1,10-Phenanthroline Inhibits the Metallopeptidase Secreted by Phialophora verrucosa and Modulates its Growth, Morphology and Differentiation

Abstract

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.

Chromoblastomycosis

“…The cytolytic effect of ATP suggests that ecto-ATPases found at the surface of F. pedrosoi may favor fungus survival in hostile environments such as the human body (209,210). Peptidases secreted by F. pedrosoi are able to cleave human plasma proteins, such as immunoglobulins and albumin, and components of the matrix, such as fibronectin, while metalloproteinase inhibitors impair conidial growth and differentiation (211) These findings were recently confirmed in Phialophora verrucosa (212). HIV aspartyl peptidase inhibitors strongly abrogate aspartyl proteolytic activity (204), greatly affect F. pedrosoi ultrastructure, diminish adhesion to epithelial cells, and increase susceptibility to killing by macrophage cells, indicating possible therapeutic use in CBM patients (184).…”

Aspartic peptidase of Phialophora verrucosa as target of HIV peptidase inhibitors: blockage of its enzymatic activity and interference with fungal growth and macrophage interaction

“…In the last years, our research group has shown that Fonsecaea pedrosoi, another aetiological agent of CBM, is able to secrete different proteolytic enzymes involved with growth, cell differentiation and fungal pathogenesis [11][12][13][14][15] . In the previous study, we detected an extracellular metallopeptidase activity on P. verrucosa and showed that this enzyme could be involved with fungal growth and cellular differentiation 16 .…”

Dual Mechanism of Action of 5-Nitro-1,10-Phenanthroline against Mycobacterium tuberculosis

“…These phenanthroline-metal complexes have been demonstrated to inhibit the growth of drug-resistant Pseudomonas aeruginosa and Clostridium difficile strains (30,31) and also inhibit biofilm formation by P. aeruginosa and Enterococcus faecalis (31,32). The 1,10-phenanthroline compounds also possess fungistatic activity by inhibiting the secretory metallopeptidase of Phialophora verrucosa (33). Therefore, we next investigated whether 5NP inhibits M. tuberculosis growth by chelating intracellular metal ions.…”