Metrics details. The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus. We evaluated the production of nitric oxide NO in macrophages, the oxidative burst and the inducible nitric oxide synthase i-NOS activity in interactions between activated murine macrophages and F. Experiments were carried out with or without tricyclazole TC treatment, a selective inhibitor of the dihydroxynaphthalene DHN -melanin biosynthesis pathway in F.
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Fonsecaea pedrosoi is a dematiaceous fungus and the main causative agent of chromoblastomycosis that is a chronic disease usually affecting the human skin and subcutaneous tissues, which causes deformations and incapacities, being frequently refractory to available therapies. A typical globe-shaped, multiseptated and pigmented cells, known as sclerotic cells, are found in the lesions of infected individuals. In the present work, we have investigated the production of aspartic-type peptidase in F.
Our data showed that sclerotic cells are able to secrete pepstatin A-sensible aspartic peptidase when grown under chemically defined conditions. Moreover, sclerotic cell-derived aspartic peptidase hydrolyzed human albumin, an important serum protein, as well as laminin, an extracellular matrix component, but not immunoglobulin G and fibronectin.
It is well-known that aspartic peptidases play important physiological roles in fungal cells. With this task in mind, the effect of pepstatin A, a classical aspartic peptidase inhibitor, on the F. Pepstatin A inhibited the fungal viability in both cellular density- and drug-concentration manners.
The detection of aspartic peptidase produced by sclerotic cells, the parasitic form of F. This dimorphic fungus produce different morphotypes including conidia reproduction structures and mycelia filamentous forms , both are usually found in its saprophytic lifestyle, as well as sclerotic cells synonymous with muriform or medlar bodies , which are the parasitic forms observed in the infected tissues Rippon, The morphology of sclerotic cell is well-known, but its physiology remains poorly studied, mainly because this tissue form is very hard to be induced in vitro and is not usually obtained in high quantities in its disarticulated state Santos et al.
Even though, there are several reports showing different procedures to induce in vitro sclerotic cell formation from chromoblastomycosis fungi, such as pH reduction, manganese deprivation, calcium or propranolol supplementation and natural culture medium formulated from tree fruits Alviano et al.
Studies conducted by our group revealed that sclerotic cells obtained in vitro were similar to those observed in vivo. The cellular morphology, ultrastructure, as well as the antigenic cross-reactivity between in vivo and in vitro sclerotic cells confirmed their similarity, showing that the latter can be used in experiments aiming to understand the physiopathology of chromoblastomycosis fungi Silva et al. In the past, sclerotic cells were mainly known as resistant forms able to survive inside the host tissues.
However, several studies have shown that sclerotic cells are active parasitic forms involved directly with F. In addition, sclerotic cells are extremely resistant to immune system attack. Dong et al. Recently, chromoblastomycosis murine model studies revealed that only sclerotic cells depend on dectin-1 recognition to be internalized, suggesting different F. Furthermore, those authors also reported that F. These reasons could explain the difficulty in treating this chronic disease, even more after considering the fact that highly melanized sclerotic cells make the fungi much more resistant to different classes of antifungal drugs Revankar and Sutton, ; Queiroz-Telles et al.
The chromoblastomycosis pathogenicity mechanisms are not well established. However, in recent years, our research group has described some enzymes involved in the physiopathology of chromoblastomycosis fungi, including peptidases Kneipp et al. It is known that proteolytic enzymes participate in infectious processes caused by a number of human pathogenic fungi, being main actors in several aspects of fungi-host interplays such as adhesion, invasion, nutrition, escape, proliferation, and differentiation Monod et al.
Over the last years, we identified and characterized the proteolytic activity secreted by F. Several studies have proposed that fungal peptidases are potential targets to develop new antifungal drugs Pozio and Morales, ; Santos, ; Santos and Braga-Silva, ; Santos et al. Furthermore, HIV-PIs treatment restrained the conidia-into-mycelia differentiation as well as reduced their adhesion to mammalian cells Palmeira et al.
For all the reasons elucidated above, in the present work, we have investigated the capability of F. Also, the effects of aspartic PIs were evaluated on fungal enzymatic activity and viability. Louis, United States. Media constituents, reagents used in electrophoresis and buffer components were purchased from Amersham Life Science Little Chalfont, United Kingdom.
All other reagents were of analytical grade. Stock cultures were maintained on Sabouraud dextrose agar under mineral oil. For all the experiments, sclerotic cells were washed three times in saline 0. The cell-free culture supernatants were fold concentrated in a 10, molecular weight cut-off Amicon micropartition system Beverly, MA, United States Palmeira et al. Protein concentration was detected by the method described by Lowry et al. Extracellular proteolytic activity was measured spectrophotometrically according to the method described by Buroker-Kilgore and Wang Alternatively, the concentrated supernatant was added to different buffers, such as 10 mM sodium citrate pH 2.
After 10 min, to allow dye binding, the plate was read on a Molecular Devices Thermomax microplate reader at an absorbance of nm. One unit of proteolytic activity was defined as the amount of enzyme that caused an increase of 0. These proteins were diluted in 10 mM sodium citrate pH 4. Then, gels were stained with silver nitrate to evidence the protein cleavage profiles.
Controls were made by replacing concentrated culture supernatants with the same volume of citrate buffer Palmeira et al. To test the possible involvement of aspartic peptidases on F. Alternatively, 10 5 sclerotic cells were also treated with different pepstatin A concentrations 0. The sclerotic cells were then harvested by centrifugation, washed twice with PBS and re-inoculated into solid medium without drugs, in order to measure the colony-forming units CFU Palmeira et al.
Methanol, the diluent of PIs, was also tested. All the experiments were repeated at least three times. All the systems were performed in triplicate, and representative images of these experiments are shown.
P- values of 0. We have shown that chromoblastomycosis fungi secrete distinct peptidases and that these enzymatic profiles are closely related with fungal morphology and cultivation conditions Palmeira et al. Studies have reported that cell shape modifications are strategies used by different fungi to survive in the environment and inside the host Wang and Lin, Considering that F.
Thus, after the growth of F. The BSA degradation was observed only in acidic pH values, reaching a maximum hydrolytic activity at pH 4.
In the current study, we showed for the first time that sclerotic cells were able to produce an extracellular peptidase that was active at extremely acidic pH, as also described for conidial and mycelial forms of this fungus Palmeira et al.
Coincidentally, filamentous forms of F. In addition, acidic peptidase produced by F. Consequently, the differential pattern of peptidase expression may be essential for fungal adaptation to various environments, including host tissues Naglik et al. Peptidase activity released by F. Dark-brown, thick-walled and spherical fungal cells exhibiting typical planate division can be observed arrows. The image was captured by Zeiss Axioplan 2 microscope using an AxioCam camera. B The influence of pH on the peptidase activity released by sclerotic cells was evaluated.
Fungal concentrated supernatant was incubated with the substrate BSA in different buffers: 10 mM sodium citrate pH 2. The reaction mixtures were measured at nm and the proteolytic activity expressed in arbitrary units AU , which was defined as the amount of enzyme that caused an increase of 0.
Subsequently, both systems were supplemented with 10 mM sodium citrate pH 4. The arrow on the left shows the molecular mass of intact BSA. The arrowheads on the right indicate the fragmentation of BSA after proteolysis.
Recently, F. Fonsecaea species and other black fungi belonging to the bantiana-clade were predicted to produce a wide repertoire of different endo- and exopeptidases Vicente et al. Members of Herpotrichiellaceae , which include Fonsecaea , presented specific and significant number of S09 prolyl oligopeptidase , S33 prolyl aminopeptidase and M38 isoaspartic dipeptidase families Teixeira et al.
Caspases, which are cysteine dependent aspartic-specific peptidase playing essential roles in programmed cell death and inflammation, occur in the Fonsecaea core genome Madeo et al. The effect of classic PIs on the acidic peptidase released by F. Taking into consideration the extremely acidic pH for peptidase activity as well as its inhibitory profile, the peptidase released by F.
Our results corroborate previous in silico studies that predicted the presence of aspartic peptidase-encoding genes in F. Aspartic peptidases are characterized in different ways, according to their catalytic properties, cellular localization and pepstatin A inhibition, for example Santos et al.
Pepstatin A was also able to block the aspartic peptidase activities produced by other pathogenic filamentous fungi, including Sporothrix schenckii, Aspergillus fumigatus, Paracoccidioides brasiliensis, Pseudallescheria boydii, Scedosporium aurantiacum , and Trichosporon asahii Tsuboi et al. Effect of proteolytic inhibitors on the peptidase activity released by sclerotic cells. BSA supplemented exclusively with buffer was used as control.
Peptidase activity was determined as described by Buroker-Kilgore and Wang For instance, amprenavir was the most potent inhibitor of the secreted aspartic peptidases Sap , the principal virulence factor produced by Candida albicans Braga-Silva et al.
Recently, Valle et al. The HIV-PIs ritonavir, indinavir and nelfinavir also inhibited the aspartic peptidase activity produced by both conidial and mycelial forms of F. These data indicated that F. Studies have reported the differential expression of aspartic peptidases during the fungal morphogenesis and that aspartic PIs might control this essential phenomenon to the establishment of fungal infection Braga-Silva et al. TABLE 1. Overview of the action of aspartic peptidase inhibitors on the peptidase activity and viability of F.
To provide more information about the aspartic peptidase secreted by F. The results revealed that the aspartic peptidase of F. The protein cleavage profiles detected herein are in contrast to those previously observed to aspartic peptidases produced by F. The ability to cleave host structural proteins was also observed in other human pathogenic fungi; for instance, A.
In addition, C. Cleavage of soluble proteinaceous substrates by aspartic peptidase activity released by sclerotic cells. The arrows on the right represent bands generated after substrates degradation.
Based on the efficacy of pepstatin A in reducing the viability of both conidial and mycelial forms of F. In this context, two relevant parameters were analyzed: distinct fungal densities Figure 4A and different concentrations of the PI Figure 4B.
In parallel, pepstatin A was also able to block the viability of sclerotic cells 10 5 fungi in a typically dose-dependent fashion Figure 4B.
Effects of pepstatin A on sclerotic cell viability. Supporting these findings, several studies have reported that pepstatin A, as well as HIV-PIs have effective antifungal action in both in vitro and in vivo assays Tsuboi et al.
Fonsecaea pedrosoi is a dematiaceous fungus and the main causative agent of chromoblastomycosis that is a chronic disease usually affecting the human skin and subcutaneous tissues, which causes deformations and incapacities, being frequently refractory to available therapies. A typical globe-shaped, multiseptated and pigmented cells, known as sclerotic cells, are found in the lesions of infected individuals. In the present work, we have investigated the production of aspartic-type peptidase in F. Our data showed that sclerotic cells are able to secrete pepstatin A-sensible aspartic peptidase when grown under chemically defined conditions. Moreover, sclerotic cell-derived aspartic peptidase hydrolyzed human albumin, an important serum protein, as well as laminin, an extracellular matrix component, but not immunoglobulin G and fibronectin.
Santos, Vanila F. Palmeira, Sonia Rozental, Lucimar F. Kneipp, Leonardo Nimrichter, Daniela S. Alviano, Marcio L. Rodrigues, Celuta S. Fonsecaea pedrosoi is the principal etiologic agent of chromoblastomycosis, a fungal disease whose pathogenic events are poorly understood. Treatment of the disease presents poor effectiveness and serious side effects.
We focus on the introduction of Fonsecaea pedrosoi here. As a genus of ascomycetous fungi, Fonsecaea is affiliated with the family Herpotrichiellaceae and comprises three sibling species, all with pathogenic potential, namely F. The species F. In the life cycle, lipase activity can be tested, but phospholipase, collagenase, and amylase are not expressed. Salgado, Fonsecaea pedrosoi is the main agent of human chromoblastomycosis, which presents typical dermal lesions, such as Cauliflower-like nodules.