Sonia Rozental

Antimicrobial and antiviral activities of polyphenolics from Cocos nucifera Linn. (Palmae) husk fiber extract

The decoction of Cocos nucifera L. husk fiber has been used in northeastern Brazil traditional medicine for treatment of diarrhea and arthritis. Water extract obtained from coconut husk fiber and fractions from adsorption chromatography revealed antimicrobial activity against Staphylococcus aureus. The crude extract and one of the fractions rich in catechin also showed inhibitory activity against acyclovir-resistant herpes simplex virus type 1 (HSV-1-ACVr). All fractions were inactive against the fungi Candida albicans, Fonsecaea pedrosoi and Cryptococcus neoformans. Catechin and epicatechin together with condensed tannins (B-type procyanidins) were demonstrated to be the components of the water extract.

Chromoblastomycosis: a retrospective study of 325 cases on Amazonic Region (Brazil).

A retrospective study of 325 cases of chromoblastomycosis diagnosed in the last 55 years in the Amazon region was carried out by the main Mycology services of the state of Pará, Brazil (Department of Tropical Pathology – UFPA and Mycology Department of the Evandro Chagas Institute/FNS). The data obtained showed that: (a) the main age group affected by the diseases range from 41 to 70 years-old, (b) 86.1% of the patients were agricultural-workers, (c) 93.2% of them were males and (d) 80.7% showed lesions on the lower limbs (feet and legs). The diagnosis of 62% of the cases was confirmed by laboratory studies considering the tissue form in histopathological analysis. In 24% of patients (78 cases), the etiological agent was isolated and identified through culture. Fonsecaea pedrosoi was present in 77 cases and Phialophora verucosa in only one case.

Melanin from Fonsecaea pedrosoi Induces Production of Human Antifungal Antibodies and Enhances the Antimicrobial Efficacy of Phagocytes

ABSTRACT Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients’ sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients’ lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans.

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.

The in vitro susceptibility ofFonsecaea pedrosoi to activated macrophages

Light and electron microscopy were used to analyze in vitro the interaction ofFonsecaea pedrosoi with in vivo activated-macrophages. Adherence of the fungi to the surface of activated macrophages triggers the respiratory burst as revealed by reduction of nitroblue tetrazolium. Transmission electron microscopy revealed NAD(P)H-oxidase activity in the portions of the macrophage plasma of membrane that were in contact with the fungus as well as within phagocytic vacuoles. Activated macrophages failed to kill ingested fungi, but they showed a fungistatic activity delaying germ tube and hyphae formation.

Effect of alkylphospholipids on Candida albicans biofilm formation and maturation

OBJECTIVES The aim of this study was to evaluate miltefosine and four synthetic compounds (TCAN26, TC19, TC106 and TC117) for their in vitro inhibitory activity against Candida albicans planktonic and biofilm cells and investigate whether these compounds are able to inhibit the biofilm formation and to reduce the viability of mature C. albicans biofilm cells. METHODS The XTT reduction assay and transmission and scanning electron microscopy were employed to determine the inhibitory effects of the test compounds in comparison with amphotericin B and fluconazole against both planktonic cells and sessile cells in biofilms. RESULTS C. albicans planktonic cells were susceptible to miltefosine, TCAN26 and TC19, all alkylphospholipid compounds. Miltefosine and TCAN26 present a fungicidal activity with similar values of MIC and minimum fungicidal concentration (MFC), ranging from 2 to 8 mg/L. Cell treatment with sub-inhibitory concentrations of alkylphospholipids induced several ultrastructural alterations. In relation to biofilms, miltefosine reduced formation (38%-71%) and mature biofilms viability (32%-44%), at concentrations of 64 mg/L. TCAN26 also reduced biofilm formation (24%-30%) and mature biofilm viability (15%-20%), at concentrations of 64 mg/L. Although amphotericin B reduced biofilm formation similarly to miltefosine (51%-74%), its activity was lower on mature biofilms (24%-30%). Miltefosine antibiofilm activity was significantly higher than amphotericin B, on both formation and mature biofilms (P<0.05 and P<0.0001, respectively). Fluconazole was the least effective compound tested. CONCLUSION Promising antibiofilm activity was displayed by miltefosine and other alkylphosphocholine compounds, which could be considered a putative option for future treatment of candidaemia associated with biofilm formation, although further evaluation in in vivo systems is required.

Phosphatase activity on the cell wall of Fonsecaea pedrosoi.

The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.

Melanin in Fonsecaea pedrosoi: a trap for oxidative radicals

BackgroundThe 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. pedrosoi. Experiments were carried out with or without tricyclazole (TC) treatment, a selective inhibitor of the dihydroxynaphthalene (DHN)-melanin biosynthesis pathway in F. pedrosoi. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. The fungal growth responses to H2O2 and to S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, were also evaluated.ResultsMelanised F. pedrosoi cells were more resistant to both H2O2 and NO. Nitrite was not detected in the supernatant of macrophages incubated with melanised fungal cells. However, i-NOS expression was unaffected by the presence of either untreated control F. pedrosoi or TC-treated F. pedrosoi. In addition, the inhibition of the DHN-melanin pathway by TC improved the oxidative burst capability of the macrophages.ConclusionThe NO-trapping ability of F. pedrosoi melanin is an important mechanism to escape the oxidative burst of macrophages.

The Aspergillus fumigatus transcription factor Ace2 governs pigment production, conidiation and virulence

Aspergillus fumigatus causes serious and frequently fatal infections in immunocompromised patients. To investigate the regulation of virulence of this fungus, we constructed and analysed an A. fumigatus mutant that lacked the transcription factor Ace2, which influences virulence in other fungi. The Δace2 mutant had dysmorphic conidiophores, reduced conidia production and abnormal conidial cell wall architecture. This mutant produced an orange pigment when grown on solid media, although its conidia had normal pigmentation. Conidia of the Δace2 mutant were larger and had accelerated germination. The resulting germlings were resistant to hydrogen peroxide, but not other stressors. Non‐neutropenic mice that were immunosuppressed with cortisone acetate and infected with the Δace2 mutant had accelerated mortality, greater pulmonary fungal burden, and increased pulmonary inflammatory responses compared with mice infected with the wild‐type or Δace2::ace2‐complemented strains. The Δace2 mutant had reduced ppoC, ecm33 and ags3 mRNA expression. It is known that A. fumigatus mutants with absent or reduced expression of these genes have increased virulence in mice, as well as other phenotypic similarities to the Δace2 mutant. Therefore, reduced expression of these genes likely contributes to the increased virulence of the Δace2 mutant.

Susceptibility of Sporothrix brasiliensis isolates to amphotericin B, azoles, and terbinafine

The in vitro activity of the antifungal agents amphotericin B (AMB), itraconazole (ITC), posaconazole (PSC), voriconazole (VRC), and terbinafine (TRB) against 32 Brazilian isolates of Sporothrix brasiliensis, including 16 isolates from a recent (2011-2012) epidemic in Rio de Janeiro state, was examined. We describe and genotype new isolates and clustered them with 16 older (from 2004 or earlier) S. brasiliensis isolates by phylogenetic analysis. We tested both the yeast and the mycelium form of all isolates using broth microdilution methods based on the reference protocols M38-A2 and M27-A3 (recommended by the Clinical and Laboratory Standards Institute). Considering minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), TRB was found to be the most active drug in vitro for both fungal forms, followed by PSC. Several isolates showed high MICs for AMB and/or ITC, which are currently used as first-line therapy for sporotrichosis. VRC displayed very low activity against S. brasiliensis isolates. The primary morphological modification observed on treated yeasts by transmission electron microscopy analysis was changes in cell wall. Our results indicate that TRB is the antifungal with the best in vitro activity against S. brasiliensis and support the use of TRB as a promising option for the treatment of cutaneous and/or lymphocutaneous sporotrichosis.