Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Paracoccidioidomycosis: Current Status and Future Trends.

Paracoccidioidomycosis (PCM), initially reported in 1908 in the city of São Paulo, Brazil, by Adolpho Lutz, is primarily a systemic and neglected tropical mycosis that may affect individuals with certain risk factors around Latin America, especially Brazil. Paracoccidioides brasiliensis sensu stricto, a classical thermodimorphic fungus associated with PCM, was long considered to represent a monotypic taxon. However, advances in molecular taxonomy revealed several cryptic species, including Paracoccidioides americana, P. restrepiensis, P. venezuelensis, and P. lutzii, that show a preference for skin and mucous membranes, lymph nodes, and respiratory organs but can also affect many other organs. The classical diagnosis of PCM benefits from direct microscopy culture-based, biochemical, and immunological assays in a general microbiology laboratory practice providing a generic identification of the agents. However, molecular assays should be employed to identify Paracoccidioides isolates to the species level, data that would be complemented by epidemiological investigations. From a clinical perspective, all probable and confirmed cases should be treated. The choice of treatment and its duration must be considered, along with the affected organs, process severity, history of previous treatment failure, possibility of administering oral medication, associated diseases, pregnancy, and patient compliance with the proposed treatment regimen. Nevertheless, even after appropriate treatment, there may be relapses, which generally occur 5 years after the apparent cure following treatment, and also, the mycosis may be confused with other diseases. This review provides a comprehensive and critical overview of the immunopathology, laboratory diagnosis, clinical aspects, and current treatment of PCM, highlighting current issues in the identification, treatment, and patient follow-up in light of recent Paracoccidioides species taxonomic developments.

Dectin-2 is critical for phagocyte function and resistance to Paracoccidioides brasiliensis in mice.

Germline-encoded pattern recognition receptors, particularly C-type lectin receptors (CLRs), are essential for phagocytes to sense invading fungal cells. Among CLRs, Dectin-2 (encoded by Clec4n) plays a critical role in the antifungal immune response as it recognizes high-mannose polysaccharides on the fungal cell wall, triggering phagocyte functional activities and ultimately determining adaptive responses. Here, we assessed the role of Dectin-2 on the course of primary Paracoccidioides brasiliensis systemic infection in mice with Dectin-2-targeted deletion. Paracoccidioides brasiliensis constitutes the principal etiologic agent of paracoccidioidomycosis, the most prominent invasive mycosis in Latin American countries. The deficiency of Dectin-2 resulted in shortened survival rates, high lung fungal burden, and increased lung pathology in mice infected with P. brasiliensis. Consistently, dendritic cells (DCs) from mice lacking Dectin-2 infected ex vivo with P. brasiliensis showed impaired secretion of several proinflammatory and regulatory cytokines, including TNF-α, IL-1ß, IL-6, and IL-10. Additionally, when cocultured with splenic lymphocytes, DCs were less efficient in promoting a type 1 cytokine pattern secretion (i.e., IFN-γ). In macrophages, Dectin-2-mediated signaling was required to ensure phagocytosis and fungicidal activity associated with nitric oxide production. Overall, Dectin-2-mediated signaling is critical to promote host protection against P. brasiliensis infection, and its exploitation might lead to the development of new vaccines and immunotherapeutic approaches.

We report a critical role of the innate immune receptor Dectin-2 during Paracoccidioides brasiliensis infection. Fungal sensing by Dectin-2 improved the survival of mice and lowered fungal burden. Further, Dectin-2 was required for cytokine production, phagocytosis, and fungal killing by phagocytes.

Systematic review of neuroparacoccidioidomycosis: The contribution of neuroimaging.

BACKGROUND: Advanced neuroimaging demonstrated that neurological involvement occurs in up to 30% of paracoccidioidomycosis (PCM) cases. Current knowledge of neuroparacoccidioidomycosis (NPCM) is based on a 2009 systematic review. However, in the last decade, several new cases have been published, with modern neuroimaging techniques. OBJECTIVES: We believe a new systematic review is needed to summarise these advances. METHODS: We searched PubMed/MEDLINE, Embase and LILACS for studies from January 2010 to May 2022. Case series and case reports of NPCM were included. We performed a metaproportion to estimate a summary proportion with 95% confidence intervals (CI). RESULTS: Thirty-four studies including 104 patients were evaluated. We combined our data with the results from the previous review that included 257 cases, totalling 361 patients. We found no new important demographic, clinical or laboratory characteristics. On magnetic resonance imaging (MRI), we found that 72% (95%CI: 38-91) had hyperintensity on T1-weighted image; 84% (95%CI: 71%-92%) had hypointensity on T2-weighted image; 80% (95%CI: 66-89) had contrast enhancement with the classical ring-enhancing pattern. All 8 patients undergoing spectroscopy presented lipid peaks. We found a 16% mortality, lower than in the previous review (44%). CONCLUSION: NPCM presents a characteristic pattern on MRI that may help to differentiate it from other causes of single or multiple brain lesions. Albeit there is a frequent pattern, it is not specific, as other granulomatous diseases may show similar findings. Advances in neuroimaging with early diagnosis and appropriate management of the disease may have contributed to reducing its mortality.

Paracoccidioides and Paracoccidioidomycosis in the 21st Century.

Paracoccidioidomycosis (PCM) defines a broad spectrum of human and animal diseases caused by Paracoccidioides species (Onygenales). In the twenty-first century, Paracoccidioides advanced from a monotypic taxon to a genus that harbors seven species, including P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, P. lutzii, P. loboi, and P. cetii. Classic PCM, acquired upon inhalation of propagules from P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii, affects the human lungs and may progress to systemic granulomatous disease with tegumentary and visceral involvement. On the other hand, PCM loboi and PCM ceti caused by the unculturable P. loboi and P. cetii are subcutaneous mycoses, typically observed as keloid lesions in humans and dolphins. Such heterogeneity highlights the importance of recognizing species boundaries in Paracoccidioides to gain insights into the ecology, evolution, clinical features, and mitigation strategies to tackle the advance of PCM.

PbGP43 Genotyping Using Paraffin-Embedded Biopsies of Human Paracoccidioidomycosis Reveals a Genetically Distinct Lineage in the Paracoccidioides brasiliensis Complex.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii. Four species were recently inferred to belong to the P. brasiliensis complex, but the high genetic diversity found in both human and environmental samples have suggested that the number of lineages may be higher. This study aimed to assess the 43-kilodalton glycoprotein genotypes (PbGP43) in paraffin-embedded samples from PCM patients to infer the phylogenetic lineages of the P. brasiliensis complex responsible for causing the infection. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with histopathological diagnosis of PCM were analyzed. DNAs were extracted and amplified for a region of the second exon of the PbGP43 gene. Products were sequenced and aligned with other PbGP43 sequences available. A haplotype network and the phylogenetic relationships among sequences were inferred. Amino acid substitutions were investigated regarding the potential to modify physicochemical properties in the proteins. Six phylogenetic lineages were identified as belonging to the P. brasiliensis complex. Two lineages did not group with any of the four recognized species of the complex, and, interestingly, one of them comprised only FFPE samples. A coinfection involving two lineages was found. Five parsimony-informative sites were identified and three of them showed radical non-synonymous substitutions with the potential to promote changes in the protein. This study expands the knowledge regarding the genetic diversity existing in the P. brasiliensis complex and shows the potential of FFPE samples in species identification and in detecting coinfections.

Paracoccin Overexpression in Paracoccidioides brasiliensis Enhances Fungal Virulence by Remodeling Chitin Properties of the Cell Wall.

BACKGROUND: The thermodimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis. Although poorly studied, paracoccin (PCN) from Paracoccidioides brasiliensis has been shown to harbor lectinic, enzymatic, and immunomodulatory properties that affect disease development. METHODS: Mutants of P. brasiliensis overexpressing PCN (ov-PCN) were constructed by Agrobacterium tumefaciens-mediated transformation. ov-PCN strains were analyzed and inoculated intranasally or intravenously to mice. Fungal burden, lung pathology, and survival were monitored to evaluate virulence. Electron microscopy was used to evaluate the size of chito-oligomer particles released by ov-PCN or wild-type strains to growth media. RESULTS: ov-PCN strains revealed no differences in cell growth and viability, although PCN overexpression favored cell separation, chitin processing that results in the release of smaller chito-oligomer particles, and enhanced virulence. Our data show that PCN triggers a critical effect in the cell wall biogenesis through the chitinase activity resulting from overexpression of PCN. As such, PCN overexpression aggravates the disease caused by P. brasiliensis. CONCLUSIONS: Our data are consistent with a model in which PCN modulates the cell wall architecture via its chitinase activity. These findings highlight the potential for exploiting PCN function in future therapeutic approaches.

Paracoccidioidesbrasiliensis induces IL-32 and is controlled by IL-15/IL-32/vitamin D pathway in vitro.

Paracoccidioidomycosis (PCM) is a systemic fungal disease caused by Paracoccidioides spp., whose clinical outcome depends on immune response. Interleukin 32 (IL-32) is a cytokine present in inflammatory and infectious diseases, including bacterial, virus and protozoan infections. Its role in fungal disease remains unclear. The axis IL-15, IL-32 and vitamin D leads to microbicidal capacity against intracellular pathogens. Thus, the aims of this study were to investigate the production of IL-32 during Paracoccidioides spp. infection and whether this cytokine and IL-15 can increase P. brasiliensis control in a vitamin D dependent manner. IL-32 was highly detected in oral lesions from patients with PCM. In addition, high production of this cytokine was intracellularly detected in peripheral blood mononuclear cells (PBMCs) from healthy donors after exposure to particulated P. brasiliensis antigens (PbAg). The IL-32γ isoform was predominantly expressed, but there was mRNA alternative splicing for IL-32α isoform. The induction of IL-32 was dependent on Dectin-1 receptor. Infection of PBMCs with P. brasiliensis yeasts did not significantly induce IL-32 production even after activation with exogenous IFN-γ or IL-15 treatments. Although IL-15 was a potent inducer of IL-32 production, treatment with this cytokine did not increase the fungal control unless vitamin D was present in high levels. In this case, both IL-15 and IL-32 increased fungicidal activity of PBMCs. Together, data showed that IL-32 is present in lesions of PCM, PbAg induces IL-32, and the axis of IL-15/IL-32/vitamin D can contribute to control fungal infection. The data suggest that exposure to molecules from P. brasiliensis, as ß-glucans, is needed to induce IL-32 production since only heat-killed and sonicated P. brasiliensis yeasts were able to increase IL-32, which was blocked by anti-Dectin-1 antibodies. This is the first description about IL-15/IL-32/vitamin D pathway role in P. brasiliensis infection.

Lung CD103+ Dendritic cells of mice infected with Paracoccidioides brasiliensis contribute to Treg differentiation.

The DC subsets that express αE integrin (CD103) have been described to exert antagonistic functions, driving T cells towards either an inflammatory (Th1/Th17) or immunosuppressive phenotype (regulatory T cells - Treg). These functions depend on the tissue they reside and microenvironment factors or stimuli that this Antigen-presenting cell (APC) subpopulation receive. In this regard, immunoregulatory phenotype has been described in small subsets of CD103+ DCs from lung and intestinal mucosa. The function of this APC subpopulation in pulmonary Paracoccidioides brasiliensis infection is poorly described. Here, we showed that lung CD103+ DCs contribute to Treg differentiation in a pulmonary P. brasiliensis infection model, which was attributed to downregulation of costimulatory molecules analyzed in these APC subtypes 21 days post-infection. Overall, this data suggests that P. brasiliensis infection caused an immunosuppression that has also been observed in patients with the most severe form of Paracoccidioidomycosis (PCM) - a sickness caused by this fungus genus. Furthermore, these results open new perspectives for knowledge of the mechanisms that underlie the higher percentage of Treg cells found in peripheral blood of PCM patients.

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.

Transcriptomic profiling identifies novel transcripts, isomorphs, and noncoding RNAs in Paracoccidioides brasiliensis.

This paper describes a transcriptomic profiling of Paracoccidioides brasiliensis (Pb) performed with the aid of an RNA-seq-based approach, aimed at characterizing the general transcriptome in this human pathogenic fungus, responsible for paracoccidioidomycosis (PCM). Results confirm that ∼75% of the genes currently annotated in the P. brasiliensis genome are, in fact, transcribed in vivo and that ∼19% of them may display alternative isomorphs. Moreover, we identified 627 transcripts that do not match any gene currently mapped in the genome, represented by 114 coding transcripts (probably derived from previously unmapped protein-coding genes) and 513 noncoding RNAs (ncRNAs), including 203 long-noncoding RNAs (lncRNAs).

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.

Association between IL-27 and Tr1 cells in severe form of paracoccidioidomycosis.

Interleukin-27, a cytokine of the IL-12 family, is secreted by antigen-presenting cells such as macrophages and dendritic cells (DCs). Recent studies suggest an anti-inflammatory role for IL-27 by inducing IL-10 producing Tr1 cells capable of inhibiting Th1 and Th17 type responses. Our study aimed to investigate the involvement of IL-27 and Tr1 cells in the immunomodulation of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Brazil. The presence of IL-27 was evaluated in serum and biopsies of patients with PCM by ELISA, immunohistochemistry, and immunofluorescence. The presence of Tr1 in peripheral blood was analyzed by flow cytometry. In vitro assays were performed to verify the ability of P. brasiliensis yeast to induce IL-27 production by DCs and macrophages, as well as the polarization of lymphocytes to the Tr1 phenotype. Patients with the acute form and severe chronic form, the most severe and disseminated forms of PCM, presented higher serum concentrations of IL-27 and higher percentage of Tr1 cells compared to patients with mild chronic form. IL-27 was also detected in lesions of patients with PCM and associated with DCs and macrophages. P. brasiliensis Pb18 yeasts were able to induce IL-27 production by both DCs and macrophages. We found that DCs pulsed with Pb18 were able to induce Tr1 lymphocytes in vitro. Our data suggest that IL-27 and Tr1 cells could contribute to the deficient immune response to P. brasiliensis that leads to severe and disseminated forms of the disease.

Linoleate diol synthase related enzymes of the human pathogens Histoplasma capsulatum and Blastomyces dermatitidis.

Histoplasma capsulatum is an ascomyceteous fungus and a human lung pathogen, which is present in river valleys of the Americas and other continents. H. capsulatum and two related human pathogens, Blasmomyces dermatitidis and Paracoccidioides brasiliensis, belongs to the Ajellomycetaceae family. The genomes of all three species code for three homologous and tentative enzymes of the linoleate diol synthase (LDS) family of fusion enzymes with dioxygenase (DOX) and cytochrome P450 domains. One group aligned closely with 8R-DOX-5,8-LDS of Aspergilli, which oxidizes linoleic acid to 5S,8R-dihydroxylinoleic acid; this group was not further investigated. The second group aligned with 10R-DOX-epoxy alcohol synthase (EAS) of plant pathogens. Expression of this enzyme from B. dermatitidis revealed only 10R-DOX activities, i.e., oxidation of linoleic acid to 10R-hydroperoxy-8E,12Z-octadecadienoic acid. The third group aligned in a separate entity. Expression of these enzymes of H. capsulatum and B. dermatitidis revealed no DOX activities, but both enzymes transformed 13S-hydroperoxy-9Z,11E-octadecadienoic acid efficiently to 12(13S)epoxy-11-hydroperoxy-9Z-octadecenoic acid. Other 13-hydroperoxides of linoleic and α-linolenic acids were transformed with less efficiency and the 9-hydroperoxides of linoleic acid were not transformed. In conclusion, a novel EAS has been found in H. capsulatum and B. dermititidis with 13S-hydroperoxy-9Z,11E-octadecadienoic acid as the likely physiological substrate.

Evaluation of antifibrotic and antifungal combined therapies in experimental pulmonary paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the Paracoccidioides genus. Most of the patients with chronic form present sequelae, like pulmonary fibrosis, with no effective treatment, leading to impaired lung functions. In the present study, we aimed to investigate the antifibrotic activity of three compounds: pentoxifylline (PTX), azithromycin (AZT), and thalidomide (Thal) in a murine model of pulmonary PCM treated with itraconazole (ITC) or cotrimoxazole (CMX). BALB/c mice were inoculated with P. brasiliensis (Pb) by the intratracheal route and after 8 weeks, they were submitted to one of the following six treatments: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC, and Thal/CMX. After 8 weeks of treatment, the lungs were collected for determination of fungal burden, production of OH-proline, deposition of reticulin fibers, and pulmonary concentrations of cytokines and growth factors. Pb-infected mice treated with PTX/ITC presented a reduction in the pulmonary concentrations of OH-proline, associated with lower concentrations of interleukin (IL)-6, IL-17, and transforming growth factor (TGF)-ß1 and higher concentrations of IL-10 compared to the controls. The Pb-infected mice treated with AZT/CMX exhibited decreased pulmonary concentrations of OH-proline associated with lower levels of TGF-ß1, and higher levels of IL-10 compared controls. The mice treated with ITC/Thal and CMX/Thal showed intense weight loss, increased deposition of reticulin fibers, high pulmonary concentrations of CCL3, IFN-γ and VEGF, and decreased concentrations of IL-6, IL-1ß, IL-17, and TGF-ß1. In conclusion, our findings reinforce the antifibrotic role of PTX only when associated with ITC, and AZT only when associated with CMX, but Thal did not show any action upon addition.

Virulence factors of Paracoccidioides brasiliensis as therapeutic targets: a review.

Paracoccidiodomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The disease requires long and complicated treatment. The aim of this review is to address the fungal virulence factors that could be the target of the development of new drugs for PCM treatment. Virulence factors favoring the process of fungal infection and pathogenicity are considered as a microbial attribute associated with host susceptibility. P. brasiliensis has some known virulence factors which are 43 kDa glycoprotein (gp 43) which is an important fungal antigen, 70 kDa glycoprotein (gp 70), the carbohydrates constituting the fungal cell wall α-1,3, glucan and ß-1,3-glucan, cell adhesion molecules and the presence of melanin pigments. The discovery and development of drugs that interact with these factors, such as inhibitors of ß-1,3-glucan, reduced synthesis of gp 43, inhibitors of melanin production, is of great importance for the treatment of PCM. The study of virulence factors favors the understanding of pathogen-host relationships, aiming to evaluate the possibility of developing new therapeutic targets and mechanisms that these molecules play in the infectious process, favoring the design of a more specific treatment for this disease.

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

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

Paracoccidioides brasiliensis 14-3-3 protein is important for virulence in a murine model.

The Paracoccidioides brasiliensis strain downregulated the expression of adhesin Pb14-3-3 (Pb14-3-3 aRNA) was evaluated in a murine model of paracoccidioidomycosis (PCM). Pb14-3-3 aRNA displays attenuated virulence and triggered the formation of fewer granulomas by lowering the fungal burden in the lungs. Additionally, the Pb14-3-3 aRNA showed more elongated yeast cells and less ability to induce pneumocytes apoptosis in vitro. Our results show that 14-3-3 is an important virulence factor in P. brasiliensis-induced pulmonary infection.

Acute-subacute paracoccidioidomycosis: A paediatric cohort of 141 patients, exploring clinical characteristics, laboratorial analysis and developing a non-survival predictor.

The acute-subacute form of paracoccidioidomycosis (PCM) is a severe systemic mycosis that affects children and adolescents from endemic regions, leading to generalised lymphadenopathy, fever, weight loss, anaemia, eosinophilia, hypoalbuminemia and hypergammaglobulinemia. The objective of this study is to describe the clinical and laboratorial characteristics of acute-subacute PCM, to determine a mortality risk factor and to propose a test for non-survival hazard related to the disease. Children and adolescents diagnosed with PCM, under 15 years were included in the study. Their epidemiological, clinical and laboratorial data were obtained from the hospital records. Descriptive analysis, comparison of means, univariate logistic regression, multivariate logistic regression and a ROC curve were performed in order to identify significant information (P < .05). Through a period of 38 years, 141 children and adolescents were diagnosed with acute-subacute PCM. The main antifungal agent used for the treatment was sulfamethoxazole-trimethoprim (SMX-TMP). The complication rate was 17%, the relapse rate was 7.8% and the mortality rate was 5.7%. A low albumin dosage was identified as a predictor factor for mortality. The cut-off for serum albumin was 2.18 g/dL, above which, the survival rate is 99.1%. Thus, simple clinical and laboratorial examinations may lead to the diagnosis of acute-subacute PCM, and the beginning of the treatment is encouraged even before the isolation of the fungus in biological samples, preventing unfavourable outcomes. Patients with an albumin dosage ≤ 2.18g/dL must receive special attention, preferably hospitalised, during the first four weeks of treatment for presenting an elevated mortality hazard.

Effects of cytochalasin E on Paracoccidioides brasiliensis.

AIMS: To determine the effects of cytochalasin E, isolated from the extremophile fungus Aspergillus felis, on the cells of Paracoccidioides brasiliensis Pb18. METHODS AND RESULTS: Cytochalasin E showed a minimal inhibitory concentration of 3·6 µmol l-1 and minimum fungicidal concentration of 7·2 µmol l-1 on P. brasiliensis by in vitro microdilution and IC50 >964·0 µmol l-1 on murine macrophages. Its selectivity index (>263) indicated that this compound has selectivity for fungal cells. Morphological alterations were determined by optical and fluorescence microscopy, as well as scanning and transmission electron microscopy. Cytochalasin E affected P. brasiliensis bud-forming pseudohyphae, cell morphology, cell walls and cell membranes; caused the release of cellular material; and resulted in the production of reactive oxygen species. In murine macrophages, it affected cytoskeletal actin and inhibited phagocytosis. CONCLUSION: Cytochalasin E may be useful as an antifungal prototype against P. brasiliensis and in studies on phagocytosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM). Treatment is prolonged to control the clinical manifestations and prevent relapse. The study on the effects of cytochalasin E in P. brasiliensis is important because it can be used as a prototype for new antifungal drugs and consequently, broadens the treatment options for PCM.