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

Fonsecaea pedrosoi Sclerotic Cells: Secretion of Aspartic-Type Peptidase and Susceptibility to Peptidase Inhibitors

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. pedrosoi sclerotic cells as well as the effect of peptidase inhibitors (PIs) on its enzymatic activity and viability. Our data showed that sclerotic cells are able to secrete pepstatin A-sensible aspartic peptidase when grown under chemically defined conditions. In addition, aspartic PIs (ritonavir, nelfinavir, indinavir, and saquinavir), which are clinically used in the HIV chemotherapy, significantly decreased the fungal peptidase activity, varying from 55 to 99%. 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. pedrosoi proliferation was evaluated. Pepstatin A inhibited the fungal viability in both cellular density- and drug-concentration manners. Moreover, HIV-PIs at 10 μM powerfully inhibited the viability (>65%) of F. pedrosoi sclerotic cells. The detection of aspartic peptidase produced by sclerotic cells, the parasitic form of F. pedrosoi, may contribute to reveal new virulence markers and potential targets for chromoblastomycosis therapy

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

Effect of aspartyl HIV PIs on the interaction between <i>Fonsecaea pedrosoi</i> conidia and epithelial cells (CHO).

<p>After growth in Czapek medium for 5 days at room temperature, the conidia were harvested, washed with PBS and 1×10⁶ cells were incubated for 1 hour in the absence or in the presence of different concentrations (50, 100 and 200 µM) of the following PIs: indinavir, saquinavir, ritonavir and nelfinavir. After that, the conidial cells were washed in PBS and the interaction process was performed as described in Experimental Procedures section. The viability of the conidial cells was not affected by the PIs treatment used in this set of experiments as judged by propidium iodide staining (data not shown). Adhesion (A) and endocytic (B) indices of the interaction after 2 h were shown. The values represent the mean±standard deviation of three independent experiments performed in triplicate. Symbols denote systems treated with PIs that had an interaction index significantly different from the control (♦, <i>P</i><0.05; ★, <i>P</i><0.001; Student's t test). Representative images of attached (a₁ and a₂) and ingested (b₁ and b₂) fungi are shown by means of light (a₁ and b₁) and transmission electron (a₂ and b₂) microscopy analyses. Note that ingested conidia are located within vacuole (b₁ and b₂). Arrows show the conidia attached (a₁) or ingested (b₁) by CHO cells. Scale bars: (a₁ and b₁), 10 µm; (a₂ and b₂), 1 µm.</p

Effect of aspartyl HIV PIs on the <i>F. pedrosoi</i> morphological transition (conidia into mycelia transformation) after the interaction with epithelial cells (CHO).

<p>In this set of experiment, conidia of <i>F. pedrosoi</i> were initially untreated (a and b) or treated (c) with 100 µM of HIV aspartyl PIs for 1 hour, washed in PBS and then interacted with CHO cells for 4 hours. Optical microscopy of the interaction showed that in the control system, some conidial cells transform in hyphae, as indicated by the arrows, showing the well-known differentiation process triggered by the contact of conidia with host cells. Conversely, when conidia were pre-treated with aspartyl PIs, especially saquinavir (c), the number of hyphae is drastically diminished. Arrowheads show the conidia. Scale bars: (a, c), 10 µm; (b), 1 µm.</p

Effect of aspartyl HIV PIs on the interaction between <i>F. pedrosoi</i> conidial cells and fibroblasts or macrophages.

<p>After growth in Czapek medium for 5 days at room temperature, the conidia were harvested, washed with PBS and 1×10⁶ cells were incubated for 1 hour in the absence or in the presence of the following PIs at 200 µM: indinavir, saquinavir, ritonavir and nelfinavir. After that, the conidial cells were washed in PBS and the interaction process was performed as described in Experimental Procedures section. The viability of the conidial cells was not affected by the PIs treatment used in this set of experiments as judged by propidium iodide staining (data not shown). Adhesion and endocytic indices of the interaction with macrophages for 1 hour or fibroblasts for 2 hours were shown. The values represent the mean±standard deviation of three independent experiments performed in triplicate. Symbols denote systems treated with PIs that had an interaction index significantly different from the control (♦, <i>P</i><0.05; ★, <i>P</i><0.001; Student's t test).</p

Effect of aspartyl PIs on the viability and killing activity of macrophage cells (RAW264.7 lineage).

<p>Initially, the macrophages (1×10⁵ cells) were incubated in a 96-well plate for 24 hours in the absence or in the presence of different concentrations (3.125, 6.25, 12.5, 25 and 50 µM) of the following PIs: indinavir, saquinavir, ritonavir and nelfinavir. After this period, the viability of the macrophage cells was determined spectrophotometrically at 490 nm by means of MTT assay. The dotted line separates the graphic in two portions: minor than and equal or major than 90% of macrophage viability (A). After that, we studied the influence of aspartyl PIs on the killing activity of macrophages. In this context, we repeated the interaction of conidia with macrophage (ratio of 10∶1, respectively) for 1 hour, followed by exhaustive washing in PBS and then incubated the interaction for additional 24 hours in the absence (control) or in the presence of indinavir, nelfinavir and ritonavir at 6.25 µM, concentration in which more than 90% of the macrophage cells were viable as demonstrated in (A). Finally, the adhered macrophages were washed, lysed with sterile cold water, and the resulting suspension was plated on Czapek solid medium to count the number of CFU (B).</p

Study of the possible synergistic effect between aspartyl HIV PI and antifungal compounds against <i>F. pedrosoi</i> development.

<p>In this experiments, conidia (1×10³ cells) were treated or not with a sub-inhibitory concentration of both PIs (saquinavir and nelfinavir at 25 µM) and antifungal drugs (itraconazol at 0.3 µg/ml and amphotericin B at 3 µg/ml), alone or in combination for 1 hour. Afterward, the conidial cells were washed in PBS and inoculated in a fresh solid Czapek medium to measure the CFU. The values represent the mean±standard deviation of three independent experiments performed in triplicate. Symbol denotes system that had a growth rate significantly different from the control (★, <i>P</i><0.001; Student's t test).</p

Biochemical properties of the secreted peptidase from conidial cells of <i>Fonsecaea pedrosoi</i>.

<p>(A) Effect of pH on the extracellularly released proteolytic activity of <i>F. pedrosoi</i> conidia. Cells were grown in Czapek chemically defined medium for 5 days at room temperature. Afterward, culture supernatant was filtered, concentrated and tested to degrade soluble BSA using 10 mM sodium citrate buffer adjusted for distinct pH's varying from 2.0 to 5.0. The values represent the mean±standard deviation of three independent experiments, which were performed in triplicate. The inset shows the effect of different PIs on the cleavage of BSA by <i>F. pedrosoi</i> conidial-derived peptidase. Concentrated supernatant was incubated for 1 hour at 37°C in 10 mM sodium citrate buffer, pH 4.0, in the absence (b) or in the presence of 1 µM pepstatin A (c), 10 µM pepstatin A (d), 1 mM PMSF (e), 1 mM E-64 (f) and 1 mM 1,10-phenanthroline (g). A control (a) in which the BSA was supplemented only with citrate buffer was used. These mixture reactions were applied on SDS-PAGE to detect the BSA hydrolysis, and the gel was stained with Coomassie blue R-250. In addition, the extracellular polypeptide profile secreted by <i>F. pedrosoi</i> conidia was analyzed by SDS–PAGE (h). In this last case, the gel was silver-stained and the numbers on the right indicate the relative molecular mass markers expressed in kilodaltons. (B) Effect of aspartyl PIs on the secretory peptidase activity from conidial forms of <i>F. pedrosoi</i>. Concentrated supernatant was incubated for 1 hour at 37°C in 10 mM sodium citrate buffer, pH 4.0, in the absence or in the presence of different concentrations of the following PIs: indinavir, saquinavir, ritonavir, nelfinavir (50, 100 and 200 µM) and pepstatin A (1, 5, 10 and 20 µM). The control (39.3±1.7 arbitrary units) was taken as 100%. The values represent the mean±standard deviation of three independent experiments performed in triplicate. Symbols denote systems treated with PIs that had a rate of substrate hydrolysis significantly different from the control (♦, <i>P</i><0.05; ★, <i>P</i><0.001; Student's t test).</p