosphorylation. The ethanol treatment increased the phagocytic activity, expression of CD206, and IL-10 production; however, reduced ROS production, fungicidal activity, caspase-1 cleavage, and IL-1ß and IL-6 production. Our data also showed that the presence of ethanol reduced the differentiation of Th1 and Th17 cells and increased the frequency of Th2 cells. Our results indicated that ethanol exposure could suppress effector function of macrophages, possibly leading to the polarization of M2 macrophages. The ethanol modulates the expression of costimulatory and antigen-presentation molecules and interferes with the NLRP3 inflammasome. Altogether, these alterations affect the development of the adaptive response, decreasing the frequency of IL-17, IL-22, and IFN- γ producing cells, and increasing the frequency of IL-4 producing cells. Therefore, exposure to ethanol can impair the capability of macrophages to exert their effector functions and activate the acquired response related to resistance to P. brasiliensis infection.

Paracoccidioides species present distinct fungal adherence to epithelial lung cells and promote different IL-8 secretion levels.

Fungi that belong to the genus Paracoccidioides are the etiologic agents of paracoccidioidomycosis, a human systemic mycosis, which occurs in Latin America. Epithelial cell is one of the first cells that interact with these fungi and responds by secreting inflammatory mediators such as cytokines. In the present study, we demonstrate that yeasts of different isolates of Paracoccidioides brasiliensis (Pb18 and Pb03) and Paracoccidioides lutzii (Pb01) distinctly promoted interleukin (IL)-8 secretion by the lung epithelial cell line A549. Depending on the isolate, this cytokine release may rely on the epithelial cell interaction with fungal secreted components or direct contact with the pathogen. In addition, adhesion of yeasts to the pulmonary epithelial cells was also different among Paracoccidioides isolates, and the highest percentage of A549 cells with adhered fungi was observed with P. lutzii. All Paracoccidioides isolates induced an expression increase of α3 and α5 integrins in A549 cells and, using small interfering RNA, we observed that the integrin silencing promoted a reduction of P. lutzii adhesion, which suggests the involvement of integrins in this event. Together, these results indicate that host epithelial cell response may depend on the isolate of Paracoccidioides.

Cross-talk between the Ras GTPase and the Hog1 survival pathways in response to nitrosative stress in Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that cause paracoccidioidomycosis (PCM), the major systemic mycosis in Latin America. The capacity to evade the innate immune response of the host is due to P. brasiliensis ability to respond and to survive the nitrosative stress caused by phagocytic cells. However, the regulation of signal transduction pathways associated to nitrosative stress response are poorly understood. Ras GTPase play an important role in the various cellular events in many fungi. Ras, in its activated form (Ras-GTP), interacts with effector proteins and can initiate a kinase cascade. In this report, we investigated the role of Ras GTPase in P. brasiliensis after in vitro stimulus with nitric oxide (NO). We observed that low concentrations of NO induced cell proliferation in P. brasiliensis, while high concentrations promoted decrease in fungal viability, and both events were reversed in the presence of a NO scavenger. We observed that high levels of NO induced Ras activation and its S-nitrosylation. Additionally, we showed that Ras modulated the expression of antioxidant genes in response to nitrosative stress. We find that the Hog1 MAP kinase contributed to nitrosative stress response in P. brasiliensis in a Ras-dependent manner. Taken together, our data demonstrate the relationship between Ras-GTPase and Hog1 MAPK pathway allowing for the P. brasiliensis adaptation to nitrosative stress.

Recovery of the Paracoccidioides brasiliensis virulence after animal passage promotes changes in the antioxidant repertoire of the fungus.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), a cause of disease in healthy and immunocompromised persons in Latin America. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. The development of the disease depends on factors associated with the host immune response and the infectious agent's characteristics, especially virulence. The oxidative stress response is an important virulence attribute in several fungi. In this study, we assessed the enzymatic repertoire of responses to oxidative stress in the Pb18 isolate with different degrees of virulence. The virulence of attenuated Pb18 (aPb18) strain was recovered after several animal passages. Virulent strain (vPb18) showed an effective fungal oxidative stress response and several genes involved in response to oxidative stress were up-regulated in this isolate. These genes expressed the same profile when we recovered the phenotypic virulence in attenuated strain aPb18. Our study demonstrated that attenuated P. brasiliensis recovered their virulence after serial animal passages (vPb18), and this process positively modulated the fungus's antioxidant repertoire.

Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects.

Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition.

Anti-apoptotic effects of decyl gallate on the induction of apoptosis in A549 pneumocytes by Paracoccidioides brasiliensis gp43.

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.

In Vivo Paracoccidioides sp. Biofilm on Vascular Prosthesis.

Paracoccidioidomycosis is an endemic mycosis caused by Paracoccidioides species limited to Latin America arising with the chronic form in 90% of cases. The capacity of microorganisms to form biofilms is considered of great importance medical since can contribute to the persistence and to the chronic state of the diseases. The ability of Paracoccidioides to form biofilm has been demonstrated in vitro. In our study, for the first time we have observed this capability in vivo on a vascular prosthesis using scanning electron microscope showing a dense network of Paracoccidioides yeasts covered by an extracellular matrix.

Osmotic stress adaptation of Paracoccidioides lutzii, Pb01, monitored by proteomics.

The ability to respond to stressful conditions is essential for most living organisms. In pathogenic organisms, this response is required for effective transition from a saprophytic lifestyle to the establishment of pathogenic interactions within a susceptible host. Hyperosmotic stress has been used as a model to study signal transduction and seems to cause many cellular adaptations, including the alteration of protein expression and cellular volume as well as size regulation. In this work, we evaluated the proteomic profile of Paracoccidioides lutzii Pb01 yeast cells during osmotic stress induced by potassium chloride. We performed a high accuracy proteomic technique (NanoUPLC-MS(E)) to identify differentially expressed proteins during osmotic shock. The data describe an osmoadaptative response of this fungus when subjected to this treatment. Proteins involved in the synthesis of cell wall components were modulated, which suggested cell wall remodeling. In addition, alterations in the energy metabolism were observed. Furthermore, proteins involved in amino acid metabolism and hydrogen peroxide detoxification were modulated during osmotic stress. Our study suggests that P. lutzii Pb01. presents a vast osmoadaptative response that is composed of different proteins that act together to minimize the effects caused by osmotic stress.

New insights into a complex fungal pathogen: the case of Paracoccidioides spp.

Paracoccidioidomycosis is a systemic mycosis endemic to Latin America, with Paracoccidioides brasiliensis and P. lutzii being the causal agents of this disorder. Several issues have been raised in the 100 years since its discovery and in this article we discuss features of this fascinating fungal pathogen, including its biology, eco-epidemiology and aspects of its pathogenicity. We also consider some of its virulence determinants, the most recent advances in the study of its metabolic pathways and the molecular and genetic research tools developed for this research. We also review the animal models used to study host-fungal interactions and how the host defence mechanisms against this pathogen work.

Identification and characterisation of elongation factor Tu, a novel protein involved in Paracoccidioides brasiliensis-host interaction.

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.

Paracoccidioides brasiliensis infection promotes thymic disarrangement and premature egress of mature lymphocytes expressing prohibitive TCRs.

BACKGROUND: Paracoccidioidomycosis, a chronic granulomatous fungal disease caused by Paracoccidioides brasiliensis yeast cells affects mainly rural workers, albeit recently cases in immunosuppressed individuals has been reported. Protective immune response against P. brasiliensis is dependent on the activity of helper T cells especially IFN-γ-producing Th1 cells. It has been proposed that Paracoccidioides brasiliensis is able to modulate the immune response towards a permissive state and that the thymus plays a major role in it. METHODS: In this paper, we show that acute infection of BALB/c mice with P. brasiliensis virulent isolate (Pb18) might cause alterations in the thymic environment as well as the prohibitive TCR-expressing T cells in the spleens. RESULTS: After seven days of infection, we found yeast cells on the thymic stroma, the thymic epithelial cells (TEC) were altered regarding their spatial-orientation and inflammatory mediators gene expression was increased. Likewise, thymocytes (differentiating T cells) presented higher migratory ability in ex vivo experiments. Notwithstanding, P. brasiliensis-infected mice showed an increased frequency of prohibitive TCR-expressing T cells in the spleens, suggesting that the selection processes that occur in the thymus may be compromised during the acute infection. CONCLUSION: In this paper, for the first time, we show that acute infection with Paracoccidioides brasiliensis yeast cells promotes thymic alterations leading to a defective repertoire of peripheral T cells. The data presented here may represent new mechanisms by which P. brasiliensis subverts the immune response towards the chronic infection observed in humans.

Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells.

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.

Paracoccidioidomycosis in patients with lymphoma and review of published literature.

This paper describes four new cases of lymphomas, two Hodgkin lymphomas and two non-Hodgkin lymphomas in patients with paracoccidioidomycosis. All had mycosis diagnosed before lymphomas with Paracoccidioides brasiliensis demonstrated in several lymph nodes, as seen in the disseminated form of the disease. When lymphoma was diagnosed, one patient was under regular paracoccidioidomycosis treatment and in clinic-serological remission for this disease, another was under regular treatment but with clinic-serological mycosis activity, one had abandoned paracoccidioidomycosis treatment 6 years earlier, and the other had not yet received any kind of antifungal drugs. Three patients received treatment for lymphomas with one remaining in remission until now, one achieving tumor remission which relapsed years later, and one having only residual lymphoma in bone marrow for a decade but clinically well. All three experienced paracoccidioidomycosis clinical remission, however, serology became negative just in one. Similar previously described cases were reviewed: five Hodgkin lymphomas, three non-Hodgkin lymphomas, and one described only as "lymphoma" without specifying type; a summary of their findings is presented. Finally, there is also a brief discussion on the possible pathophysiological mechanisms involved in the concomitance of these two disorders.

Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components.

BACKGROUND: Paracoccidioides spp is a fungi genus and the agent of paracoccidioidomycosis. The strategies of infection used by these pathogens involve the expression of proteins related to adaptation to the host, particularly regarding the uptake of micronutrients. This study analyzed the adhesion of Paracoccidioides lutzii during conditions of copper (Cu) and iron (Fe) deprivation, while also evaluating the proteins expressed in conditions of Cu depletion in the presence of four extracellular matrix (ECM) components (laminin, fibronectin and types I and IV collagen). RESULTS: We cultured the P. lutzii in a chemically defined media without Cu and Fe. The fungus was then placed in contact with different ECM components and adhesion was evaluated. A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression. A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components. The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction. CONCLUSIONS: This study is important for determining prospective proteins that may be involved in the interaction of Paracoccidioides with a host. Understanding the adaptive response to different growth conditions, elucidating the processes of adhesion and cell invasion, and identifying the proteins that are differentially expressed during the fungus-host interaction may help elucidate mechanisms used for survival and growth of Paracoccidioides in various human tissues.

Molecular and morphological data support the existence of a sexual cycle in species of the genus Paracoccidioides.

The genus Paracoccidioides includes the thermodimorphic species Paracoccidioides brasiliensis and P. lutzii, both of which are etiologic agents of paracoccidioidomycosis, a systemic mycosis that affects humans in Latin America. Despite the common occurrence of a sexual stage among closely related fungi, this has not been observed with Paracoccidioides species, which have thus been considered asexual. Molecular evolutionary studies revealed recombination events within isolated populations of the genus Paracoccidioides, suggesting the possible existence of a sexual cycle. Comparative genomic analysis of all dimorphic fungi and Saccharomyces cerevisiae demonstrated the presence of conserved genes involved in sexual reproduction, including those encoding mating regulators such as MAT, pheromone receptors, pheromone-processing enzymes, and mating signaling regulators. The expression of sex-related genes in the yeast and mycelial phases of both Paracoccidioides species was also detected by real-time PCR, with nearly all of these genes being expressed preferentially in the filamentous form of the pathogens. In addition, the expression of sex-related genes was responsive to the putative presence of pheromone in the supernatants obtained from previous cocultures of strains of two different mating types. In vitro crossing of isolates of different mating types, discriminated by phylogenetic analysis of the α-box (MAT1-1) and the high-mobility-group (HMG) domain (MAT1-2), led to the identification of the formation of young ascocarps with constricted coiled hyphae related to the initial stage of mating. These genomic and morphological analyses strongly support the existence of a sexual cycle in species of the genus Paracoccidioides.

Paracoccidioidomycosis mimicking squamous cell carcinoma on the dorsum of the tongue and review of published literature.

INTRODUCTION: Paracoccidioidomycosis is a disease that is endemic to southern and southeastern Brazil, caused by the fungus Paracoccidioides brasiliensis. The most common clinical oral manifestation is the presence of multiple granulomatous ulcers with hemorrhagic dots, located mainly on the lips, palate, and buccal mucosa. However, the disease can manifest atypically as a single ulcer, mimicking oral squamous cell carcinoma (SCC) or tuberculosis. CASE REPORT: A 65-year-old male patient presented with a complaint of a single ulcerated lesion on the dorsum of the tongue; the lesion had evolved over 6 months. The diagnostic hypotheses were SCC and oral manifestation of tuberculosis. An incisional biopsy was performed, and histopathological analysis of the specimen revealed pseudoepitheliomatous hyperplasia, a granulomatous structure of epithelioid histiocytes, multinucleated giant cells, and lymphocytes in the connective tissue. Grocott staining confirmed the presence of the fungus in the lesion, and a diagnosis was made of paracoccidioidomycosis. The patient was treated with 200 mg/day of itraconazole for 12 months and now shows no signs or symptoms of recurrence of the disease. CONCLUSION: Correct diagnosis is essential for a successful therapeutic approach and resolution of the lesion.

Involvement of the 90 kDa heat shock protein during adaptation of Paracoccidioides brasiliensis to different environmental conditions.

HSP90 is a molecular chaperone that participates in folding, stabilization, activation, and assembly of several proteins, all of which are key regulators in cell signaling. In dimorphic pathogenic fungi such as Paracoccidioides brasiliensis, the adaptation to a higher temperature, acid pH and oxidative stress, is an essential event for fungal survival and also for the establishing of the infectious process. To further understand the role of this protein, we used antisense RNA technology to generate a P. brasiliensis isolate with reduced PbHSP90 gene expression (PbHSP90-aRNA). Reduced expression of HSP90 decreased yeast cell viability during batch culture growth and increased susceptibility to acid pH environments and imposed oxidative stress. Also, PbHSP90-aRNA yeast cells presented reduced viability upon interaction with macrophages. The findings presented here suggest a protective role for HSP90 during adaptation to hostile environments, one that promotes survival of the fungus during host-pathogen interactions.

Hsp90 regulates Paracoccidioides brasiliensis proliferation and ROS levels under thermal stress and cooperates with calcineurin to control yeast to mycelium dimorphism.

Paracoccidioidomycosis is a systemic human mycosis in Latin America caused by Paracoccidioides brasiliensis, a dimorphic pathogenic fungus that lives as a mold in the environment and as yeast during infections of human lungs. In this work, we provide evidence that the inhibition of Hsp90 by geldanamycin (GDA) impairs the proliferation of the yeast, but has no effect on mycelial development. Treatment with cyclosporin A (CsA), an inhibitor of the Hsp90 client protein calcineurin, did not increase the effect of GDA. In contrast, GDA prevented mycelial to yeast differentiation through a mechanism partially dependent on calcineurin, whereas differentiation from yeast to mycelia occurred independent of GDA or CsA. A significant increase in reactive oxygen species (ROS) levels was detected in GDA-treated yeast at 42°C. However, the levels of ROS remained unchanged in GDA-treated yeast or mycelia incubated at 37°C, suggesting that Hsp90 plays different roles under normal and thermal stress conditions. We propose that Hsp90 strengthens the stress response of P. brasiliensis at 37°C through a mechanism that does not involve ROS. Moreover, we suggest that Hsp90 has calcineurin-dependent functions in this organism.

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen.

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.

The hydrolase PbHAD32 participates in the adherence of Paracoccidioides brasiliensis conidia to epithelial lung cells.

Adherence of the dimorphic pathogenic fungus Paracoccidioides brasiliensis to lung epithelial cells is considered an essential event for the establishment of infection. We have previously shown that the PbHAD32 hydrolase is important in this early stage of the host-P. brasiliensis yeast cells interaction. The aim of this study was to further elucidate the role of PbHAD32 in conidial thermodimorphism and their interaction with lung epithelial cells. Analysis of the PbHAD32 gene expression revealed higher mRNA levels during the conidia to mycelia (C-M) germination when compared to the conidia to yeast (C-Y) transition. Moreover, PbHAD32 was consistently expressed at higher levels upon infection of lung epithelial cells, but to a greater extent when conidia germinated to produce mycelia. Interestingly, at this particular transitional stage, more conidia adhered to epithelial cells than when they were transiting to the yeast form. Altogether our data further corroborates the importance of PbHAD32 during initial adherence to host cells and suggest that the 32-KDa hydrolase may also participate at different stages of the C-M and C-Y conversions.