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.

Melanin: Quantification and protection against oxidative stress in chromoblastomycosis agents.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous infection caused by melanized fungal species. We quantified the extractable melanin of 77 strains of CBM agents distributed within five genera. Moreover, resistance to oxidative stress was evaluated in strains exposed or not to the melanin inhibitor tricyclazole. The median percentage of melanin mass extracted from dry fungal mass varied from 0.69 (Rhinocladiella similis) to 3.81 (Phialophora americana). Inhibition of melanin synthesis decreased survival rates to hydrogen peroxide. Together, these data highlight the importance of melanin in CBM agents.

Combination of Amphotericin B and Terbinafine against Melanized Fungi Associated with Chromoblastomycosis.

Our in vitro studies showed that a combination of amphotericin B and terbinafine had synergistic effects against the majority of melanized fungi associated with chromoblastomycosis (CBM) and similar infections, including those with Cladophialophora carrionii, Cladophialophora arxii, Exophialadermatitidis, Exophialaspinifera, Fonsecaea monophora, Fonsecaea nubica, Fonsecaea pedrosoi, and Phialophora verrucosa. This drug combination could provide an option for the treatment of severe or unresponsive cases of CBM, particularly in cases due to species of Fonsecaea and Cladophialophora.

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 and their combinations against Phialophora verrucosa.

The in vitro activities of nine antifungal drugs and their combinations against 31 clinical and 15 environmental Phialophora verrucosa strains were tested. The MIC90/90% minimum effective concentration (MIC/MEC90) values (µg/ml) across all strains were as follows: for terbinafine, 0.25; for posaconazole, 0.5; for voriconazole, 1; for itraconazole, 2; for amphotericin B, 4; for caspofungin and micafungin, 16; and for fluconazole and flucytosine, 64. The highest synergy was shown by the combination of itraconazole plus caspofungin (with synergy against 100% of the 31 clinical strains), followed by amphotericin B plus flucytosine (45.2%) and itraconazole plus terbinafine or micafungin (25.8% or 12.9%, respectively).

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.

Cytotoxic and antimicrobial activities of aporphine alkaloids isolated from Stephania venosa (Blume) Spreng.

The cytotoxic activity of five alkaloids, namely 4,5-dioxo-dehydrocrebanine (1), dehydrocrebanine (2), crebanine (3), oxostephanine (4), and thailandine (5) isolated from the tuber and leaves of Stephania venosa (Blume) Spreng was investigated. Thailandine showed the strongest activity against lung carcinoma cells (A549) (IC50 of 0.30 µg/mL) with very low cytotoxicity against normal embryonic lung cells (MRC-5). Thailandine also demonstrated strong activity against Plasmodium falciparum, K1 strain (IC50 of 20 ng/mL), and Mycobacterium tuberculosis H(37)Ra (MIC of 6.25 µg/mL) as well as gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. Oxostephanine exhibited strong activity against breast cancer (BC) and acute lymphoblastic leukemia cells (MOLT-3) with an IC50 of 0.24 and 0.71 µg/mL, respectively, and exhibited very low cytotoxicity against MRC-5 cells. Dehydrocrebanine demonstrated strong activity against promyelocytic leukemia cells (HL-60) with an IC50 of 2.14 µg/mL whereas crebanine showed weak activity against cancer cell lines. However, both of them showed cytotoxicity against MRC-5 cells.

Isolation, characterization, and sensitivity to 2,4-diacetylphloroglucinol of isolates of Phialophora spp. from Washington wheat fields.

Dark pigmented fungi of the Gaeumannomyces-Phialophora complex were isolated from the roots of wheat grown in fields in eastern Washington State. These fungi were identified as Phialophora spp. on the basis of morphological and genetic characteristics. The isolates produced lobed hyphopodia on wheat coleoptiles, phialides, and hyaline phialospores. Sequence comparison of internal transcribed spacer regions indicated that the Phialophora isolates were clearly separated from other Gaeumannomyces spp. Primers AV1 and AV3 amplified 1.3-kb portions of an avenacinase-like gene in the Phialophora isolates. Phylogenetic trees of the avenacinase-like gene in the Phialophora spp. also clearly separated them from other Gaeumannomyces spp. The Phialophora isolates were moderately virulent on wheat and barley and produced confined black lesions on the roots of wild oat and two oat cultivars. Among isolates tested for their sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), the 90% effective dose values were 11.9 to 48.2 microg ml(-1). A representative Phialophora isolate reduced the severity of take-all on wheat caused by two different isolates of Gaeumannomyces graminis var. tritici. To our knowledge, this study provides the first report of an avenacinase-like gene in Phialophora spp. and demonstrated that the fungus is significantly less sensitive to 2,4-DAPG than G. graminis var. tritici.

In vitro interaction of terbinafine with itraconazole and amphotericin B against fungi causing chromoblastomycosis in China.

The combined effects of terbinafine with itraconazole and amphotericin B against Cladophialophora carrionii, Phialophora verrucosa and Fonsecaea pedrosoi were evaluated in vitro by the checker-board method and expressed as a fractional inhibitory concentration (FIC) index. Synergy was observed with the combination of terbinafine and itraconazole against one isolate of C. carrionii and no antagonism was observed. When amphotericin B was combined with terbinafine or itraconazole, no synergy or antagonism was noted with all isolates included in this investigation.

Atypical eumycetoma caused by Phialophora parasitica successfully treated with itraconazole and flucytosine.

Phialophora species are occasional pathogens causing subcutaneous and invasive disease. We report the first case of eumycetoma caused by P. parasitica in an otherwise healthy U.K. resident who visited India. She failed to respond to surgical excision and itraconazole, 400 mg daily, but responded to itraconazole, 400 mg daily, and flucytosine, 1 g three times daily, for 12 months. In vitro susceptibility testing predicted a response.

[Diagnosis, clinical aspects and therapy of early chromoblastomycosis in a case example]. / Diagnostik, Klinik und Therapie der frühen Chromoblastomykose an einem Fallbeispiel.

Despite the availability of modern antimycotics, which produce high cure rates in early infections, the therapy of advanced chromoblastomycosis is still unsatisfactory. An initial chromoblastomycosis caused by a hitherto unidentified species of the genus Phialophora was diagnosed in a 46-year-old teacher. The organism was isolated twice at an interval of 6 weeks from a partly psoriasiform, partly verrucous lesion on the 4th toe. The infection was apparently acquired 4 years ago during a holiday at Cape Verde. Treatment with itraconazole (Sempera). 200 mg/day, and amphotericin B (Ampho-Moronal) cream for 6 weeks initially resulted in rapid regression. However, 4 weeks after cessation of therapy, the Phialophora species was cultured again from skin scrapings. Complete healing was achieved after re-treatment with itraconazole for 20 weeks at the same dosage in combination with topical amorolfine and local hyperthermia. Until now, no relapse has occurred. The present case demonstrates that this rare disease, which mainly occurs as a traumatic mycosis in the rural population of tropical regions, must be included in the differential diagnosis of psoriasiform or verrucous skin lesions and also included in the list of diseases which may be acquired while on vacation in exotic locations.

Sex hormone effects on Phialophora verrucosa in vitro and characterization of progesterone receptors.

To determine the effects of sex steroid hormones on the growth of an aetiologic agent of chromoblastomycosis, we studied the dematiaceous fungus Phialophora verrucosa. The in vitro growth of this species on culture media containing either progesterone, testosterone or oestradiol at various concentrations was assessed. Both progesterone and testosterone inhibited the growth of P. verrucosa, whereas oestradiol did not. In other experiments, fungal cytosolic fractions were obtained and steroid binding assays were performed. These studies showed that the presence of progesterone receptors possessed two binding sites as determined by Scatchard analysis, one of which has a high affinity to progesterone (Kd = 6.02 x 10(-8) M) with total binding sites of 120 fmol micrograms-1 protein. These findings suggest that the growth of P. verrucosa is regulated by steroid hormones and that the effect of progesterone could be mediated through fungal intracellular progesterone receptors.

Calcium regulates in vitro dimorphism in chromoblastomycotic fungi.

Cladosporium carrionii, Fonsecaea pedrosoi and Phialophora verrucosa, the three most important agents of chromoblastomycosis, produced large numbers of sclerotic bodies at 25 degrees C, and greater numbers at 37 degrees C, after inoculation into a defined pH 2.5 medium containing 0.1 mmol l-1 Ca2+. Higher concentrations of Ca2+ reversed this tendency and promoted maintenance of hyphal growth. Addition of the Ca2+ chelator EGTA to the same medium buffered at pH 6.5 also induced sclerotic bodies, but in a more concentration-dependent fashion. EGTA at 0.5-1.0 mmol l-1 induced maximum numbers of sclerotic bodies in Cl. carrionii, whereas 2 and 8 mmol l-1 concentrations were required for the same results with F. pedrosoi and P. verrucosa, respectively. These findings suggest that Ca2+ concentrations in human tissue may play a paramount role in the dimorphic switching between hyphae and sclerotic bodies among chromoblastomycotic agents during infection.

Early experience with itraconazole in vitro and in patients: pharmacokinetic studies and clinical results.

The efficacy of itraconazole, a new triazole antifungal agent, was studied in vitro and assessed in patients. The MICs of itraconazole for 16 strains of Aspergillus fumigatus were in the same range as those of amphotericin B: less than 0.09-0.36 microgram/ml vs. less than 0.09-0.78 microgram/ml, respectively. Eight adult patients with systemic fungal infections were treated orally with 100-200 mg of itraconazole two times a day. A patient with relapsing histoplasmosis (Histoplasma capsulatum var. duboisii) was cleared of the infection; a patient with arthritis of the knee due to Phialophora parasitica did not respond to treatment; four patients with semiinvasive pulmonary aspergillosis improved dramatically and were considered clinically cured; and two patients with aspergilloma improved. The duration of follow-up was one to nine months. Levels of itraconazole in body fluids were measured by a bioassay. Levels of drug in knee fluid were about 30% of the simultaneous levels in plasma. A progressive decrease in the level of itraconazole in plasma occurred in two patients, and a progressive increase in the levels occurred in five patients.

Cell-cell recognition of host surfaces by pathogens. The adsorption of maize (Zea mays) root mucilage by surfaces of pathogenic fungi.

The adsorption of radioactive mucilage by pathogenic fungi was shown to be dependent upon time, the composition of mucilage, the type of fungal surface (conidia, hyphae, hyphal apices), fungal species, pH and bivalent cations. All fungal adhesins were inactivated by either proteinase or polysaccharase treatments. Adsorption was not inhibited by the numberous mono-, di- and oligo-saccharides that were tested individually, but it was inhibited absolutely by several polysaccharides. This suggested that adsorption of mucilage by pathogens involved conformational and ionic interactions between plant and fungal polymers but not fungal lectins bound to sugar residues of mucilage. Several fractionation schemes showed that pathogens bound only the most acidic of the variety of polymers that comprise mucilage. There was not any absolute distinction between ability to bind radioactive mucilage and type of pathogen or non-pathogen. However, there were notable differences in characteristics of adsorption between two types of pathogen. Differences were revealed by comparison of the adsorption capacities of conidia and germinant conidia and chromatography of radioactive mucilage on germinant conidia. An ectotrophic root-infecting fungus (a highly specialized pathogen) bound a greater proportion of mucilage than did a vascular-wilt fungus (of catholic host and tissue range) with more than one class of site for adsorption. In contrast with the vascular-wilt fungus, sites for adsorption on the specialized pathogen were present solely on surfaces formed by germination.

Fungal peritonitis caused by Lecythophora mutabilis.

Fungal peritonitis caused by Lecythophora mutabilis, a mold rarely isolated from humans, is described. A patient on continuous peritoneal dialysis developed clinical, microbiological, and serological evidence for peritonitis due to this fungus. In vitro susceptibility testing of the fungus revealed marked differences in the activities of various antifungal agents. Although initially responding to treatment with oral ketoconazole, intraperitoneal miconazole, and catheter replacement, the patient had a documented relapse. The patient was eventually cured following the removal of a second catheter in association with prolonged imidazole treatment.

Antimycotic azoles. 7. Synthesis and antifungal properties of a series of novel triazol-3-ones.

A series of novel triazol-3-ones have been synthesized, and their in vitro and in vivo antifungal properties are reported. Compound 68 (itraconazole), which displays a pronounced oral activity against vaginal candidosis in rats and against microsporosis in guinea pigs, has been selected for clinical evaluation.

Phialophora richardsiae isolated from infected human bone: morphological, physiological and antifungal susceptibility studies.

A dematiaceous fungus, Phialophora richardsiae (Nannf.) Conant, was isolated from human bone. In culture the fungus produced no yeast forms and was less pigmented than two other P. richardsiae isolates. While growth rates were similar, colonial forms differed. Phialides were of two kinds. While both had broad bases and tapered at the tips, only one terminated with a cupulate or rarely a saucer-shaped collarette. Most phialides were hyaline with a few lightly pigmented ones in older cultures. Broth dilution susceptibility testing of the isolates against amphotericin B, miconazole, ketoconazole, clotrimazole, and 5-fluorocytosine showed the fungus was susceptible to miconazole, ketoconazole and amphotericin B at achievable serum levels and resistant to 5-fluorocytosine and clotrimazole. The other isolates were reported to differ in their resistance to miconazole and amphotericin B. Enzyme and salinity studies showed minor difference among the isolates.