Lucimar Ferreira Kneipp

Phosphatase activity on the cell wall of Fonsecaea pedrosoi.

The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.

Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV‐positive and HIV‐negative isolates

OBJECTIVE This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. METHODS The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. RESULTS Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. CONCLUSION These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

Beneficial effects of HIV peptidase inhibitors on Fonsecaea pedrosoi: promising compounds to arrest key fungal biological processes and virulence.

Background Fonsecaea pedrosoi is the principal etiologic agent of chromoblastomycosis, a fungal disease whose pathogenic events are poorly understood. Current therapy for chromoblastomycosis is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the fact that endemic countries and regions are economically poor. Purpose and Principal Findings In the present work, we have investigated the effect of human immunodeficiency virus (HIV) peptidase inhibitors (PIs) on the F. pedrosoi conidial secreted peptidase, growth, ultrastructure and interaction with different mammalian cells. All the PIs impaired the acidic conidial-derived peptidase activity in a dose-dependent fashion, in which nelfinavir produced the best inhibitory effect. F. pedrosoi growth was also significantly reduced upon exposure to PIs, especially nelfinavir and saquinavir. PIs treatment caused profound changes in the conidial ultrastructure as shown by transmission electron microscopy, including invaginations in the cytoplasmic membrane, disorder and detachment of the cell wall, enlargement of fungi cytoplasmic vacuoles, and abnormal cell division. The synergistic action on growth ability between nelfinavir and amphotericin B, when both were used at sub-inhibitory concentrations, was also observed. PIs reduced the adhesion and endocytic indexes during the interaction between conidia and epithelial cells (CHO), fibroblasts or macrophages, in a cell type-dependent manner. Moreover, PIs interfered with the conidia into mycelia transformation when in contact with CHO and with the susceptibility killing by macrophage cells. Conclusions/Significance Overall, by providing the first evidence that HIV PIs directly affects F. pedrosoi development and virulence, these data add new insights on the wide-spectrum efficacy of HIV PIs, further arguing for the potential chemotherapeutic targets for aspartyl-type peptidase produced by this human pathogen.

Microwave-assisted synthesis of new N₁,N₄-substituted thiosemicarbazones

We present an efficient procedure for the synthesis of thirty-six N1,N4-substituted thiosemicarbazones, including twenty-five ones that are reported for the first time, using a microwave-assisted methodology for the reaction of thiosemicarbazide intermediates with aldehydes in the presence of glacial acetic acid in ethanol and under solvent free conditions. Overall reaction times (20–40 min when ethanol as solvent, and 3 min under solvent free conditions) were much shorter than with the traditional procedure (480 min); satisfactory yields and high-purity compounds were obtained. The thiosemicarbazide intermediates were obtained from alkyl or aryl isothiocyanates and hydrazine hydrate or phenyl hydrazine by stirring at room temperature for 60 min or by microwave irradiation for 30 min, with lower yields for the latter. The preliminary in vitro antifungal activity of thiosemicarbazones was evaluated against Aspergillus parasiticus and Candida albicans.

Surface phosphatase in Rhinocladiella aquaspersa: biochemical properties and its involvement with adhesion

Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.

1,10-Phenanthroline-5,6-Dione-Based Compounds Are Effective in Disturbing Crucial Physiological Events of Phialophora verrucosa

Phialophora verrucosa is a dematiaceous fungus able to cause chromoblastomycosis, phaeohyphomycosis and mycetoma. All these fungal diseases are extremely difficult to treat and often refractory to the current therapeutic approaches. Therefore, there is an urgent necessity to develop new antifungal agents to combat these mycoses. In this context, the aim of the present work was to investigate the effect of 1,10-phenanthroline-5,6-dione (phendione) and its metal-based derivatives [Ag(phendione)2]ClO4 = ([Ag(phendione)2]+) and [Cu(phendione)3](ClO4)2.4H2O = ([Cu(phendione)3]2+) on crucial physiological events of P. verrucosa conidial cells. Using the CLSI protocol, we have shown that phendione, [Ag(phendione)2]+ and [Cu(phendione)3]2+ were able to inhibit fungal proliferation, presenting MIC/IC50 values of 12.0/7.0, 4.0/2.4, and 5.0/1.8 μM, respectively. [Cu(phendione)3]2+ had fungicidal action and when combined with amphotericin B, both at sub-MIC (½ × MIC) concentrations, significantly reduced (~40%) the fungal growth. Cell morphology changes inflicted by phendione and its metal-based derivatives was corroborated by scanning electron microscopy, which revealed irreversible ultrastructural changes like surface invaginations, cell disruption and shrinkages. Furthermore, [Cu(phendione)3]2+ and [Ag(phendione)2]+ were able to inhibit metallopeptidase activity secreted by P. verrucosa conidia by approximately 85 and 40%, respectively. Ergosterol content was reduced (~50%) after the treatment of P. verrucosa conidial cells with both phendione and [Ag(phendione)2]+. To different degrees, all of the test compounds were able to disturb the P. verrucosa conidia-into-mycelia transformation. Phendione and its Ag+ and Cu2+ complexes may represent a promising new group of antimicrobial agents effective at inhibiting P. verrucosa growth and morphogenesis.

Antifungal activities of thiosemicarbazones and semicarbazones against mycotoxigenic fungi

Mycotoxigenic fungi can compromise the quality of food, exposing human and animal health at risk. The antifungal activity of eight thiosemicarbazones (1-8) and nine semicarbazones (9-17) was evaluated against Aspergillus flavus, A. nomius, A. ochraceus, A. parasiticus and Fusarium verticillioides. Thiosemicarbazones had MIC values of 125-500 µg/ml. The thiosemicarbazones 1 and 2 exerted fungistatic activity against Aspergillus spp., and thiosemicarbazone 2 exerted fungicidal activity against F. verticillioides. Compound 2 showed an iron chelating effect of 63%. The ergosterol content of A. parasiticus had a decrease of 28 and 71% for the 31.2 and 62.5 µg/ml concentrations of thiosemicarbazone 2 compared to the control. The obtained results of antifungal activity revealed that thiosemicarbazone class was more active when compared to semicarbazone class and, the thiosemicarbazone 2 was the most active compound, specially, against Aspergillus spp.

Aspartic Proteolytic Inhibitors Induce Cellular and Biochemical Alterations in Fungal Cells

Although fungal infections contribute substantially to human morbidity and mortality, the impact of these diseases on human health is not widely appreciated. Diagnosis and treatment of fungal infections remain a challenge in medicine despite recent major advances. The search for novel pharmacological compounds with antifungal action is a real requirement. Taking it into consideration, research groups have investigated the effects of aspartic peptidase inhibitors (PIs) on the development of human fungal pathogens such as Candida spp, mainly Candida albicans and Candida parapsilosis, Cryptococcus neoformans, Pneumocystis jiroveci and Fonsecaea pedrosoi, based on the following premises: (1) this class of hydrolytic enzymes performs multiple relevant roles in pathophysiological events associated to the fungal infections and (2) the introduction of human immunodeficiency virus (HIV) PIs in the clinical arena drastically reduced the opportunistic infections caused by fungi in this population. As expected, the blockage of one of these physiological/pathological processes should help in containing the fungal infection. Corroborating this hypothesis, both in vitro and in vivo studies have reported that classical aspartic PIs (e.g., pepstatin A) as well as HIV PIs (e.g., nelfinavir, saquinavir, ritonavir, indinavir, amprenavir, lopinavir and tipranavir) have induced several cellular and biochemical alterations on fungal cells. Some of the metabolic perturbations are extremely drastic to the fungal cells, which culminate in arresting nutrition, growth, proliferation, differentiation, adhesion, invasion and dissemination. In the present chapter, we will describe the beneficial effects of aspartic PIs against some human fungal pathogens, reporting in details their mechanisms capable in disturbing the fungal homeostasis.

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics.