Prospecção de substâncias anti histoplasma capsulatum nas formas planctônica e biofilme e análise proteômica

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Prospecção de protótipos antifúngicos contra biofilmes de Histoplasma capsulatum e Paracoccidioides brasiliensis

A paracoccidioidomicose é uma doença de grande impacto social, sendo a sétima causa de morte entre as doenças crônicas infecciosas no Brasil, que responde por mais de 80% dos casos relatados mundialmente. A histoplasmose é uma micose endêmica em determinadas regiões das Américas, onde causa surtos frequentes. Sabe-se que os dois fungos dimórficos causadores destas doenças, Paracoccioides brasiliensis (Pb) e Histoplasma capsulatum (Hc), respectivamente, são capazes de formar biofilme, uma forma de organização que os torna muito mais perigosos, difíceis de serem combatidos e que é essencial para o desenvolvimento de infecção. Além disso não existem muitas opções terapêuticas antifúngicas que não sejam tóxicas com o uso prolongado disponíveis no mercado atualmente. Estes fatos, deixam claro a necessidade de pesquisar novas formas de combater esses microrganismos, com o intuito de desenvolver fármacos menos tóxicos e mais eficientes. Uma nova possibilidade de opções terapêuticas com novas substância antifúngica são as chalconas, que foi o objeto de estudo desta pesquisa. Foi estudada a ação antifúngica de diversas moléculas de chalconas contra as formas planctônica e de biofilme dos dois fungos em questão. As chalconas que apresentaram menor valor de CIM90 durante os ensaios com a forma planctônica foram selecionadas para o teste contra biofilme fúngico, estas chalconas foram T3, T24, T19, J4 e A20. Foi observado que estas substâncias possuem atividade antifúngica capaz de inibir 90% do crescimento fúngico do biofilme de Pb. Em relação ao biofilme de Hc, não obtivemos uma inibição de 90% com nenhuma molécula, a chalcona T3 foi a que apresentou a maior inibição atingindo 70%. Também foi estudado a citotoxicidade destas chalconas e algumas delas apresentaram bons valores de seletividade para o Pb, como a T3, T24, T19 e A20 porém, em relação ao Hc, apenas a T3 apresentou um valor de seletividade...

Prospecção de protótipos antifúngicos contra biofilmes de Histoplasma capsulatum e Paracoccidioides brasiliensis

A paracoccidioidomicose é uma doença de grande impacto social, sendo a sétima causa de morte entre as doenças crônicas infecciosas no Brasil, que responde por mais de 80% dos casos relatados mundialmente. A histoplasmose é uma micose endêmica em determinadas regiões das Américas, onde causa surtos frequentes. Sabe-se que os dois fungos dimórficos causadores destas doenças, Paracoccioides brasiliensis (Pb) e Histoplasma capsulatum (Hc), respectivamente, são capazes de formar biofilme, uma forma de organização que os torna muito mais perigosos, difíceis de serem combatidos e que é essencial para o desenvolvimento de infecção. Além disso não existem muitas opções terapêuticas antifúngicas que não sejam tóxicas com o uso prolongado disponíveis no mercado atualmente. Estes fatos, deixam claro a necessidade de pesquisar novas formas de combater esses microrganismos, com o intuito de desenvolver fármacos menos tóxicos e mais eficientes. Uma nova possibilidade de opções terapêuticas com novas substância antifúngica são as chalconas, que foi o objeto de estudo desta pesquisa. Foi estudada a ação antifúngica de diversas moléculas de chalconas contra as formas planctônica e de biofilme dos dois fungos em questão. As chalconas que apresentaram menor valor de CIM90 durante os ensaios com a forma planctônica foram selecionadas para o teste contra biofilme fúngico, estas chalconas foram T3, T24, T19, J4 e A20. Foi observado que estas substâncias possuem atividade antifúngica capaz de inibir 90% do crescimento fúngico do biofilme de Pb. Em relação ao biofilme de Hc, não obtivemos uma inibição de 90% com nenhuma molécula, a chalcona T3 foi a que apresentou a maior inibição atingindo 70%. Também foi estudado a citotoxicidade destas chalconas e algumas delas apresentaram bons valores de seletividade para o Pb, como a T3, T24, T19 e A20 porém, em relação ao Hc, apenas a T3 apresentou um valor de seletividade...

Evaluation of the genetic diversity of Histoplasma capsulatum var. capsulatum isolates from north-eastern Brazil

Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceara, a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceara, through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1 b and 1 c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1 -5.8S-ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceara have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.

Phenotypic variation and intracellular parasitism by Histoplasma capsulatum

The success of Histoplasma capsulatum as an intracellular pathogen depends completely on successful conversion of the saprophytic mycelial (mold) form of this fungus to a parasitic yeast form. It is therefore not surprising that yeast phase-specific genes and gene products are proving to be important for survival and proliferation of H. capsulatum within macrophages. In this study, we have focused on the role and regulation of two yeast-specific characteristics: α-(1,3)-glucan, a cell wall polysaccharide modulated by cell-density (quorum) sensing, and a secreted calcium-binding protein (CBP) that is essential for pathogenicity.

Mitochondrial function in the yeast form of the pathogenic fungus Paracoccidioides brasiliensis

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I-V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD(+)-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast`s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.

Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.

DNAhsp65 vaccination induces protection in mice against Paracoccidioides brasiliensis infection

Heat-shock proteins are Molecules with extensive data showing their potential as immunomodulators of different types of diseases, The gene of HSP65 from Mycobacterium leprae has shown prophylactic and immunotherapeutic effects against a broad arrays of experimental models including tuberculosis, leishmaniasis, arthritis and diabetes. With this in mind, we tested the DNAhsp65 vaccine using an experimental model of Paraccocidiodomycosis, an important endemic mycosis in Latin America. The intramuscular immunization with DNAhsp65 induced, in BALB/c mice, an increase of Th1-levels cytokines and a reduction of fungal burdens resulted in a marked reduction of collagen and lung remodeling. DNAhsp65 may be an attractive candidate for prevention, therapy and as an adjuvant for mycosis treatment. (C) 2008 Elsevier Ltd. All rights reserved.

Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB(4) induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB(4) is also important in early effector T cell recruitment that is mediated by LTB(4) receptor 1, the high-affinity receptor for LTB(4). The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB(4) and IFN-gamma production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase(-/-), a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-beta as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase(-/-)-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis. The Journal of Immunology, 2008,181: 8544-8551.

Type 1 and Type 2 cytokines: from basic science to fungal infections

At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.

Characterization of cytotoxic immune response in skin and mucosal lesions of paracoccidioidomycosis

Background: CD8+ T cells and natural killer (NK) cells are involved in the immune response against some pathogens. For this purpose, we investigated the in situ paracoccidioidomycosis (PCM) immune response addressing the participation of NK cells, CD8+ T cells, perforin and granzyme B expression. Methods: Sixty biopsies of PCM skin and mucosa were classified according to the presence of compact granulomas (G1), poorly organized granulomas (G2) and both kinds in the same lesion (G3). CD8+ T cells, NK cells, perforin and granzyme B were showed by immunohistochemistry. Results: CD8+ T cells were increased over NK cells in cutaneous G1 and G2 lesions. There was no difference regarding such cells in G3 lesions, although they were abundant in such lesions. In mucosa, CD8+ T cells were increased in number over NK cells in all groups. Granzyme B in skin increased in G2 and G3. The number of granzyme did not differ in mucosal lesions in the three groups. Conclusions: CD8+ T cells and NK cells play a role in PCM cutaneous and mucosal lesions. The predominance of CD8+ T cells over NK cells may represent an effective response against the fungi. Moreover, the high number of granzyme B expressing cells corroborates this possibility.

HSP65 DNA as therapeutic strategy to treat experimental paracoccidioidomycosis

The conventional treatment for paracoccidioidomycosis, the most prevalent mycosis in Latin America, involves long periods of therapy resulting in sequels and high frequency of relapses. The search for new alternatives of treatment is necessary. Previously, we have demonstrated that the hsp65 gene from Mycobacterium leprae shows prophylactic effects against murine paracoccidioidomycosis. Here, we tested the DNAhsp65 immunotherapy in BALB/c mice infected with Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. We observed an increase of Th1 cytokines accompanied by a reduction in fungal burden and pulmonary injury. These results provide new prospects for immunotherapy of paracoccidioidomycosis and other mycoses. (C) 2009 Elsevier Ltd. All rights reserved.

Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii

We earlier demonstrated that nitric oxide (NO) is a fungicidal molecule against Sporothrix schenckii in vitro. In the present study we used mice deficient in inducible nitric oxide synthase (iNOS(-/-)) and C57BL/6 wild-type (WT) mice treated with N omega-nitro-arginine (Nitro-Arg-treated mice), an NOS inhibitor, both defective in the production of reactive nitrogen intermediates, to investigate the role of endogenous NO during systemic sporotrichosis. When inoculated with yeast cells of S. schenckii, WT mice presented T-cell suppression and high tissue fungal dissemination, succumbing to infection. Furthermore, susceptibility of mice seems to be related to apoptosis and high interleukin-10 and tumour necrosis factor-alpha production by spleen cells. In addition, fungicidal activity and NO production by interferon-gamma (IFN-gamma) and lipopolysaccharide-activated macrophages from WT mice were abolished after fungal infection. Strikingly, iNOS(-/-) and Nitro-Arg-treated mice presented fungal resistance, controlling fungal load in tissues and restoring T-cell activity, as well as producing high amounts of IFN-gamma Interestingly, macrophages from these groups of mice presented fungicidal activity after in vitro stimulation with higher doses of IFN-gamma. Herein, these results suggest that although NO was an essential mediator to the in vitro killing of S. schenckii by macrophages, the activation of NO system in vivo contributes to the immunosuppression and cytokine balance during early phases of infection with S. schenckii.

Associação da rifampicina à anfotericina B no tratamento da paracoccidioidomicose: resultados em três pacientes tratados

Trabalhos experimentais demonstraram que a anfotericina B, desorganizando funcionalmente a membrana celular fúngica, permite a penetração da rifampicina no citoplasma e sua conseqüente ação contra Histoplasma capsulatum, Blastomyces dermatitidis e Candida albicans. Com metade das doses habituais' de anfotericina B associada à rifampicina conseguem-se melhores resultados do que com a anfotericina B isoladamente em doses plenas. Os Autores discutem as possíveis aplicações desta associação no tratamento da paracoccidioidomi-cose e apresentam 3 casos desta micose em que a inatividade clínica e micológica só foi obtida após o emprego combinado destas drogas.

Histoplasma com derrame pleural: relato de um caso

Efusões pleurais surgem raramente em associação com a histoplasmose capsu-lata, ocorrendo em geral nas formas agudas da doença. Relatamos e discutimos um caso clínico em que um histoplasmoma subpleural acompanhou-se de dor pleurítica, hidrotórax e pleurite fibrosante.