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.

Immunomodulatory activity of ß-glucan-containing exopolysaccharides from Auricularia auricular in phagocytes and mice infected with Cryptococcus neoformans.

Current antifungal drugs present poor effectiveness and there is no available vaccine for fungal infections. Thus, novel strategies to treat or prevent invasive mycosis, such as cryptococcosis, are highly desirable. One strategy is the use of immunomodulators of polysaccharide nature isolated from mushrooms. The purpose of the present work was to evaluate the immunostimulatory activity of ß-(1,3)-glucan-containing exopolysaccharides (EPS) from the edible mushrooms Auricularia auricula in phagocytes and mice infected with Cryptococcus neoformans. EPS triggered macrophages and dendritic cell activation upon binding to Dectin-1, a pattern recognition receptor of the C-type lectin receptor family. Engagement of Dectin-1 culminated in pro-inflammatory cytokine production and cell maturation via its canonical Syk-dependent pathway signaling. Furthermore, upon EPS treatment, M2-like phenotype macrophages, known to support intracellular survival and replication of C. neoformans, repolarize to M1 macrophage pattern associated with enhanced production of the microbicidal molecule nitric oxide that results in efficient killing of C. neoformans. Treatment with EPS also upregulated transcript levels of genes encoding products associated with host protection against C. neoformans and Dectin-1 mediated signaling in macrophages. Finally, orally administrated ß-glucan-containing EPS from A. auricular enhanced the survival of mice infected with C. neoformans. In conclusion, the results demonstrate that EPS from A. auricula exert immunostimulatory activity in phagocytes and induce host protection against C. neoformans, suggesting that polysaccharides from this mushroom may be promising as an adjuvant for vaccines or antifungal therapy.

Upregulation of glyoxylate cycle genes upon Paracoccidioides brasiliensis internalization by murine macrophages and in vitro nutritional stress condition.

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a facultative intracellular human pathogen that can persist within macrophage phagolysosomes, indicating that the fungus has evolved defense mechanisms in order to survive under nutritionally poor environments. The analysis of P. brasiliensis transcriptome revealed several virulence factor orthologs of other microorganisms, including the glyoxylate cycle genes. This cycle allows the utilization of two-carbon (C2) compounds as carbon source in gluconeogenesis. Semiquantitative RT-PCR analyses revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any additional in vitro growth. The induction of this cycle, in response to macrophage microenvironments, was shown to be coordinated with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase gene. In addition, assays employing RNA extracted from P. brasiliensis grown in a medium with acetate instead of glucose also showed increased levels of glyoxylate cycle transcripts. Our main results suggest that P. brasiliensis uses the glyoxylate cycle as an important adaptive metabolic pathway.

Virulence insights from the Paracoccidioides brasiliensis transcriptome

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.