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.

Mesoionic compounds with antifungal activity against Fusarium verticillioides.

BACKGROUND: Fungi contaminate the food of humans and animals, are a risk to health, and can cause financial losses. In this work, the antifungal activities of 16 mesoionic compounds (MI 1-16) were evaluated against mycotoxigenic fungi, including Aspergillus spp., Fusarium verticillioides and Penicillium citrinum. Furthermore, the decreased ergosterol in the total lipid content of Fusarium verticillioides was investigated. RESULTS: F. verticillioides was the most sensitive fungus to the mesoionic compounds. Among the evaluated compounds, MI-11 and MI-16 presented higher antifungal effects against F. verticillioides, with MIC values of 7.8 µg/ml, and MI-2 and MI-3 followed, with MICs of 15.6 µg/ml. The most active compounds were those with heterocyclic ring phenyl groups substituted by electron donor moieties (MI-11 and MI-16). Among some compounds with higher activity (MI-2, MI-11 and MI-16), decreased ergosterol content in the total lipid fraction of F. verticillioides was demonstrated. MI-2 reduced the ergosterol content approximately 40% and 80% at concentrations of 7.8 µg/ml and 15.6 µg/ml, respectively, and MI-11 and MI-16 decreased the content by 30% and 50%, respectively, when at a concentration of 7.8 µg/ml. CONCLUSION: These findings indicate that mesoionic compounds have significant antifungal activity against F. verticillioides.

1,10-phenanthroline inhibits the metallopeptidase secreted by Phialophora verrucosa and modulates its growth, morphology and differentiation.

Phialophora verrucosa is one of the etiologic agents of chromoblastomycosis, a fungal infection that affects cutaneous and subcutaneous tissues. This disease is chronic, recurrent and difficult to treat. Several studies have shown that secreted peptidases by fungi are associated with important pathophysiological processes. Herein, we have identified and partially characterized the peptidase activity secreted by P. verrucosa conidial cells. Using human serum albumin as substrate, the best hydrolysis profile was detected at extreme acidic pH (3.0) and at 37 °C. The enzymatic activity was completely blocked by classical metallopeptidase inhibitors/chelating agents as 1,10-phenanthroline and EGTA. Zinc ions stimulated the metallo-type peptidase activity in a dose-dependent manner. Several proteinaceous substrates were cleaved, in different extension, by the P. verrucosa metallopeptidase activity, including immunoglobulin G, fibrinogen, collagen types I and IV, fibronectin, laminin and keratin; however, mucin and hemoglobin were not susceptible to proteolysis. As metallopeptidases participate in different cellular metabolic pathways in fungal cells, we also tested the influence of 1,10-phenanthroline and EGTA on P. verrucosa development. Contrarily to EGTA, 1,10-phenanthroline inhibited the fungal viability (MIC 0.8 µg/ml), showing fungistatic effect, and induced profound morphological alterations as visualized by transmission electron microscopy. In addition, 1,10-phenanthroline arrested the filamentation process in P. verrucosa. Our results corroborate the supposition that metallopeptidase inhibitors/chelating agents have potential to control crucial biological events in fungal agents of chromoblastomycosis.

Proteomic analysis of the secretions of Pseudallescheria boydii, a human fungal pathogen with unknown genome.

Pseudallescheria boydii is a filamentous fungus that causes a wide array of infections that can affect practically all the organs of the human body. The treatment of pseudallescheriosis is difficult since P. boydii exhibits intrinsic resistance to the majority of antifungal drugs used in the clinic and the virulence attributes expressed by this fungus are unknown. The study of the secretion of molecules is an important approach for understanding the pathogenicity of fungi. With this task in mind, we have shown that mycelial cells of P. boydii were able to actively secrete proteins into the extracellular environment; some of them were recognized by antibodies present in the serum of a patient with pseudallescheriosis. Additionally, molecules secreted by P. boydii induced in vitro irreversible damage in pulmonary epithelial cells. Subsequently, two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of secreted proteins from P. boydii mycelial cells. The two-dimensional map showed that most of the proteins (around 100 spots) were focused at pH ranging from 4 to 7 with molecular masses ranging from 14 to >117 kDa. Fifty spots were randomly selected, of which 30 (60%) were consistently identified, while 20 (40%) spots generated peptides that showed no resemblance to any known protein from other fungi and/or MS with low quality. Notably, we identified proteins involved in metabolic pathways (energy/carbohydrate, nucleotide, and fatty acid), cell wall remodeling, RNA processing, signaling, protein degradation/nutrition, translation machinery, drug elimination and/or detoxification, protection against environmental stress, cytoskeleton/movement proteins, and immunogenic molecules. Since the genome of this fungus is not sequenced, we performed enzymatic and immunodetection assays in order to corroborate the presence of some released proteins. The identification of proteins actively secreted by P. boydii provides important new information for understanding immune modulation and provides important new perspectives on the biology of this intriguing fungus.

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.

Phospholipase and esterase production by clinical strains of Fonsecaea pedrosoi and their interactions with epithelial cells.

Fonsecaea pedrosoi is the major etiologic agent of chromoblastomycosis. The virulence of F. pedrosoi is a meagerly explored phenomenon. The ability to interact with host cells and the production of hydrolytic enzymes are thought to be important virulence mechanisms of fungal pathogens. Here, we measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by three clinical strains of F. pedrosoi isolated from chromoblastomycosis lesions, as well as their capabilities to interact with epithelial cells. All the strains were excellent esterase producers, generating elevated hydrolytic halos after 5 days of growth. Conversely, phospholipase activity was detected only after 10 days, except for the most recent strain of F. pedrosoi (Magé) in which measurable phospholipase activity was detected on day 5. The ability to interact with epithelial cells was also investigated. Regarding the adhesion capability, an indirect connection was observed in relation to the adaptation time of each strain in axenic culture, in which Magé strain showed the best adhesion ability followed by LDI 11428 and 5VPL strains. Both 5VPL and Magé strains were also detected inside the epithelial cells, while the LDI 11428 strain was rarely detected in cytoplasmatic vacuolar compartments. Moreover, these F. pedrosoi strains were able to cause injury in epithelial cells.

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae.

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.

Secretory aspartyl peptidase activity from mycelia of the human fungal pathogen Fonsecaea pedrosoi: effect of HIV aspartyl proteolytic inhibitors.

Fonsecaea pedrosoi is the principal causative agent of chromoblastomycosis, which is a chronic, often debilitating, suppurative and granulomatous mycosis. Very little is known about the hydrolytic enzymes produced by this human fungal pathogen. In the present study, we have identified extracellular proteolytic activity from F. pedrosoi mycelial forms when grown in chemically defined conditions. Secretory aspartyl peptidase activity was measured during 15 days of fungal growth in vitro using bovine serum albumin (BSA) as the soluble substrate and extreme acidic pH (2.0). This activity was totally inhibited by pepstatin A, a classic aspartyl peptidase inhibitor. Conversely, metallo (o-phenanthroline), cysteine (E-64) and serine (PMSF) proteolytic inhibitors failed to restrain proteolytic activity. We also evaluated the effect of four distinct HIV aspartyl peptidase inhibitors on the secretory proteolytic activity of F. pedrosoi mycelia. Indinavir, ritonavir and nelfinavir powerfully inhibited extracellular aspartyl proteolytic activity by approximately 97, 96 and 87%, respectively, whereas saquinavir did not significantly interfere with BSA hydrolysis. Mycelial-derived secretory aspartyl peptidase activity cleaved other proteinaceous substrates, including human albumin, fibrinogen, fibronectin, laminin and type I collagen. As previously reported by our group, conidia also produce secretory aspartyl peptidase. In this sense, we investigated the effect of pepstatin A on F. pedrosoi development. Pepstatin A was able to inhibit the growth of conidium and its transformation into mycelium. Taken together, our results suggest a possible participation of aspartyl peptidases in the essential fungal processes, such as growth, differentiation, nutrition and cleavage of relevant host proteinaceous components.

The major chromoblastomycosis fungal pathogen, Fonsecaea pedrosoi, extracellularly releases proteolytic enzymes whose expression is modulated by culture medium composition: implications on the fungal development and cleavage of key's host structures.

We investigated the possible secretion of peptidases by F. pedrosoi, when conidial cells were cultured in two distinct media. Aspartyl proteolytic activity was detected on the Czapeck-Dox-derived supernatant, which was blocked by pepstatin, and only active in extremely acidic conditions. The supernatant obtained after conidia growth in Kauffman medium presented metallopeptidase activity, which was active over a broad pH range and sensitive to 1,10-phenanthroline and EGTA. Additionally, both culture supernatants were able to cleave a wide range of proteinaceous substrates, including important human serum proteins (e.g. albumin and immunoglobulin G) and extracellular matrix components (e.g. fibronectin and laminin). As peptidases participate in different cellular metabolic pathways, we also tested the influence of proteolytic inhibitors on the F. pedrosoi conidia development in vitro. The metallopeptidase inhibitors, 1,10-phenanthroline, EGTA and EDTA, strongly abrogated the growth of conidial forms by approximately 95%, 85% and 60%, respectively. Moreover, 1,10-phenanthroline blocked the differentiation process from conidia to mycelia, an essential step during the F. pedrosoi life cycle. Phenylmethanesulfonyl fluoride, a serine peptidase inhibitor, slightly reduced the conidial growth, whereas proteolytic inhibitors of cysteine (E-64) and aspartic (pepstatin) type peptidases did not alter conidial developmental behavior. In summary, our results showed for the first time the expression of extracellular proteolytic activity by F. pedrosoi conidial cells.

Caracterização bioquímica e funcional de ecto-fosfatases em Fonsecaea pedrosoi / Biochemical and function characterization of ecto-fphosphatases in Fonsecaea pedrosoi

As diferentes formas de F. pedrosoi, agente etiológico da cromoblastomicose, apresentam atividades fosfatásicas de superfície, como demonstrado pela capacidade das células fúngicas de hidrolisar diferentes substratos fosforilados. Para caracterização enzimática foram utilizadas as formas de conidio e micélio de F. pedrosoi. As atividades ecto-fosfatásicas foram inibidas com o aumento do pH, o que indicou o carácter acídico dessas enzimas. Essas atividades foram também sensíveis a imbidores clássicos de fosfatases ácidas como ortovanadato de sódio, molibdato de sódio e fluoreto de sódio, sendo insensíveis ao levamizol e tartarato de sódio. O efeito de diferentes cátions nas atividades fosfatásicas foi testado. A atividade das formas micelianas foi estimulada por Zn2+, enquanto que os conídios apresentaram maiores níveis de atividade fosfatásica quando incubados na presença de Fe 2+. A localização citoquímica de atividade enzimática indicou a presença da fosfatase ácida na parede celular de formas micelianas de F. pedrosoi. A atividade fosfatásica de superfície de F. pedrosoi parece estar associada com o parasitismo, uma vez que as células escleróticas, encontradas na lesão tecidual, apresentaram maior atividade enzimática, quando comparada ao micélio e conídios. Além disso, indutores de diferenciação celular de F. pedrosoi foram capazes de estimular a atividade ecto-fosfatásica de células micelianas, sugerindo uma associação entre atividade ecto-fosfatásica e morfogênese fúngica. Constatamos ainda que recente isolado humano de F. pedrosoi apresentou atividade fosfatásica superior à detectada em amostra submetida a várias passagens em meio sintético, sugerindo que a expressão de fosfatases de superficie no F. pedrosoi pode ser modulada por interações com o hospedeiro...