Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development.

Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.

Antibody- Based Immunotherapy Combined With Antimycotic Drug TMP- SMX to Treat Infection With Paracoccidioides brasiliensis.

Monoclonal antibodies (mAbs) are promising alternatives to treat infectious diseases, especially given their potential for applications in combination therapies with antimicrobial drugs to enhance the antifungal efficacy. Protection mediated by mAbs used to treat experimental paracoccidioidomycosis (PCM) has been demonstrated previously. Our aim in the present work was to characterize a monoclonal antibody (mAbF1.4) raised against a cell wall glycoconjugate fraction of Paracoccidioides spp. and to analyze its efficacy combined with trimethoprim-sulfamethoxazole (TMP/SMX) as treatment for experimental PCM. We demonstrated that the epitope recognized by mAbF1.4 is consistent with branched glucose residues present on a cell wall ß-glucan polymer. In vitro, mAbF1.4 increased the phagocytic capacity and nitric oxide concentration induced by the macrophage cell line J774.1A, and this resulted in a significant reduction in the viability of the opsonophagocytized yeasts. In vivo, we detected a significant reduction in pulmonary fungal burdens of mice treated with mAbF1.4 in association with TMP/SMX, which correlated with increased pulmonary concentrations (determined by ELISA) of IFN- Î³, TNF-α, IL-10 and IL-17. In parallel, we observed a decrease in IL-4, suggesting that the treatment was associated with a mixed Th1-Th17 type immune response. Histopathology of lung segments from mice receiving the combination therapy showed a significant reduction in granulomas, which were well-defined, and improved maintenance of lung architecture. These findings demonstrate that mAbF1.4 + TMP/SMX therapy is a promising approach to combat PCM as well as decrease disease sequelae and highlights the potential benefits of immune mediators in PCM combined immunotherapy.

Protective Response in Experimental Paracoccidioidomycosis Elicited by Extracellular Vesicles Containing Antigens of Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a systemic disease caused by Paracoccidioides spp. PCM is endemic in Latin America and most cases are registered in Brazil. This mycosis affects mainly the lungs, but can also spread to other tissues and organs, including the liver. Several approaches have been investigated to improve treatment effectiveness and protection against the disease. Extracellular vesicles (EVs) are good antigen delivery vehicles. The present work aims to investigate the use of EVs derived from Paracoccidioides brasiliensis as an immunization tool in a murine model of PCM. For this, male C57BL/6 were immunized with two doses of EVs plus adjuvant and then infected with P. brasiliensis. EV immunization induced IgM and IgG in vivo and cytokine production by splenocytes ex vivo. Further, immunization with EVs had a positive effect on mice infected with P. brasiliensis, as it induced activated T lymphocytes and NKT cell mobilization to the infected lungs, improved production of proinflammatory cytokines and the histopathological profile, and reduced fungal burden. Therefore, the present study shows a new role for P. brasiliensis EVs in the presence of adjuvant as modulators of the host immune system, suggesting their utility as immunizing agents.

Invariant Natural Killer T Cells as Key Players in Host Resistance against Paracoccidioides brasiliensis.

Invariant Natural Killer T (iNKT) cells are key players in the immunity to several pathogens; however, their involvement in the resistance to Paracoccidioides brasiliensis infection remains unknown. Using splenocytes from CD1d (CD1d-/-) and iNKT-deficient (Jα18-/-) mice, we found that iNKT cells are the innate source of IFN-γ after P. brasiliensis infection and are required to potentiate macrophage oxidative burst and control fungal growth. To determine whether iNKT cells contribute in vivo to host resistance against P. brasiliensis infection, we infected intratracheally wild-type and Jα18-/- C57BL/6 mouse strains with the virulent Pb18 isolate. iNKT cell deficiency impaired the airway acute inflammatory response, resulting in decreased airway neutrophilia and reduced IFN-γ, KC, and nitric oxide (NO) production. The deficient innate immune response of Jα18-/- mice to Pb18 infection resulted in increased fungal burden in the lungs and spleen. Besides, the activation of iNKT cells in vivo by administration of the exogenous iNKT ligand α-galactosylceramide (α-GalCer) improved host resistance to P. brasiliensis infection. Although the mechanisms responsible for this phenomenon remain to be clarified, α-GalCer treatment boosted the local inflammatory response and reduced pulmonary fungal burden. In conclusion, our study is the first evidence that iNKT cells are important for the protective immunity to P. brasiliensis infection and their activation by an exogenous ligand is sufficient to improve the host resistance to this fungal infection.

Paracoccidioidomycosis due to P lutzii: The importance of neutrophil/lymphocyte ratio in the symptomatic and asymptomatic phases in severe cases.

BACKGROUND: PCM is a neglected systemic mycosis endemic in Brazil. The middle-west region of Brazil has shown the highest number of PCM by Paracoccidioides lutzii (P lutzii) cases. Differentiating cases of severe PCM from non-severe ones should be a concern at the bedside. Diagnosis of severe PCM by P lutzii is based on the subjectivity of clinical manifestations, which can result in a delay in starting its treatment and, consequently evolution to severe sequelae. There is not laboratory biomarker available to support the early diagnosis of severe PCM that is feasible for all the realities that coexist in Brazil. OBJECTIVES: The aim of this study was to investigate the usefulness of laboratory biomarkers as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and neutrophil/lymphocyte ratio (NLR) in the diagnosis of severe PCM. PATIENTS/METHODS: ESR, CRP and NLR were analysed for 44 patients with PCM by P lutzii and a Receiver Operation Characteristic (ROC) curve were generated to identify the NLR cut-off point and point out the presence of severe PCM. RESULTS: Sixteen (36.4%) had severe PCM and 28 (63.6%) had non-severe PCM. The mean NLR was higher and statistically significant among patients with severe PCM than among those with non-severe PCM. The area under the ROC curve was 0.859 for the diagnosis of severe PCM. The cut-off point for NLR for the diagnosis of severe PCM was 3.318 (sensitivity of 100%, specificity of 77%). CONCLUSIONS: According to results, it is plausible to conclude that NLR represents a potential biomarker for the diagnosis of severe PCM.

An update on the occurrence of Paracoccidioides species in the Midwest region, Brazil: Molecular epidemiology, clinical aspects and serological profile of patients from Mato Grosso do Sul State.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii. METHODOLOGY AND MAIN FINDINGS: This study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome. CONCLUSIONS: This study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented.

Zymosan enhances in vitro phagocyte function and the immune response of mice infected with Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the major etiologic agent of Paracoccidioidomycosis (PCM), the most frequent human deep mycosis in Latin America. It is proposed that masking of ß-glucan in P. brasiliensis cell wall is a critical virulence factor that contributes to the development of a chronic disease characterized by a long period of treatment, which is usually toxic. In this context, the search for immunomodulatory agents for therapeutic purposes is highly desirable. One strategy is to use pattern recognition receptors (PRRs) ligands to stimulate the immune response mediated by phagocytes. Here, we sought to evaluate if Zymosan, a ß-glucan-containing ligand of the PRRs Dectin-1/TLR-2, would enhance phagocyte function and the immune response of mice challenged with P. brasiliensis. Dendritic cells (DCs) infected with P. brasiliensis and treated with Zymosan showed improved secretion of several proinflammatory cytokines and expression of maturation markers. In addition, when cocultured with splenic lymphocytes, these cells induced the production of a potential protective type 1 and 17 cytokine patterns. In macrophages, Zymosan ensued a significant fungicidal activity associated with nitric oxide production and phagolysosome acidification. Importantly, we observed a protective effect of Zymosan-primed DCs delivered intranasally in experimental pulmonary PCM. Overall, our findings support the potential use of ß-glucan-containing compounds such as Zymosan as an alternative or complementary antifungal therapy. LAY SUMMARY: We report for the first time that Paracoccidioides brasiliensis-infected phagocytes treated with Zymosan (cell wall extract from bakers' yeast) show enhanced cytokine production, maturation, and fungal killing. Also, Zymosan-primed phagocytes induce a protective immune response in infected mice.

Paracoccidioides brasiliensis activates mesenchymal stem cells through TLR2, TLR4, and Dectin-1.

Numerous researchers have described the potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of various infectious and inflammatory diseases. However, contrary to what has been reported, the transplantation of BM-MSCs in a mouse model of Paracoccidioides brasiliensis-induced pulmonary fibrosis exacerbated the inflammatory process and fibrosis, worsening the course of the infection. The aim of this work was to determine whether P. brasiliensis exerts an immunomodulatory effect on BM-MSCs. The results indicate that P. brasiliensis can activate BM-MSCs through a mechanism dependent on TLR2, TLR4 and Dectin-1. In addition, it was found that these fungal cells can adhere and internalize within BM-MSCs. Nonetheless, this process did not affect the survival of the fungus and on the contrary, triggered the expression of inflammatory mediators such as IL-6, IL-17, TNF-α, and TGF-ß. The present findings correlate with the loss of a fungicidal effect and poor control of the fungus, evidenced by the count of the colony-forming units. Previously reported in vivo results are thus confirmed, showing that P. brasiliensis induces an inflammatory profile in BM-MSCs when producing pro-inflammatory molecules that amplify such response. Numerous researchers have described the potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of various infectious and inflammatory diseases. However, contrary to what has been reported, the transplantation of BM-MSCs in a mouse model of Paracoccidioides brasiliensis-induced pulmonary fibrosis exacerbated the inflammatory process and fibrosis, worsening the course of the infection. The aim of this work was to determine whether P. brasiliensis exerts an immunomodulatory effect on BM-MSCs. The results indicate that P. brasiliensis can activate BM-MSCs through a mechanism dependent on TLR2, TLR4 and Dectin-1. In addition, it was found that these fungal cells can adhere and internalize within BM-MSCs. Nonetheless, this process did not affect the survival of the fungus and on the contrary, triggered the expression of inflammatory mediators such as IL-6, IL-17, TNF-α, and TGF-ß. The present findings correlate with the loss of a fungicidal effect and poor control of the fungus, evidenced by the count of the colony-forming units. Previously reported in vivo results are thus confirmed, showing that P. brasiliensis induces an inflammatory profile in BM-MSCs when producing pro-inflammatory molecules that amplify such response.

IgG reactivity profile to Paracoccidioides spp. antigens in people with asymptomatic Paracoccidioidomycosis.

Introduction. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. As the disease is known to affect mostly men over 40 years old who previously worked handling soil, some cities of agricultural economy in endemic regions may have more cases of paracoccidioidal infection.Gap statement. The true frequency of PCM cannot be established in Brazil because it is not a disease of mandatory reporting. The detection of paracoccidioidal infection may assist in the planning of health services, in order to provide early detection of the disease and to prevent its worsening or even progression to death. In addition, little is described about sera reactivity with antigens from different species of Paracoccidiodes, especially P. lutzii.Aim. Current research was conducted in an inland municipality of southern Brazil, in order to assess infection rate within this endemic region of PCM disease.Methodology. ELISA was employed to evaluate 359 sera from random volunteers from Guarapuava, Paraná, Brazil, to detect IgG against cell-free antigens (CFA) from P. restrepiensis B339, P. americana LDR3 and P. lutzii LDR2. Confirmatory ELISA employed gp43 from B339. Reduction of cross-reactions was sought by treatment with sodium metaperiodate (SMP-CFA, SMP-gp43). Immunoblot was performed with 37 selected sera among those reactive in ELISA. Epidemiological profile was assessed by questionnaire.Results. ELISA reactivity was: CFA/SMP-CFA in general 37.3/17.8 %, B339 25.3/14.5 %, LDR3 24.5/1.4 %, LDR2 8.3/5.8 %; gp43/SMP-gp43 7.2/4.7 %. There were sera reactive with multiple CFAs. In immunoblot, five sera showed the same reaction profile with P. lutzii's antigens as PCM disease sera. Rural residence and soil-related professions were risk factors for paracoccidioidal infection.Conclusion. The low prevalence is in accordance with previous reports of lower PCM disease endemicity in Guarapuava than in other areas of Paraná. Although P. brasiliensis seems to be the prevalent strain of the region, 21 sera from people who only lived in Guarapuava reacted with P. lutzii LDR2. CFA-ELISA with whole antigens seems a good option for serological screening in epidemiological surveys.

Polymorphism in the Promoter Region of the IL18 Gene and the Association With Severity on Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is an important endemic, systemic disease in Latin America caused by Paracoccidioides spp. This mycosis has been associated with high morbidity and sequels, and its clinical manifestations depend on the virulence of the infecting strain, the degree and type of immune response, infected tissues, and intrinsic characteristics of the host. The T helper(Th)1 and Th17/Th22 cells are related to resistance and control of infection, and a Th2/Th9 response is associated with disease susceptibility. In this study, we focused on interleukin(IL)-12p35 (IL12A), IL-18 (IL18), and IFN-γ receptor 1 (IFNGR1) genetic polymorphisms because their respective roles have been described in human PCM. Real-time PCR was employed to analyze IL12A-504 G/T (rs2243115), IL18-607 C/A (rs1946518), and IFNGR1-611 A/G (rs1327474) single nucleotide polymorphisms (SNP). One hundred forty-nine patients with the acute form (AF), multifocal chronic (MC), or unifocal chronic (UC) forms of PCM and 110 non-PCM individuals as a control group were included. In the unconditional logistic regression analysis adjusted by ethnicity and sex, we observed a high risk of the IL18-607 A-allele for both AF [p = 0.015; OR = 3.10 (95% CI: 1.24-7.77)] and MC groups [p = 0.023; OR = 2.61 (95% CI: 1.14-5.96)] when compared with UC. The IL18-607 A-allele associated risk for the AF and MC groups as well as the protective role of the C-allele in UC are possibly linked to higher levels of IL-18 at different periods of the course of the disease. Therefore, a novel role of IL18-607 C/A SNP is shown in the present study, highlighting its importance in the outcome of PCM.

Unraveling the susceptibility of paracoccidioidomycosis: Insights towards the pathogen-immune interplay and immunogenetics.

Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis caused by Paracoccidioides spp. This disease comprises three clinical forms: symptomatic acute and chronic forms (PCM disease) and PCM infection, a latent form without clinical symptoms. PCM disease differs markedly according to severity, clinical manifestations, and host immune response. Fungal virulence factors and adhesion molecules are determinants for entry, latency, immune escape and invasion, and dissemination in the host. Neutrophils and macrophages play a paramount role in first-line defense against the fungus through the recognition of antigens by pattern recognition receptors (PRRs), activating their microbicidal machinery. Furthermore, the clinical outcome of the PCM is strongly associated with the variability of cytokines and immunoglobulins produced by T and B cells. While the mechanisms that mediate susceptibility or resistance to infection are dictated by the immune system, some genetic factors may alter gene expression and its final products and, hence, modulate how the organism responds to infection and injury. This review outlines the main findings relative to this topic, addressing the complexity of the immune response triggered by Paracoccidioides spp. infection from preclinical investigations to studies in humans. Here, we focus on mechanisms of fungal pathogenesis, the patterns of innate and adaptive immunity, and the genetic and molecular basis related to immune response and susceptibility to the development of the PCM and its clinical forms. Immunogenetic features such as HLA system, cytokines/cytokines receptors genes and other immune-related genes, and miRNAs are likewise discussed. Finally, we point out the occurrence of PCM in patients with primary immunodeficiencies and call attention to the research gaps and challenges faced by the PCM field.

Paracoccidioides brasiliensis infection increases regulatory T cell counts in female C57BL/6 mice infected via two distinct routes.

Studies that show an overview of the peripheral immune response in a model of Paracoccidioides brasiliensis (Pb) infection in females are scarce in the literature. We sought to characterize the innate and adaptive immune responses in female C57BL/6 mice infected with Pb through two distinct routes of administration, intranasal and intravenous. In addition to the lung, P. brasiliensis yeast cells were observed in liver and brain tissues of females infected intravenously. To our knowledge, our study is the first to prove the presence of this pathogenic fungus in the cerebral cortex of female mice. During the initial stages of infection, augmented expression of both MHCII and CD86 was observed on the surface of CD11c+ pulmonary antigen-presenting cells (APCs) in intranasally and intravenously infected females. However, CD40 expression was downregulated in these cells. Concomitantly with increasing serum IL-10 levels, we noted that splenic dendritic cells (DCs) from both intravenously- and intranasally-infected female mice had acquired an immature phenotype. Further, increased T regulatory cell counts were observed in female mice infected via both routes, along with an increase in the infiltration of IL-10-producing CD8+ T cells into the lungs. Moreover, we noted that P. brasiliensis infection resulted in enhanced IL-10 production - by CD11c+ APCs in the lung tissue - and induction of Th17 polarization. Taken together, our results suggest that P. brasiliensis could modulates the immune response in female mice by influencing the balance between regulatory T cells (Tregs) and Th17 polarization.

Expression of Hsp60 and its cell location in Paracoccidioides brasiliensis.

Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis.

Paracoccin: Purification and Validation of Its Lectin and Enzymatic Properties.

Studies on the effects of components derived from the human pathogenic fungi Paracoccidioides brasiliensis have identified paracoccin (PCN), as a bifunctional protein with lectin (GlcNAc-binding) and enzymatic (chitinase) activities, able to induce modulation of host immune response. Endogenous PCN acts as a fungal virulence factor, whereas exogenous purified PCN, administered to the host, confers protective immunity in a murine model of paracoccidioidomycosis. The immunomodulation induced by purified-PCN injection has characterized it as an agent applicable in the therapy and vaccine against paracoccidioidomycosis. This section describes methods for PCN purification and validation of its lectin and enzymatic activities. It includes detailed protocols to obtain homogeneous PCN from P. brasiliensis yeasts, as well as to purify recombinant PCN from transformed heterologous microorganisms.

Evaluation of (1 → 3)-ß-D-glucan assay for diagnosing paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis (PCM) is highly prevalent in Latin America, but no commercial system is available for diagnosing this endemic mycosis. OBJECTIVES: To check the performance of (1 â†’ 3)-ß-D-glucan assay (BDG) for diagnosing  PCM in 29 patients with proven fungal disease and compared with double immunodiffusion assay for detecting anti-Paracoccidioides antibodies. PATIENTS AND METHODS: We selected 52 serum samples sequentially obtained from 29 patients with active PCM (12 chronic and 17 acute form). Samples were collected at baseline, and for 16 patients, additional serum levels were obtained after 3 and 6 months of antifungal treatment. Detection of BDG in serum was performed by using the Fungitell® assay. For the double immunodiffusion assay, Paracoccidioides exoantigen was used in latex agglutination tests to detect serum anti-Paracoccidioides antibodies. RESULTS: Despite exhibiting good sensitivity in the diagnosis of patients with PCM, we failed to demonstrate any correlation between the postdiagnosis kinetic profile of BDG serum levels and clinical response to antifungal therapy. This finding may be related to the maintenance of quiescent foci of fungal infection in several organs and tissues, a phenomenon that has been previously reported by other authors and helps to understand why so many relapses are documented in patients treated for short periods of time. Finally, we did not find any correlation between BDG quantification and specific anti-P brasiliensis antibodies serum titres in patients with PCM. CONCLUSIONS: In conclusion, BDG is detected in serum samples of most patients with PCM but is probably not useful for predicting clinical response to antifungal therapy.

43 kDa Glycoprotein (gp43) from Paracoccidioides brasiliensis Induced IL-17A and PGE2 Production by Human Polymorphonuclear Neutrophils: Involvement of TLR2 and TLR4.

The glycoprotein gp43 is the major antigenic/diagnostic component of Paracoccidioides brasiliensis, one of the etiologic agents of paracoccidioidomycosis (PCM). Gp43 has protective roles in mice, but due to adhesive properties, this glycoprotein has also been associated with immune evasion mechanisms. The present study evaluated gp43 interaction in vitro with Toll-like receptors 2 and 4 (TLR2 and TLR4) present in polymorphonuclear neutrophils (PMNs) from healthy human individuals and the consequent modulation of the immune response through the expression and release of cytokines and eicosanoids. PMNs were incubated in the absence or presence of monoclonal antibodies anti-TLR2 and anti-TLR4 (individually or in combination) before gp43 stimulation. Then, PMNs were analyzed for the expression of both surface receptors and the detection of intracytoplasmic IL-17A and IL-4 using flow cytometry, while the production of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α was evaluated in the supernatants by enzyme-linked immunosorbent assay (ELISA). Our results showed that gp43 increased TLR2 and TLR4 expression by PMNs and induced PGE2 and IL-17A via TLR4 and TLR2, respectively. Thus, our data suggest that gp43 from P. brasiliensis might modulate host susceptibility to the fungal infection by affecting PGE2 and IL-17A production.

Therapeutic effect of DNA vaccine encoding the 60-kDa-heat shock protein from Paracoccidoides brasiliensis on experimental paracoccidioidomycosis in mice.

Paracoccidioidomycosis (PCM) is a systemic mycosis autochthonous to Latin America and endemic to Brazil, which has the majority of the PCM cases. PCM is acquired through the inhalation of propagules of fungi from genus Paracoccidioides spp. and mainly affects the lungs. We have previously shown that P. brasiliensis-infected mice treated with single-dose of recombinant 60-kDa-heat shock protein from P. brasiliensis (rPbHsp60) had a worsening infection in comparison to animals only infected. In this study, we investigate whether the treatment of infected mice with PB_HSP60 gene cloned into a plasmid (pVAX1-PB_HSP60) would result in efficient immune response and better control of the disease. The harmful impact of single-dose therapy with protein was not seen with plasmid preparations. Most importantly, three doses of pVAX1-PB_HSP60 and protein induced a beneficial effect in experimental PCM with a reduction in fungal load and lung injury when compared with infected mice treated with pVAX1 or PBS. The increase of the cytokines IFN-γ, TNF, and IL-17 and the decrease of IL-10 observed after treatment with three doses of pVAX1-PB_HSP60 appears to be responsible for the control of infection. These results open perspectives of the therapeutic use of Hsp60 in PCM.

Caspase-11-dependent IL-1α release boosts Th17 immunity against Paracoccidioides brasiliensis.

The granulomatous lesion resulting from infection with the fungus Paracoccidioides brasiliensis is characterized by a compact aggregate of mature cells, surrounded by a fibroblast- and collagen-rich content. Granuloma formation requires signaling elicited by inflammatory molecules such as members of the interleukin-1 family. Two members of this family have been thoroughly studied, namely IL-1α and IL-1ß. In this study, we addressed the mechanisms underlying IL-1α secretion and its functional role on the host resistance to fungal infection. We found that, the expression of caspase-11 triggered by P. brasiliensis infection of macrophages depends on IFN-ß production, because its inhibition reduced procaspase-11 levels. Curiously, caspase-11 deficiency did not impair IL-1ß production, however caspase-11 was required for a rapid pore-mediated cell lysis. The plasma membrane rupture facilitated the release of IL-1α, which was necessary to induce NO production and restrict fungal replication. Furthermore, P. brasiliensis-infected macrophages required IL-1α to produce optimal levels of IL-6, a major component of Th17 lymphocyte differentiation. Indeed, IL-1α deficiency accounted for a significant reduction of Th17 lymphocytes in lungs of infected mice, correlating with diminished neutrophil infiltration in the lungs. Strikingly, we identified that IL-1α directly reprograms the transcriptional profile of Th17-committed lymphocytes, increasing cellular proliferation, as for boosting IL-17 production by these cells. Beyond neutrophil chemotaxis in vivo, IL-17 also amplified IL-1α production by infected macrophages in vitro, endorsing a critical amplification loop of the inflammatory response. Therefore, our data suggest that the IFN-ß/caspase-11/IL-1α pathway shapes a protective antifungal Th17 immunity, revealing a molecular mechanism underlying the cross-talk between innate and adaptive immunity.

Proteomic analysis of Paracoccidioides brasiliensis complex isolates: Correlation of the levels of differentially expressed proteins with in vivo virulence.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis commonly found in Latin America that is caused by distinct species of Paracoccidioides genus: Paracoccidioides brasiliensis complex (S1, PS2, PS3 and PS4) and Paracoccidioides lutzii. Its pathobiology has been recently explored by different approaches to clarify the mechanisms of host-pathogen interactions underpinning PCM. The diversity of clinical forms of this disease has been attributed to both host- and fungus-related factors. METHODOLOGY/PRINCIPAL FINDINGS: For better understanding of the molecular underpinnings of host-fungus interactions, we evaluated in vivo virulence of nine Paracoccidioides brasiliensis complex isolates and correlated it to protein expression profiles obtained by two-dimensional gel electrophoresis. Based on the recovery of viable fungi from mouse organs, the isolates were classified as those having low, moderate, or high virulence. Highly virulent isolates overexpressed proteins related to adhesion process and stress response, probably indicating important roles of those fungal proteins in regulating the colonization capacity, survival, and ability to escape host immune system reaction. Moreover, highly virulent isolates exhibited enhanced expression of glycolytic pathway enzymes concomitantly with repressed expression of succinyl-CoA ligase beta chain, a protein related to the tricarboxylic acid cycle. CONCLUSIONS/SIGNIFICANCE: Our findings may point to the mechanisms used by highly virulent P. brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host.

Structural and immunological characterization of a new nucleotidyltransferase-like antigen from Paracoccidioides brasiliensis.

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αßαßαß topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.