Biosynthesis of silver nanoparticles by the endophytic fungus Epicoccum nigrum and their activity against pathogenic fungi

There is an enormous interest in developing safe, cost-effective and environmentally friendly technologies for nano-materials synthesis. In the present study, extracellular biosynthesis of silver nanoparticles was achieved by Epicoccum nigrum, an endophytic fungus isolated from the cambium of Phellodendron amurense. The reduction of the silver ions was monitored by UV–visible spectrophotometry, and the characterization of the Ag NPs was carried out by X-ray diffraction and transmission electron microscopy. The synthesized Ag NPs were exceptionally stable. It was found that an alkaline pH favored the formation of Ag NPs and elevated temperature accelerated the reduction process. Furthermore, the antifungal activity of the Ag NPs was assessed using a microdilution method. The biosynthesized Ag NPs showed considerable activity against the pathogenic fungi. The current research opens a new path for the green synthesis of Ag NPs and the process is easy to scale up for biomedical applications.     

Biosynthesis and function of melanins in hepatic pigmentary system

In this report we show the most important results obtained from our study of the pigment liver cells of Amphibia and Reptilia. Contrary to the cutaneous pigment cells that derive from the neural crest, the liver pigment cells, instead, derive from the Kupffer cells: this can be seen from the results obtained from labelled precursor incorporation experiments, not only in vivo but also in vitro, using surviving liver slices or isolated melanosomes. Chemical analysis of both liver and cutaneous melanosomes reveal a great difference in their chemical composition, and this is in agreement with the different origins of these cells. We therefore propose that: liver pigment cells of Amphibia and Reptilia should be classified as "Extra Cutaneous Pigment Cells from Histocytic Origin". As regards the function of melanins, we show that O2 is trapped by these substances. Moreover, in my laboratory we have shown in several animal species, that the superoxide dismutase activity is inversely proportional to the quantity of melanin present; thus, we think that melanin could mime SOD activity.     

Inhibition of the lysis of fungi by melanins

Evidence is presented that the resistance of Aspergillus nidulans hyphae to lysis by a β-(1→3) glucanase-chitinase mixture results from the presence of melanin in the fungal walls. The resistance of the walls to digestion was directly correlated with the melanin content of the mycelium. A melanin-less mutant of A. nidulans was highly susceptible to hydrolysis by the enzyme mixture. Preincubation of a synthetic melanin with the glucanase, chitinase, and a protease, before addition of the substrate, resulted in a marked inhibition of the rate of substrate hydrolysis. Melanin also appeared to combine with and protect at least certain substrates from decomposition, as indicated by the direct relationship between the extent of inhibition of casein hydrolysis by a bacterial protease and the length of time the protein was incubated with the melanin prior to addition of the enzyme. Melanin was found to be highly resistant to microbial degradation, a likely requirement for the polyaromatic to be effective in protecting fungal structures from lysis or decomposition by natural communities of microorganisms.     

Inositol phosphoryl transferases from human pathogenic fungi

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.     

Screening of micro-organisms for decolorization of melanins produced by bluestain fungi

A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate. On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747. The four fungi were more efficient on nitrogen-limited medium than on complete medium. The melanin-decolorizing activity of G. geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.     

Sugars in hydrolysates of fungal melanins and soil humic acids

Humic acids from four Brazilian topsoils of different origin and four fungal melanins, synthesized under two cultural conditions were subjected to a two step hydrolysis procedure and the released monosaccharides qualitatively and quantitatively determined by gas-liquid chromatography. The neutral sugars, glucose, galactose, mannose, arabinose, xylose, fucose, rhamnose and the alcohol sugar inositol, were detected in most of the soil humic acid samples. The fungal melanins showed the presence of glucose, galactose, mannose and arabinose. Ribose was present in two out of the eight samples tested. Some quantitative differences in the two types of humic polymers were noted and expected considering their origins. However, similarities were more apparent than differences and give further indication that melanic fungi may play a significant role in the formation of soil humic acids.     

Phytotoxicity of pathogenic fungi and their mycotoxins to cereal seedling viability

Aspergillus flavus, Alternaria alternata and Fusarium oxysporum were found to be the pathogenic fungi mostly reducing cereal (barley, sorghum and wheat) seedlings. The pathogens have the ability to produce aflatoxin B1 and G1, diacetoxyscirpenol, kojic acid and tenuazonic acid that reduced seedling viability. The inhibition dose for 50% reduction (LD50) was recorded by aflatoxins at 0.83 mg L-1 for barley, 1.74 mg L-1 for wheat and 2.75 mg L-1 for sorghum. Diacetoxyscirpenol produced its inhibition at 1.26 mg L-1 for barley, 3.98 mg L-1 for wheat and 10 mg L-1 for sorghum. Kojic acid induced 50% inhibition at 63 mg L-1 for barley, 105 mg L-1 for wheat and 251 mg L-1 for sorghum. However, tenuazonic acid was less toxic where, the toxicity was ranged between 79-550 mg L-1. The inhibition in germination was more pronounced in barley followed by wheat and negligible in sorghum to all tested mycotoxins. This inhibition attributed to the reduction in seedling amylase activity. Amylase was also reduced in the same trend: barley > wheat > sorghum. Grain treatment with carboxin-captan and thiophanatemethyl-thiram at 1 g kg-1 grain increased seedlings vigour of wheat in sterilized soil by 45 and 22%, barley by 24 and 33% and sorghum by 15 and 30%, respectively. These fungicides have also a positive effect on cereal when soil was inoculated with A. flavus, A. alternata and F. oxysporum.     

Phytotoxicity of pathogenic fungi and their mycotoxins to cereal seedling viability

Aspergillus flavus, Alternaria alternata and Fusarium oxysporum were the pathogenic fungi most reduced cereal (barley, sorghum and wheat) seedlings. The pathogens have the ability to produce aflatoxin B₁ and G₁, diacetoxyscirpenol, kojic acid and tenuazonic acid that reduced seedling viability. The inhibition dose for 50% reduction (LD₅₀) was recorded by aflatoxins at 0.83 mg L^-1 for barley, 1.74 mg L^-1 for wheat and 2.75 mg L^-1 for sorghum. Diacetoxyscirpenol produced its inhibition at 1.26 mg L^-1 for barley, 3.98 mg L^-1 for wheat and 10 mg L^-1 for sorghum. Kojic acid induced 50% inhibition at 63 mg L^-1 for barley, 105 mg L^-1 for wheat and 251 mg L^-1 for sorghum. However, tenuazonic acid was less toxic where the toxicity ranged between 79–550 mg L^-1. The germination inhibition was more pronounced in barley followed by wheat and was negligible in sorghum for all tested mycotoxins. This inhibition was attributed to the reduction in the seedling amylase activity, where amylase was also reduced in the same trend: barley > wheat > sorghum. Grain treated with carboxin-captan and thiophanatemethyl-thiram at 1 g kg^-1 grain increased the seedlings vigour of wheat in sterilized soil by 45 and 22%, barley by 24 and 33% and sorghum by 15 and 30%, respectively. These fungicides also had a positive effect on cereal when the soil was inoculated with A. flavus, A. alternata and F. oxysporum, but the improvement was still below normal. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interactions between pathogenic fungi and human epithelial and endothelial surfaces

The adhesion of fungi to host cells is an important area of study. Knowledge of the molecular mechanisms involved in these interactions can be used to devise methods to interfere with them. Similar to many pathogens, loss of fungal adhesion to epithelial or endothelial cell surfaces results in a marked decrease in virulence when evaluated in both in vivo and in vitro disease models. This review emphasizes literature from the past year and focuses on the molecular mechanisms by which fungi in the genera Candida, Cryptococcus, Sporothrix, Pneumocystis, and Aspergillus adhere to epithelial and/or endothelial host surfaces. The methodologies used to conduct these studies are also discussed.     

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.     

Identification and analysis of cation channel homologues in human pathogenic fungi

Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K(+)), calcium (Ca(2+)) and transient receptor potential (Trp) channels, but not sodium (Na(+)) channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K(+) and Trp channel subunits, and genes encoding novel homologues of voltage-gated K(v) channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca(2+) channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca(2+) channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca(2+) uniporter (MCU). In contrast to humans, which express many K(+), Ca(2+) and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K(+), Ca(2+) and Trp channel homologues. Furthermore, the sequences of fungal K(+), Ca(2+), Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.     

Host-microbe interactions: fungi invasive human fungal opportunistic infections

Jean-Paul's research interest is focused on the analysis of the structure and biosynthesis of the cell wall of Aspergillus fumigatus and its interaction with the host. The A. fumigatus genome will now be used to understand multifactorial systems such as fungal virulence of an opportunistic fungus in an immunocompromised host and assembly and regulation of cell wall polymer rearrangement under the control of the environment.     

Survey of soils for human pathogenic fungi from the Emilia-Romagna region of Italy

Summary A group of 166 soil samples collected in the Emilia-Romagna region of Italy from avian and chiropteran habitats was examined for the presence of keratinophilic fungi. One hundred forty-four isolates, classified among 14 genera, were obtained. The majority of the fungi were members of the family Gymnoascaceae. The significance of these findings was discussed.     

Icicles as carriers of yeast-like fungi potentially pathogenic to human

Most of the area of the globe is characterized by a low temperature, and the ice being formed constitutes a specific habitat for the growth of psychrotolerant microbes. They are accompanied by contaminants dripping from flat surfaces or falling down from the atmosphere. The objective of this study was, therefore, to detect the presence of potentially pathogenic fungi in ice formations found at the height of up to 2 m. The collected ice formations were transported to the laboratory and left for free melting. Yeast-like fungi were isolated with the use of a standard research procedure, typical of diagnostic mycological laboratories. The icicles examined were found to contain a high number of fungi, reaching 15,180 cfu/dm³ that originated from 12 species belonging to 2 genera: Debaryomyces and Candida. The predominating fungi were these of the genus Candida??constituting as much as 83.33% of the isolates. The study revealed also potential pathogens??Candida krusei, C. tropicalis, and C. utilis. The presence of fungi in the material examined allows ice formations to be treated as a reservoir and a vector of pathogenic factors and a temporary air filter. It additionally enforces the significance of health-promoting education that plays a key role in developing epidemiological awareness of the society.     

Study of photochemical and surface-active melanins of some representatives of black fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < lambda < 400 nm) and in the blue-violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm2. The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.