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

Phialophora verrucosa causes several fungal human diseases, mainly chromoblastomycosis, which is extremely difficult to treat. Several studies have shown that human immunodeficiency virus peptidase inhibitors (HIV-PIs) are attractive candidates for antifungal therapies. This work focused on studying the action of HIV-PIs on peptidase activity secreted by P. verrucosa and their effects on fungal proliferation and macrophage interaction. We detected a peptidase activity from P. verrucosa able to cleave albumin, sensitive to pepstatin A and HIV-PIs, especially lopinavir, ritonavir and amprenavir, showing for the first time that this fungus secretes aspartic-type peptidase. Furthermore, lopinavir, ritonavir and nelfinavir reduced the fungal growth, causing remarkable ultrastructural alterations. Lopinavir and ritonavir also affected the conidia-macrophage adhesion and macrophage killing. Interestingly, P. verrucosa had its growth inhibited by ritonavir combined with either itraconazole or ketoconazole. Collectively, our results support the antifungal action of HIV-PIs and their relevance as a possible alternative therapy for fungal infections.

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.

In vitro activities of nine antifungal drugs against 81 Phialophora and Cyphellophora isolates.

Cyphellophora guyanensis (n = 15), other Cyphellophora species (n = 11), Phialophora europaea (n = 43), and other Phialophora species (n = 12) were tested in vitro against nine antifungal drugs. The MIC(90)s across all of the strains (n = 81) were, in increasing order, as follows: posaconazole, 0.063 µg/ml; itraconazole, 0.5 µg/ml; voriconazole, 1 µg/ml; micafungin, 1 µg/ml; terbinafine, 2 µg/ml; isavuconazole, 4 µg/ml; caspofungin, 4 µg/ml; fluconazole, 8 µg/ml; amphotericin B, 16 µg/ml.

Phialophora section Catenulatae disassembled: New genera, species, and combinations and a new family encompassing taxa with cleistothecial ascomata and phialidic asexual states.

The Pleuroascaceae (Leotiomycetes) is introduced for Phialophora hyalina (section Catenulatae) and its closest relatives based on analyses of DNA sequences of five gene regions and the comparison of cultural and micromorphological characters. The family is resolved as a strongly supported clade that encompasses Pleuroascus and the new anamorph genera Entimomentora and Venustampulla. The latter includes V. parva, a species placed formerly in Scopulariopsis, and V. echinocandica, which is established for the echinocandin-producing isolate BP-5553. Entimomentora includes E. hyalina, a species based on the ex-type strain of Ph. hyalina. Additional isolates identified as Ph. hyalina are distantly related to the Pleuroacaceae and include Psychrophila antarctica (Arachnopezizaceae) and Cryonesomyces dreyfussii, the sole member of the new genus Cryonesomyces (incertae sedis). Isolates identified or deposited as Ph. alba are also not closely related; they include a species for which we propose the name Neobulgaria koningiana (Gelatinodiscaceae) and a second psychrophilic species that we describe as Psychrophila lagodekhiensis. Of the 13 isolates assessed for in vitro antifungal activity, only V. echinocandica inhibited the growth of Candida albicans.

Molecular identification of Phialophora oxyspora as the cause of mycetoma in a horse.

CASE DESCRIPTION: An 18-year-old mare was evaluated for an oral mass that developed after extraction of a broken incisor. CLINICAL FINDINGS: An ulcerated, firm, darkly pigmented, approximately 5-cm-diameter spherical mass involved the gingiva lateral and dorsal to the right first to third maxillary incisors. Osteolysis of the roots of the first and second right maxillary incisors and periosteal proliferation of the adjacent premaxilla margins were apparent on radiographs. Histologic examination of the mass revealed multiple coalescing and ramifying foci of abscess formation, each containing a well-defined, discrete, black mass (2 to 7 mm in diameter). Myriad fungal hyphae enmeshed in a black, granular, cementlike material were within each of the black structures. Mycetoma was the histologic diagnosis. The causative agent could not be identified via culture because of lack of distinguishing characteristics. Fungal DNA was isolated from frozen fungal cultures and paraffin sections. The D1/D2 domains of the large subunit P gene rDNA were amplified and sequenced. The sequences of the D1/D2 domains of both isolates were 96% homologous with those of Phialophora oxyspora. TREATMENT AND OUTCOME: The mass was surgically excised, the local area curetted, and the wound allowed to heal by second intention. Postoperative treatment consisted of administration of phenylbutazone and IV administration of sodium iodide followed by oral administration of potassium iodide. There was no evidence of recurrence 1 year later. CLINICAL RELEVANCE: Mycetomata should be a differential diagnosis for equine gingival masses. Identification of the fungal agent can be critical for selection of optimal treatments. Molecular methods may permit definitive identification when standard phenotypic-based identification criteria are inconclusive.

Lipolytic activity of chromoblastomycosis agents measured by infrared spectroscopy and chemometric methods.

The lipase activity of nine strains of six chromoblastomycosis agents (Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora bantianum, Cladophialophora carrionii, Rhinocladiela aquaspersa and Exophiala jeanselmei) grown on solid medium was investigated using Fourier transform infrared spectroscopy and hierarchical clustering analysis. The data was quantified by p-nitrophenyl palmitate assay using partial least squares (PLS) regression. These methods allowed the correlation of six genera and species within the 1230-1650 and 2800+3000 cm(-1) spectral ranges among strains grown for 14 days from their respective lipolytic activity with RMSEV=0.048 and R2val=0.95 and ten latent variables. The lipolytic activity also was predicted using PLS models with 1230-1650 and 2800-3000 cm(-1) and 900-1450 cm(-1) spectral ranges for strains grown for 21 days. The separate analysis of F. pedrosoi strains yielded a prediction model for biomass at 21 days with RMSEV=0.065 and R2val=0.95 with eight latent variables using (1100-1300)+(1330-1460)+(1550-1650) cm(-1) spectral regions The best model obtained with the clustering of P. verrucosa, C. bantianum, C. carrionii, R. aquaspersa and E. jeanselmei strains was constructed with the same spectral ranges, but with RMSEV=0.074 and R2val=0.94 and ten latent variables. Infrared spectroscopy is suitable for the quantitation of extracellular lipase activity linked to the biomass of chromoblastomycosis agents.

Phagocytosis, production of nitric oxide and pro-inflammatory cytokines by macrophages in the presence of dematiaceous [correction of dematiaceus] fungi that cause chromoblastomycosis.

Chromoblastomycosis is characterized by the slow development of polymorphic skin lesions (nodules, verrucas, tumores, plaques and scar tissue). Inside the host, infectious propagules adhere to epithelial cells and differentiate into sclerotic forms, which effectively resist destruction by host effector cells and allow onset of chronic disease. A cellular immune response against fungi is essential to control infection. Amongst the cells of the immune system, macrophages play the most important role in controlling fungal growth. In this study, we show that the fungicidal characteristic of macrophages is dependent on the fungal species that causes chromoblastomycosis. We began by observing that the phagocytic index was higher for Fonsecaea pedrosoi and Rhinocladiella aquaspersa compared with that of other fungi. Complement-mediated phagocytosis was more important for Phialophora verrucosa and R. aquaspersa and was inhibited by mannan when F. pedrosoi and R. aquaspersa conidia were phagocytosed by macrophages. We showed that macrophages killed significantly only R. aquaspersa. We also found that the phagocytosis of fungi has functional consequences for macrophages as phagocytosis resulted in down-modulation of MHC-II and CD80 expression as well as in the inhibition of the basal liberation of NO. However, the inhibition of the basal liberation of NO nor the down-modulation of MHC and co-stimulatory molecules were observed in the presence of R. aquaspersa.

Budding in the dimorphic fungus Phialophora dermatitidis.

Ultrastructural comparisons of yeast and hyphal bud formation in Phialophora dermatitidis reveal that bud initiation is characterized by a blastic rupture of the outer portion of the yeast or hyphal wall and the emergence of a bud protuberance through the resulting opening. The wall of the emerging bud is continuous, with only an inner wall layer of the parental yeast or hypha. The outer, ruptured portion of the parental wall typically forms a collar around the constricted emergence region of the developing bud. The cytoplasm within the very young emerging bud invariably contains a small number of membrane-bound vesicles. The septum formed between the daughter bud and the parental yeast or hypha is a complete septum devoid of a septal pore, septal pore plug, or any associated Woronin bodies characteristic of simple septa of the moniliform or true hyphae. These observations suggest that yeast bud formation and lateral hyphal bud formation in the dimorphic fungus P. dermatitidis involve a growth process which occurs identically in both the yeast and mold phase of this human pathogenic organism.

Conjugation in the dimorphic chromomycosis fungus Phialophora dermatitidis.

Phase-contrast microscope observations of the dimorphic fungus Phialophora dermatitidis revealed that thick-walled yeasts often tended to form aggregates and then to conjugate. Fusions were also observed among hyphae derived from the thick-walled yeasts.

[Relations between the fungi Gaeumannomyces graminis and Phialophora radicicola when growing together outside the host (author's transl)]. / Beziehungen zwischen den Pilzen Gaeumannomyces graminis und Phialophora radicicola bei gemeinsamem Wachstum ausserhalb der Wirtspflanze.

In laboratory model experiments, numerous hyphal adhesions were found to take place between a severe wheat pathogen, G. graminis var. tritici, and a weak wheat pathogen, P. radicicola var, radicicola, and therefore the linear growth of the both fungi is inhibited. When both fungi occur concurrently side by side on celophane, the hyphae of one fungus do not occupy the place colonized before by the other fungus, due to mutual contact. It can be presumed that also direct interaction between hyphae of both fungi participates in cross protection mechanism of the take-all fungus G. graminis with P. radicicola.

Morphologica and structural changes during the yeast-to mold conversion of Phialophora dermatitidis.

The details of the morphological and structural events occurring during yeast-to-mold conversion of the human pathogenic fungus Phialophora dermatitidis as seen by phase-contrast microscopy and electron microscopy are described and illustrated. Budding yeasts growing exponentially were observed to have thin walls and a cytoplasm exhibiting the characteristics of rapidly growing cells including numerous mitochondria, abundant ribosomes, few vacuoles, and little accumulation of storage material. In contrast, thick-walled yeasts were characterized by less apparent or significantly fewer mitochondria and ribosomes and the presence of considerable amounts of storage materials. Microscope observations of yeast-to-mold conversion revealed that only thick-walled yeasts having prominent lipid bodies in their cytoplasm converted to hyphal forms. Typically, the thick-walled yeast formed two to a number of moniliform hyphal cells which in turn often produced true hyphae. The results indicated that yeasts of P. dermatitidis must acquire spore-like characteristics by becoming thick-walled and by accumulating considerable endogenous substrate reserves before they convert and produce hyphae.

Gluteal abscess caused by Phialophora hoffmannii and review of the role of this organism in human mycoses.

Infections caused by members of the Phialophora hoffmannii (Beyma) Schol-Schwarz fungal aggregate (a group of related taxa, especially species of uncertain circumscription) are not reported frequently. This case report concerns the development of a gluteal abscess after multiple intramuscular injections of antimicrobial agents. Microbiological examination of the abscess material yielded a pure growth of the mold P. hoffmannii. Hyphal elements in the purulent exudate were only demonstrated when the specimen was digested by an N-acetyl cysteine-NaOH digestion-decontamination procedure or in stained paraffin block sections of the exudate. Other cases of disease caused by members of this fungal aggregate and the status of the mycology of this organism are reviewed.

Phialophora cyanescens sp. nov. with Phaeosclera-like synanamorph, causing white-grain mycetoma in man.

The human-pathogenic fungus, Phialophora cyanescens sp. nov. is characterized by the abundant production of chlamydospore-like cell aggregates resembling the form-genus Phaeosclera Sigler et al., and by elongate phialides with ellipsoidal conidia. A brown pigment and a blue, acid-labile pigment may be present.

Details of the fungistatic effect of Phialophora cinerescens. / Particularités de l'action fongistatique du Phialophora cinerescens

We have previously isolated two chlorinated antibiotics from Phialophora asteris and P. asteris f. sp. helianthi, vascular pathogens on plants. P. cinerescens, pathogen on carnation, also presents a fungitoxic activity but the nature of the antibiotic compound is still unknown. We point out that this activity can be lost by sectorial variations on the growing margin and that the antibiotic product shows a striking photolability.

Phialophora repens, an emerging agent of subcutaneous phaeohyphomycosis in humans.

A 63-year-old Japanese man had phaeohyphomycosis that occurred as a solitary subcutaneous nodule on the dorsal aspect of his left hand. In the nodule there were foci of mixed granulomatous and suppurative infiltrations circumscribed by thick fibrous tissue reaction. The foci contained short septate hyphae and occasionally small rounded aggregates of irregularly branched septate hyphae, both of which were nonpigmented or rarely weakly pale brown. Fungal culture from the nodule was positive for a dematiaceous mold. The mycologic features of the mold were typical of Phialophora repens. The infection was successfully treated by excision of the nodule. This is the second reported case of infection due to P. repens.

Phialophora verrucosa infection in an AIDS patient.

Phialophora verrucosa is one of several pathogenic dematiaceous fungi associated with chromomycosis and occasionally phaeohyphomycosis. Infection appears to be increasing in frequency in both immunocompromised and presumably healthy patients. Medical therapy is often difficult, and a wide variety of antifungal agents have been tried with varying degrees of success. We report a patient with acquired immunodeficiency syndrome (AIDS) and extensive cutaneous fungal infection due to Phialophora verrucosa. The disease failed to respond to ketoconazole, but regression of the lesions was obtained with itraconazole.

Pyrene degradation by two fungi in a freshwater sediment and evaluation of fungal biomass by ergosterol content.

Mucor racemosus var. sphaerosporus and Phialophora alba were investigated for their abilities to degrade pyrene in a freshwater sediment, with or without glucose supply as nutrient or carbon source, during 90 days. The ergosterol contents in sediment were quantified to estimate fungal biomass and to assess the correlation between fungal activity and biodegradation of pyrene. Results showed that, in an heterogeneous environment, these fungi presented different abilities to degrade pyrene. P. alba increased the degree of pyrene degradation by 9%, compared to the native micro-organisms, but a supply of glucose acted as an inhibitor to pyrene disappearance. M. racemosus var. sphaerosporus was not efficient at sediment bioremediation (with or without glucose added), because it reduced the rate of pyrene degradation by the native microflora. In any case, there was no increase of ergosterol in boxes during bioremediation experiments. In our experimental conditions, ergosterol content could not be correlated to pyrene degradation.