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1.  The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide 
Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4+-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund’s adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM.
PMCID: PMC3343455  PMID: 22586420
Paracoccidioides brasiliensis; paracoccidioidomycosis; P10; adjuvants; dioctadecyl-dimethylammonium bromide; FliC flagellin; aluminum hydroxide; complete Freund’s adjuvant
2.  T Helper 1–Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis 
Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1–stimulating adjuvant in P. brasiliensis–infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.
Author Summary
P. brasiliensis is a thermally dimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), the most prevalent human systemic mycosis in Latin America, whose drug therapy is usually prolonged and associated with toxic side effects and relapses. Although immunostimulatory therapy is a promising approach to improving the treatment of fungal infections as PCM, few studies have been reported. In the current study, we verified that a single-dose administration of an adjuvant that induces T helper (Th) 1 immune response (complete Freund's adjuvant [CFA]) in P. brasiliensis–infected mice was sufficient to break the lack of immune response to the fungus observed in infected mice. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by preserved lung structure and small fungal burden, whereas control mice that had been treated with incomplete Freund's adjuvant presented many granulomatous lesions and high fungal burden. The immunotherapy with Th1-inducing adjuvant might be a valuable tool in the treatment of PCM and potentially useful for faster and efficient cure of PCM in humans.
PMCID: PMC2263123  PMID: 18335066
3.  rPbPga1 from Paracoccidioides brasiliensis Activates Mast Cells and Macrophages via NFkB 
PLoS Neglected Tropical Diseases  2015;9(8):e0004032.
The fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients.
Methodology/Principal Findings
Endogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1.
The results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for additional studies on the pathogenesis of P. brasiliensis.
Author Summary
Paracoccidioidomycosis (PCM), one of the most prevalent mycoses in Latin America, is caused by the thermodimorphic fungus Paracoccidioides brasiliensis. P. brasiliensis is thought to infect the host through the respiratory tract. Cell wall components of P. brasiliensis interact with host cells producing granulomas, thus influencing the pathogenesis of PCM. PbPga1 is an O-glycosylated, GPI-anchored protein that is localized on the yeast cell surface and is up-regulated in the pathogenic yeast form. GPI anchored proteins are involved in cell-cell and cell-tissue adhesion and have a key role in the interaction between fungal and host cells. In the present study, the authors show that both macrophages and mast cells are associated with the P.brasiliensis granulomas. Furthermore, recombinant PbPga1 was able to activate both alveolar macrophages and mast cells via the transcription factor NFkB to release inflammatory mediators. The results of this study indicate that the surface antigen, PbPga1, may play an important role in PCM pathogenesis by activating macrophages and mast cells. Additionally, PbPga1 may be a target for new strategies for detecting and treating PCM.
PMCID: PMC4552726  PMID: 26317855
4.  Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro 
PLoS Neglected Tropical Diseases  2015;9(9):e0004037.
Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.
Author Summary
Paracoccidioidomycosis (PCM) is an infectious disease caused by fungi of genus Paracoccidioides (P. brasiliensis and P. lutzii). PCM is endemic in Latin America, with a greater incidence in Brazil, Colombia, and Argentina. Over the last years, studies are focusing on neutrophils’ (PMNs) actions against P. brasiliensis, due to the capacity of these cells to develop different defense strategies against pathogens. and especially due to constant presence of inflammatory infiltrates full of PMNs in the granuloma of the disease. As PMN release of both granular and nuclear material, identified as Neutrophil Extracellular Traps (NETs), is a spectacular action mechanism against microbes, we seek to identify whether this process would be an important mechanism triggered against P. brasiliensis. Thus, we showed for the first time the identification of NETs in tegumentary lesions of patients with PCM by viewing the individual components of NETs. Beyond that, we demonstrated the entrapment of P. brasiliensis in vitro by these structures released from human PMNs of patients with PCM and healthy donors, with different patterns, in a dependence of the evaluated strain. Our data provides important new information regarding the role of PMNs against P. brasiliensis, opening new avenues for the research on immunity of PCM.
PMCID: PMC4556621  PMID: 26327485
5.  Serology of Paracoccidioidomycosis Due to Paracoccidioides lutzii 
Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. In western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.
Author Summary
Tropical diseases, such as paracoccidioidomycosis, are the most common type of neglected diseases. From 1980 to 1995, 3,181 deaths from paracoccidioidomycosis occurred in Brazil, representing the eighth most common cause of death from predominantly chronic or recurrent types of infectious and parasitic diseases, showing considerable magnitude and low visibility. Paracoccidioidomycosis is traditionally assumed to be caused solely by Paracoccidioides brasiliensis, but a new species, Paracoccidioides lutzii, was discovered in the Central-Western region of Brazil. Thus, new antigenic preparations and tests for an accurate differential diagnosis between these two species appear to be needed. This study aimed to develop new antigenic preparations from P. lutzii isolates to improve the diagnosis of paracoccidioidomycosis. We used patient serum samples predominantly from the Central-Western region of Brazil. Various antigenic preparations were tested, and a cell free antigen derived from P. lutzii was an excellent antigen for serological diagnosis and able to diagnose 100% of sera from patients with PCM due to P. lutzii.
PMCID: PMC4102441  PMID: 25032829
6.  Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis 
Mycopathologia  2012;174(1):1-10.
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients’ sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.
PMCID: PMC3368115  PMID: 22249604
gp75; Paracoccidioides brasiliensis; Paracoccidioidomycosis; Peptides; Phage display
7.  Pulmonary Abnormalities in Mice with Paracoccidioidomycosis: A Sequential Study Comparing High Resolution Computed Tomography and Pathologic Findings 
Human paracoccidioidomycosis (PCM) is an endemic fungal disease of pulmonary origin. Follow-up of pulmonary lesions by image studies in an experimental model of PCM has not been previously attempted. This study focuses on defining patterns, topography and intensity of lung lesions in experimentally infected PCM mice by means of a comparative analysis between High Resolution Computed Tomography (HRCT) and histopathologic parameters.
Male BALB/c mice were intranasally inoculated with 3×106 Paracoccidioides brasiliensis (Pb) conidia (n = 50) or PBS (n = 50). HRCT was done every four weeks to determine pulmonary lesions, quantify lung density, reconstruct and quantify lung air structure. Lungs were also analyzed by histopathology and histomorphometry.
Three different patterns of lesions were evidenced by HRCT and histopathology, as follows: nodular-diffuse, confluent and pseudo-tumoral. The lesions were mainly located around the hilus and affected more frequently the left lung. At the 4th week post-challenge HRCT showed that 80% of the Pb-infected mice had peri-bronchial consolidations associated with a significant increase in upper lung density when compared with controls, (−263±25 vs. −422±10 HU, p<0.001). After the 8th and 12th weeks, consolidation had progressed involving also the middle regions. Histopathology revealed that consolidation as assessed by HRCT was equivalent histologically to a confluent granulomatous reaction, while nodules corresponded to individual compact granulomas. At the 16th week of infection, confluent granulomas formed pseudotumoral masses that obstructed large bronchi. Discrete focal fibrosis was visible gradually around granulomas, but this finding was only evident by histopathology.
This study demonstrated that conventional HRCT is a useful tool for evaluation and quantification of pulmonary damage occurring in experimental mouse PCM. The experimental design used decreases the need to sacrifice a large number of animals, and serves to monitor treatment efficacy by means of a more rational approach to the study of human lung disease.
Author Summary
Paracoccidioidomycosis (PCM) is a fungal infection caused by the dimorphic fungus Paracoccidioides brasiliensis. It occurs preferentially in rural workers in whom the disease is severe and may cause incapacitating pulmonary sequelae. Assessment of disease progression and treatment outcome normally includes chest x-rays or CT studies. Existing experimental PCM models have focused on several aspects, but none has done a radiologic or image follow-up evaluation of pulmonary lesions considered as the fungus primary target. In this study, the lungs of mice infected with fungal conidia were studied sequentially during the chronic stage of their experimental mycosis by noninvasive high resolution medical computed tomography, and at time of sacrifice, also by histopathology to characterize pulmonary abnormalities. Three basic lung lesion patterns were revealed by both techniques: nodular-diffuse, confluent and pseudo-tumoral which were located mainly around the hilus thus accurately reflecting the situation in human patients. The experimental design of this study decreases the need to sacrifice a large number of animals, and serves to monitor treatment efficacy by means of a more rational approach to the study of human pulmonary diseases. The findings we are reporting open new avenues for experimental research, increase our understanding of the mycosis pathogenesis and consequently have repercussions in patients' care.
PMCID: PMC2894136  PMID: 20614019
8.  Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis 
Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.
Author Summary
Paracoccidioidomycosis (PCM) is the predominant systemic mycosis in Latin America causing half of the total deaths among systemic fungal infectious diseases in Brazil. Chemotherapy is the standard treatment, but the long time required, severe cases of immunosuppression and frequent relapses indicate that additional methods should be introduced such as immunotherapy combined with antifungal drugs. Previously, the protective activity of P10, a peptide derived from the major diagnostic antigen gp43, was demonstrated, alone or combined with chemotherapy. P10 elicited a vigorous IFN-γ mediated Th-1 immune response. Presently, the reduction of fungal load, and even sterilization, was attempted using a specific DNA vaccine encoding P10. Plasmid pcDNA3 expression vector with P10 insert was tested as a vaccine in intratracheally infected BALB/c and B10.A mice. Our results showed that vaccination with pP10 induced a significant reduction of the fungal burden in the lung. Co-vaccination of pP10 with a plasmid encoding mouse IL-12 proved to be even more effective in the elimination of the fungus with virtual sterilization in a long term infection and treatment assay system. The data suggest that immunization with these plasmids, without the need of an adjuvant, could be used in the prevention and treatment of PCM in human patients.
PMCID: PMC3289603  PMID: 22389734
9.  Additive Effect of rPb27 Immunization and Chemotherapy in Experimental Paracoccidioidomycosis 
PLoS ONE  2011;6(3):e17885.
Paracoccidioidomycosis, PCM, the major systemic mycosis in Latin America, is caused by the termally dimorphic fungus Paracoccidioides brasiliensis and requires extended periods of chemotherapy with a significant frequency of relapsing disease. The search for new alternatives of treatment is necessary. rPb27 is an antigenic protein from P. brasiliensis that already showed a significant protective activity as a vaccine for PCM in experimental models. The cDNA of rPb27 was subcloned into a pET-DEST 42 plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. Immunization with this recombinant protein and chemotherapy were used together in an attempt to improve treatment of PCM. For this, BALB/c mice were challenged with pathogenic P. brasiliensis strain and after immunized with rPb27, in the presence of Corynebacterium parvum and Al(OH)3, some groups were also treated with fluconazole. After 40 days of treatment, the combined drug/rPb27 administration controlled PCM in the liver and spleen, with long lasting protection, and largely preserved tissues structures of these organs. Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions. At the same time, the levels of TNF-α were higher than infected-only mice. Moreover, significant levels of anti-rPb27 specific IgG1, IgG2a and IgG2b isotypes were detected in the sera of mice immunized with rPb27 fluconazole treated or not. These results showed an additive protective effect of rPb27 immunization and chemotherapy, suggesting that an rPb27-based vaccine can be used to enhance PCM antifungal treatment.
PMCID: PMC3053394  PMID: 21423771
10.  Detrimental Effect of Fungal 60-kDa Heat Shock Protein on Experimental Paracoccidioides brasiliensis Infection 
PLoS ONE  2016;11(9):e0162486.
The genus Paracoccidioides comprises species of dimorphic fungi that cause paracoccidioidomycosis (PCM), a systemic disease prevalent in Latin America. Here, we investigated whether administration of native 60-kDa heat shock protein of P. brasiliensis (nPbHsp60) or its recombinant counterpart (rPbHsp60) affected the course of experimental PCM. Mice were subcutaneously injected with nPbHsp60 or rPbHsp60 emulsified in complete’s Freund Adjuvant (CFA) at three weeks after intravenous injection of P. brasiliensis yeasts. Infected control mice were injected with CFA or isotonic saline solution alone. Thirty days after the nPbHsp60 or rPbHsp60 administration, mice showed remarkably increased fungal load, tissue inflammation, and granulomas in the lungs, liver, and spleen compared with control mice. Further, rPbHsp60 treatment (i) decreased the known protective effect of CFA against PCM and (ii) increased the concentrations of IL-17, TNF-α, IL-12, IFN-γ, IL-4, IL-10, and TGF-β in the lungs. Together, our results indicated that PbHsp60 induced a harmful immune response, exacerbated inflammation, and promoted fungal dissemination. Therefore, we propose that PbHsp60 contributes to the fungal pathogenesis.
PMCID: PMC5012565  PMID: 27598463
11.  Low-level Laser Therapy to the Mouse Femur Enhances the Fungicidal Response of Neutrophils against Paracoccidioides brasiliensis 
PLoS Neglected Tropical Diseases  2015;9(2):e0003541.
Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.
Author Summary
PCM triggers a typical granulomatous inflammatory reaction with PMN playing a major role; these inflammatory cells are crucial in the initial stages of PCM, participating in the innate immune reaction and also directing the acquired immune response in the later stages. In some PCM patients, these immune mechanisms are insufficient to eradicate the infection, and need to be boosted with antifungal drugs that have to be administered for long periods and can show serious side-effects. We aimed to develop a novel and safe way to activate PMN through low-level laser irradiation of the bone marrow in the mouse femoral medulla. LLLT increased PMN viability and activation, shown by a significantly greater production of protein and ROS, as well as a higher fungicidal capacity; PMN even retained their higher metabolic activity and fungicidal ability after a second exposure to the pathogenic fungus in vitro. This is the first time that LLLT has been shown to increase the immune response against a fungal infection, and could be a promising and safe technique to be used with antifungal drugs in PCM.
PMCID: PMC4326423  PMID: 25675431
12.  Characterization of gp70 and Anti-gp70 Monoclonal Antibodies in Paracoccidioides brasiliensis Pathogenesis  
Infection and Immunity  2003;71(11):6534-6542.
Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis whose agent is Paracoccidioides brasiliensis. In the culture supernatant, the fungus expresses glycoproteins of from 13 to 148 kDa. A cell surface glycoprotein of 43 kDa is the major antigenic component of P. brasiliensis. Another expressed glycoprotein, gp70, is recognized by 96% of sera from PCM patients and is able to induce lymphoproliferation. Since, little is known about this glycoprotein, we produced monoclonal antibodies (MAbs) against gp70 to isolate the molecule from total fungus extracts and to investigate its possible role in the pathogenesis of PCM. Using these MAbs, it was observed by confocal microscopy that gp70 is located mainly in the intracellular compartment of the fungus, although it was also detected in the culture supernatant. Based on observations showing that gp43 has a down-regulatory effect on mouse peritoneal macrophages, we tested the effects of gp70 on their phagocytic ability. Purified gp70 was able to inhibit the activity of macrophages through the mannose receptors and also through the Fc receptors; the latter effect was not observed with gp43. gp70 inhibits NO and H2O2 liberation by peritoneal macrophages in vitro, as does gp43. Results obtained with gp43 led us to hypothesize that gp70 could act as an escape mechanism for fungal establishment in primary infections. To corroborate this hypothesis, we analyzed the effect of passive immunization of mice during infection with P. brasiliensis using anti-gp70 MAbs. This treatment almost completely abolished granuloma formation in the lungs, suggesting that the protein facilitates fungal establishment and progression of lesions in primary infection.
PMCID: PMC219562  PMID: 14573675
13.  Therapeutic Administration of Recombinant Paracoccin Confers Protection against Paracoccidioides brasiliensis Infection: Involvement of TLRs 
Paracoccin (PCN) is an N-acetylglucosamine-binding lectin from the human pathogenic fungus Paracoccidioides brasiliensis. Recombinant PCN (rPCN) induces a T helper (Th) 1 immune response when prophylactically administered to BALB/c mice, protecting them against subsequent challenge with P. brasiliensis. In this study, we investigated the therapeutic effect of rPCN in experimental paracoccidioidomycosis (PCM) and the mechanism accounting for its beneficial action.
Methodology/Principal Findings
Four distinct regimens of rPCN administration were assayed to identify which was the most protective, relative to vehicle administration. In all rPCN-treated mice, pulmonary granulomas were less numerous and more compact. Moreover, fewer colony-forming units were recovered from the lungs of rPCN-treated mice. Although all therapeutic regimens of rPCN were protective, maximal efficacy was obtained with two subcutaneous injections of 0.5 µg rPCN at 3 and 10 days after infection. The rPCN treatment was also associated with higher pulmonary levels of IL-12, IFN-γ, TNF-α, nitric oxide (NO), and IL-10, without IL-4 augmentation. Encouraged by the pulmonary cytokine profile of treated mice and by the fact that in vitro rPCN-stimulated macrophages released high levels of IL-12, we investigated the interaction of rPCN with Toll-like receptors (TLRs). Using a reporter assay in transfected HEK293T cells, we verified that rPCN activated TLR2 and TLR4. The activation occurred independently of TLR2 heterodimerization with TLR1 or TLR6 and did not require the presence of the CD14 or CD36 co-receptors. The interaction between rPCN and TLR2 depended on carbohydrate recognition because it was affected by mutation of the receptor's N-glycosylation sites. The fourth TLR2 N-glycan was especially critical for the rPCN-TLR2 interaction.
Based on our results, we propose that PCN acts as a TLR agonist. PCN binds to N-glycans on TLRs, triggers regulated Th1 immunity, and exerts a therapeutic effect against P. brasiliensis infection.
Author Summary
Paracoccidioides brasiliensis is a pathogenic fungus that causes paracoccidioidomycosis (PCM) in humans, a debilitating fungal infection that mainly affects the lungs and is widespread in Latin America. Paracoccin (PCN) is a sugar-binding protein produced by this fungus. Previous studies have shown that PCN contributes to the colonization of host tissues by the fungus and induces the production of inflammatory factors (i.e., cytokines and nitric oxide) by immune cells such as macrophages. Here we investigated the therapeutic efficacy of recombinant PCN (rPCN) on the course of P. brasiliensis infection in mice. Histopathological analysis of lungs of animals treated with rPCN showed much lower inflammation in comparison to untreated, control mice. In addition, fewer infective P. brasiliensis yeast forms were recovered from the lung of rPCN-treated animals than from that of control animals. Administration of rPCN was associated with a profile of pro- and anti-inflammatory factors in the lung that was conducive to host protection. These effects were associated with PCN binding to sugar chains linked to innate immunity receptors, namely Toll-like receptors 2 and 4. These findings reveal a mechanism by which rPCN confers protection against PCM.
PMCID: PMC4256291  PMID: 25474158
14.  Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin▿  
Infection and Immunity  2009;77(4):1700-1707.
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4+ T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.
PMCID: PMC2663153  PMID: 19204092
15.  Characterization of the Paracoccidioides Hypoxia Response Reveals New Insights into Pathogenesis Mechanisms of This Important Human Pathogenic Fungus 
PLoS Neglected Tropical Diseases  2015;9(12):e0004282.
Hypoxic microenvironments are generated during fungal infection. It has been described that to survive in the human host, fungi must also tolerate and overcome in vivo microenvironmental stress conditions including low oxygen tension; however nothing is known how Paracoccidioides species respond to hypoxia. The genus Paracoccidioides comprises human thermal dimorphic fungi and are causative agents of paracoccidioidomycosis (PCM), an important mycosis in Latin America.
Methodology/Principal Findings
In this work, a detailed hypoxia characterization was performed in Paracoccidioides. Using NanoUPLC-MSE proteomic approach, we obtained a total of 288 proteins differentially regulated in 12 and 24 h of hypoxia, providing a global view of metabolic changes during this stress. In addition, a functional characterization of the homologue to the most important molecule involved in hypoxia responses in other fungi, the SREBP (sterol regulatory element binding protein) was performed. We observed that Paracoccidioides species have a functional homologue of SREBP, named here as SrbA, detected by using a heterologous genetic approach in the srbA null mutant in Aspergillus fumigatus. Paracoccidioides srbA (PbsrbA), in addition to involvement in hypoxia, is probable involved in iron adaptation and azole drug resistance responses.
In this study, the hypoxia was characterized in Paracoccidioides. The first results can be important for a better understanding of the fungal adaptation to the host and improve the arsenal of molecules for the development of alternative treatment options in future, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi.
Author Summary
The genus Paracoccidioides is composed of species that are causative agents of paracoccidioidomycosis (PCM), a neglected human granulomatous mycosis, endemic in Latin America. To survive in the human host, fungi must tolerate and overcome in vivo micro environmental stress conditions, including low oxygen levels. Paracoccidioides spp. depicts differential responses to several stresses such as iron/zinc deprivation, oxidative and nitrosative stresses and carbon starvation. In addition, Paracoccidioides yeast cells recovered from liver of infected mice demonstrated adaptability to the host conditions. Mechanisms by which fungi sense oxygen levels have been characterized, although this is the first description in Paracoccidioides spp. Little is known about hypoxia in thermally dimorphic fungi and nothing has been studied in Paracoccidioides genus, one of the representatives of this group of pathogens. A detailed characterization of the hypoxia responses was performed using proteomic and heterologous genetics approaches. Paracoccidioides genus have a functional homologue of the key regulator of hypoxia adaptation in fungi, SrbA, a SREBP (sterol regulatory element binding protein) orthologue. The proteome during hypoxia provided a global view of metabolic changes during this stress and species of the Paracoccidioides genus have a functional SrbA. Our study provides a better understanding of the fungal adaptation to the host and it can improve the arsenal of molecules for the development of alternative treatment options to paracoccidioidomycosis, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi.
PMCID: PMC4686304  PMID: 26659387
16.  Loss- and Gain-of-Function Approaches Indicate a Dual Role Exerted by Regulatory T Cells in Pulmonary Paracoccidioidomycosis 
PLoS Neglected Tropical Diseases  2015;9(10):e0004189.
Paracoccidioidomycosis (PCM), is a pulmonary fungal disease whose severity depends on the adequate development of T cell immunity. Although regulatory T (Treg) cells were shown to control immunity against PCM, deleterious or protective effects were described in different experimental settings. To clarify the function of Treg cells in pulmonary PCM, loss-and gain-of-function approaches were performed with Foxp3GFP knock-in mice and immunodeficient Rag1-/- mice, respectively, which were intratracheally infected with 106 yeast cells. The activity of Foxp3-expressing Treg cells in pulmonary PCM was determined in Foxp3GFP transgenic mice. First, it was verified that natural Treg cells migrate to the lungs of infected mice, where they become activated. Depletion of Treg cells led to reduced fungal load, diminished pathogen dissemination and increased Th1/Th2/Th17 immunity. Further, adoptive transfer of diverse T cell subsets to Rag1-/- mice subsequently infected by the pulmonary route demonstrated that isolated CD4+Foxp3+ Treg cells were able to confer some degree of immunoprotection and that CD4+Foxp3- T cells alone reduced fungal growth and enhanced T cell immunity, but induced vigorous inflammatory reactions in the lungs. Nevertheless, transfer of Treg cells combined with CD4+Foxp3- T cells generated more efficient and balanced immune Th1/Th2/Th17 responses able to limit pathogen growth and excessive tissue inflammation, leading to regressive disease and increased survival rates. Altogether, these loss- and gain-of-function approaches allow us to clearly demonstrate the dual role of Treg cells in pulmonary PCM, their deleterious effects by impairing T cell immunity and pathogen eradication, and their protective role by suppressing exacerbated tissue inflammation.
Author Summary
Paracoccidioidomycosis (PCM), the most relevant deep mycosis in Latin America, is caused by the fungus Paracoccidioides brasiliensis. The involvement of regulatory T (Treg) cells in the immunity against PCM has been previously demonstrated, however its underlying mechanisms are still to be elucidated. Using Foxp3GFP transgenic mice, we found that natural Tregs migrate to the lungs of infected mice, where they become activated. Depletion of Treg cells led to reduced fungal load, diminished pathogen dissemination and increased Th1/Th2/Th17 immunity. Further, after performing adoptive transfer experiments using immunodeficient Rag1-/- mice, we demonstrated that isolated CD4+Foxp3+ Treg cells were able to confer some degree of immunoprotection and that CD4+Foxp3- T cells alone contributed to fungal elimination and enhanced T cell immunity, but induced vigorous inflammatory reactions in the lungs. Nevertheless, transfer of Tregs combined with CD4+Foxp3- T cells generated more efficient and balanced immune responses able to limit pathogen growth and excessive tissue inflammation, leading to regressive disease and higher survival rates. Altogether, these loss- and gain-of-function approaches allow us to propose a dual role for Treg cells in pulmonary PCM, their deleterious effects by impairing T cell immunity and pathogen eradication, and their protective role by suppressing exacerbated tissue inflammation.
PMCID: PMC4626087  PMID: 26512987
17.  In Pulmonary Paracoccidioidomycosis IL-10 Deficiency Leads to Increased Immunity and Regressive Infection without Enhancing Tissue Pathology 
Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM.
Methodology/Principal Findings
Wild type (WT) and IL-10−/− C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10−/− mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10−/− and WT mice were i.t. infected with 1×106 Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10−/− mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10−/− mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4+ and CD8+ T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10−/− mice.
Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.
Author Summary
Paracoccidioidomycosis, the most important deep mycosis from Latin America, is acquired by inhalation of fungal spores. The pulmonary infection can remain as a quiescent infection or evolve to overt, life-threatening disease. Immunoprotection is mainly mediated by Th1 lymphocytes secreting IFN- γ, the most important macrophage activating cytokine. It is well established that the severe forms of infection are associated with elevated production of anti-inflammatory or suppressive cytokines such as IL-10. However, direct approaches investigating the role of this cytokine in pulmonary paracoccidioidomycosis were never employed. This led us to investigate the innate and adaptive aspects of immunity in pulmonary paracoccidioidomycosis using IL-10-deficient mice in comparison with their IL-10-normal counterparts. We verified that IL-10 absence leads to a regressive disease, resulting in reduced mortality rates of infected mice. This better disease outcome was associated with precocious and enhanced mechanisms of innate and adaptive immunity that allow the control of fungal growth without excessive inflammatory reactions and harmful tissue pathology. These evidences on the detrimental effects of IL-10 to pulmonary paracoccidioidomycosis suggest that therapeutic measures aimed to control IL-10 production or activity could exert a protective effect to this severe fungal pathology.
PMCID: PMC3812093  PMID: 24205424
18.  Expression of Antibodies Directed to Paracoccidioides brasiliensis Glycosphingolipids during the Course of Paracoccidioidomycosis Treatment▿  
Clinical and Vaccine Immunology  2006;14(2):150-156.
Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfβ1-6(Manα1-3)Manβ1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM.
PMCID: PMC1797792  PMID: 17135452
Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There are few reports in the literature about the disease damages during pregnancy and the consequences to the fetuses and breeding. This study evaluated the implications of PCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats were submitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 days after the infection date. Immediately after birth, rats and neonates were sacrificed to obtain organs for standard histological examination, morphometric analysis, fungi recovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were no stillbirths or miscarriages, however, the fetuses from infected pregnant rats had lower body and organ weight but the fertility rate was 100%. The largest number of CFU was recovered from the organ of pregnant rats, the pathological examination revealed more severe infection in the same group, further on the largest number of granulomas and fungal field. It can be concluded that the PCM was more severe in the group of pregnant rats, with implications to the weight of offspring.
PMCID: PMC4727139  PMID: 27049707
Paracoccidioidomycosis; Pregnant; Paracoccidioides brasiliensis
20.  Environmental Mapping of Paracoccidioides spp. in Brazil Reveals New Clues into Genetic Diversity, Biogeography and Wild Host Association 
PLoS Neglected Tropical Diseases  2016;10(4):e0004606.
Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques.
Methods/Principal Findings
Aerosol (n = 16) and soil (n = 34) samples from armadillo burrows, as well as armadillos (n = 7) were collected in endemic and non-endemic areas of PCM in the Southeastern, Midwestern and Northern regions of Brazil. Both P. brasiliensis and P. lutzii were detected in soil (67.5%) and aerosols (81%) by PCR of Internal Transcribed Spacer (ITS) region (60%), and also by in situ hybridization (83%). Fungal isolation from armadillo tissues was not possible. Sequences from both species of P. brasiliensis and P. lutzii were detected in all regions. In addition, we identified genetic Paracoccidioides variants in soil and aerosol samples which have never been reported before in clinical or armadillo samples, suggesting greater genetic variability in the environment than in vertebrate hosts.
Data may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts.
Author Summary
Paracoccidioides brasiliensis and Paracoccidioides lutzii are the fungal species responsible for one of the most important mycoses of Latin America, Paracoccidioidomycosis (PCM). These fungi can grow in soil from forests, deforested areas, sugarcane, coffee, and rice plantations, as well as pasturelands, and they are strongly associated to armadillo burrows, which can explain their frequent isolation from this mammal’s tissues. The environmental detection of these pathogens in endemic and non-endemic areas of PCM is important for mapping risk areas, as well as for understanding the infection ability and clinical manifestations of these fungi. These pieces of information are not provided by isolates obtained from human patients, because these fungi have a long latency period and the human host can migrate, leading to a misinterpretation of the actual geographic distribution of these pathogens. By using two different molecular methodologies (Nested PCR and in situ fluorescence), we detected both species of P. brasiliensis and P. lutzii in soil and in aerosol samples, even in areas where PCM is only associated to one of these two species. These data might indicate different habitat maintenance strategies between the species, which means that the infection ability may change according to the climatic and soil conditions. Despite contributing new information about the ecology of these important fungal pathogens, our molecular approach for the environmental detection of Paracoccidioides species may also be applied for their detection and differentiation in clinical samples, improving the diagnosis of this important systemic mycosis.
PMCID: PMC4821608  PMID: 27045486
21.  The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes▿ 
Eukaryotic Cell  2011;10(3):343-351.
Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.
PMCID: PMC3067469  PMID: 21216942
22.  Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis 
PLoS ONE  2012;7(9):e44792.
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.
PMCID: PMC3443090  PMID: 23024763
23.  Immunological response to cell-free antigens of Paracoccidioides brasiliensis: relationship with clinical forms of paracoccidioidomycosis. 
Journal of Clinical Microbiology  1993;31(3):671-676.
Sera from patients with the acute (AF) and chronic (CF) forms of paracoccidioidomycosis (PCM) were tested against Paracoccidioides brasiliensis cell-free antigens by Western blot (immunoblot). The CFA preparation contained components ranging in molecular mass from 18 to 102 kDa. The immunoglobulin G (IgG) reactivity profiles were similar for patients with both forms of the disease, and the 43-kDa component was recognized by 100% of the sera. IgM antibodies from the AF- and the CF-PCM sera recognized 21 and 20 components, respectively, the AF-PCM sera reacting preferentially with components with molecular masses above 50 kDa. None of the AF-PCM sera (IgM) reacted with the 43-kDa component, and only 10% of the CF-PCM sera recognized this molecule. The IgA response was more significant in the CF-PCM group than in the AF-PCM group, and the 43- and 74-kDa components were the most reactive ones (about 40% each). Our results showed that the cell-free antigen preparation is very appropriate for the immunoblotting analysis of PCM sera, and they also showed that the detection of IgG anti-gp43 is the best marker for the diagnosis and the following up of patients with the acute or the chronic form of the disease.
PMCID: PMC262839  PMID: 8458961
24.  NLRP3 Inflammasome Activation by Paracoccidioides brasiliensis 
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1β that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1β in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL-1 receptor, we demonstrated that IL-1β signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus.
Author Summary
Paracoccidioidomycosis is a systemic disease that has an important mortality and morbidity impact in Latin America. It mainly affects rural workers of Argentina, Colombia, Venezuela and Brazil. Upon host infection, one of the most important aspects that contribute to the disease outcome is the initial interaction of the Paracoccidioides brasiliensis fungus with the phagocytic cells and the induction of the inflammatory process. Among several inflammatory mediators, the cytokine interleukin-1β is of pivotal importance in this complex process. Here, we demonstrate that P. brasiliensis is sensed by the NLRP3 inflammasome, a cytoplasmatic multiprotein complex that lead to the processing and secretion of IL-1β. In addition, we described the intracellular perturbations that may be associated with NLRP3 activation such as potassium efflux, production of reactive oxygen species, and lysosomal damage. Finally, our work provides evidence for the protective role of IL-1β during fungal infection of murine macrophages.
PMCID: PMC3855149  PMID: 24340123
25.  Structural and Topographic Dynamics of Pulmonary Histopathology and Local Cytokine Profiles in Paracoccidioides brasiliensis Conidia-Infected Mice 
Paracoccidioidomycosis (PCM), an endemic systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), usually results in severe lung damage in patients.
Methods and Findings
Considering the difficulties to sequentially study the infection in humans, this work was done in mice inoculated intranasally with infective Pb-conidia. Lungs of control and Pb-infected mice were studied after 2-hours, 4, 8, 12 and 16-weeks post-infection (p.i) in order to define histopathologic patterns of pulmonary lesions, multiplex-cytokine profiles and their dynamics during the course of this mycosis. Besides the nodular/granulomatous lesions previously informed, results revealed additional non-formerly described lung abnormalities, such as periarterial sheath inflammation and pseudotumoral masses. The following chronologic stages occurring during the course of the experimental infection were defined: Stage one (2-hours p.i): mild septal infiltration composed by neutrophils and macrophages accompanied by an intense “cytokine burst” represented by significant increases in IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL12p70, IL-13, IL-17, Eotaxin, G-CSF, MCP1, MIP1α, GM-CSF, IFN-γ, MIP1β and TNFα levels. Stage two (4-weeks p.i): presence of nodules, evidence of incipient periarterial- and intense but disperse parenchymal- inflammation, abnormalities that continued to be accompanied by hyper-secretion of those cytokines and chemokines mentioned in the first stage of infection. Stages three and four (8 and 12-weeks p.i.): fungal proliferation, inflammation and collagenesis reached their highest intensity with particular involvement of the periarterial space. Paradoxically, lung cytokines and chemokines were down-regulated with significant decreases in IL-2,IL-3,IL-5,IL-9,IL-13,IL-15,GM-CSF,IFN-γ,MIP1β and TNFα. Stage five (16-weeks p.i.): inflammation decreased becoming limited to the pseudotumoral masses and was accompanied by a “silent” cytokine response, except for PDGF, MIG, RANTES and IL12p40 which remained up-regulated for the duration of the experiment.
Results of this study identified both classic and novel patterns corresponding to histopathologic and immunologic responses occurring during the course of experimental PCM.
Author Summary
Paracoccidioidomycosis (PCM), an endemic fungal infection of pulmonary origin resulting in severe disseminated disease, occurs in rural areas of most South American countries and presents several clinical forms. The infection is acquired by inhalation of specific fungal propagules, called conidia. Considering the difficulties encountered when studying the infection in humans, this work was done in mice infected by inhalation of infective fungal conidia thus mimicking the human natural infection. The lungs of mice were sequentially studied by histopathological and multiplex cytokine methods from 2 h to 16 weeks after infection to verify the course of the disease. The mycosis presented different morphologic aspects during the course of time, affecting several pulmonary compartments. Otherwise and based on the analysis of 30 cytokines, the immune response also showed heterogeneous responses, which were up or down regulated depending on the time of infection. By recognizing the different stages that correspond to the evolution of pulmonary lesions, the severity (benign, chronic or fibrotic) of the disease could be predicted and the probable prognosis of the illness be inferred.
PMCID: PMC3134433  PMID: 21765962

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