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Judit Kucsera - One of the best experts on this subject based on the ideXlab platform.
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Comparison of killer toxin-producing and non-producing strains of Filobasidium capsuligenum: proposal for two varieties.
Microbiological research, 2008Co-Authors: Andrea Keszthelyi, Ilona Pfeiffer, Zsuzsanna Hamari, Csaba Vágvölgyi, Judit KucseraAbstract:Summary The basidiomycetous yeast, Filobasidium capsuligenum, produces killer toxin against the opportunistic pathogen Cryptococcus neoformans. Not every strain isolated so far is able to produce the anti cryptococcal toxin. The aim of the present work was to study the relationship between the toxins and the toxin-producing and nonproducing isolates. The toxin was coded on chromosomal DNA in each producing strain as molecular analysis revealed. In addition, both the killing spectra and biochemical properties of the toxins proved to be identical, thus intraspecific variation in the toxin was not found. For molecular typing of the isolates, the D1/D2 region of 26S rDNA, partial sequences of internal transcribed spacer (ITS) regions, PCR fingerprinting RAPD and mtDNA-RFLP patterns were examinated. Phylogenetic analyses based on the different approaches showed that strains with the ability of killer-toxin production and those without it differ significantly and cluster into two distinct groups. The differences between the two groups and the similarity within them suggest the authority to separate the species into varieties. & 2008 Elsevier GmbH. All rights reserved.
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Patogén Crytococcusok ellen hatásos killer toxin izolálása, jellemzése és a kódoló gén lokalizálása, klónozása Filobasidium capsulogenumban = Isolation and characterization of killer toxin active against pathogen Cryptococcus, localization and clonin
2007Co-Authors: Judit Kucsera, Ilona Pfeiffer, Zsuzsanna Hamari, Ferenc Kevei, Zsuzsanna Buzás, András Patthy, József Antal, Attila GácserAbstract:A basidiomycota Filobasidium capsuligenum egy olyan feherjet szekretal, amely elpusztitja a patogen Cryptococcus neoformanst. Palyazatunkban ezen killer toxin genetikai determinansat deritettuk fel, a toxin feherje izolalasat, jellemzeset valositottuk meg, ill a toxin hatasmechanizmusat is feltartuk. Az erzekenysegi tesztek alapjan az FC-1 toxin specifikusan, csak a C. neoformans-ra hatott. Bebizonyitottuk, hogy e hatas citocidikus. A toxin protein termeszetű: hő hatasara elbomlik, aktivitasanak optimuma pH 4-6. A toxin receptorakent az erzekeny sejtek sejtfalanak beta-1,6-glukanjat hataroztuk meg. E specificitast arra hasznaltuk fel, hogy a toxint affinitaskromatografiaval tisztitsuk. A pusztulan-Sepharose 6B oszlop legaktivabb frakciojaval SDS-PAGE-t vegeztunk, es 3 protein savot talaltunk a 19 - 51 kDa tartomanyban. Tovabbi kiserletek szuksegesek a killer aktivitasu protein azonositasara. A F. capsuligenum sejtek minilizatainak agaroz gelelektroforezisevel kideritettuk, hogy az FC-1 proteint kromoszomalis gen kodolja. A toxin hatasmodjanak tanulmanyozasara a cellularis DNS mennyisegenek valtozasat a sejtciklus fuggvenyeben (DNS szintezis gatlas?) ill. a toxin kezelt sejtek FITC festődeset (sejtfal bioszintezis gatlas?) vizsgaltuk. Eredmenyeink szerint a killing mechanizmus sem a sejtciklustol, sem pedig a sejtfal bioszintezistől nem fugg. Sokkal inkabb ionofor proteinkent hat, amely a citoplazma membranfunkciojat rombolja. | The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the pathogen Cryptococcus neoformans. The goal of this project was to characterize the toxin, determine the genetic trait coding for it, and study its effect on C. neoformans cells. It was demonstrated that FC-1 toxin was specific for C. neoformans. The toxin had a cytocidal effect. FC-1 proved to be a protein: sensitive for heat and proteolytic enzymes. The optimal pH for the activity was within the range of pH 4-6. As receptor site for the killer protein beta-1,6-glucan in the cell wall of sensitive cells was determined. The specificity was used for purification of the toxin through affinity chromatography. The most active fraction from a pustulan-Sepharose 6B column was subjected to SDS-PAGE, and three protein bands, in molecular mass of 19 - 51 kDa, were detected. Additional investigations are necessary to identify the protein with killer property. Agarose gel electrophoresis of F. capsuligenum cell minilysate revealed that FC-1 protein is coded by the chromosomal DNA. Analysis of cellular DNA (inhibition of DNA synthesis?) by laser scanning cytometry and FITC staining (inhibition of cell wall biosynthesis?) of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it might act as an ionophoric protein that disrupts the cytoplasmic membrane function.
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patogen crytococcusok ellen hatasos killer toxin izolalasa jellemzese es a kodolo gen lokalizalasa klonozasa Filobasidium capsulogenumban isolation and characterization of killer toxin active against pathogen cryptococcus localization and cloning of
2007Co-Authors: Judit Kucsera, Ilona Pfeiffer, Zsuzsanna Hamari, Ferenc Kevei, Zsuzsanna Buzás, András Patthy, József Antal, Attila GácserAbstract:A basidiomycota Filobasidium capsuligenum egy olyan feherjet szekretal, amely elpusztitja a patogen Cryptococcus neoformanst. Palyazatunkban ezen killer toxin genetikai determinansat deritettuk fel, a toxin feherje izolalasat, jellemzeset valositottuk meg, ill a toxin hatasmechanizmusat is feltartuk. Az erzekenysegi tesztek alapjan az FC-1 toxin specifikusan, csak a C. neoformans-ra hatott. Bebizonyitottuk, hogy e hatas citocidikus. A toxin protein termeszetű: hő hatasara elbomlik, aktivitasanak optimuma pH 4-6. A toxin receptorakent az erzekeny sejtek sejtfalanak beta-1,6-glukanjat hataroztuk meg. E specificitast arra hasznaltuk fel, hogy a toxint affinitaskromatografiaval tisztitsuk. A pusztulan-Sepharose 6B oszlop legaktivabb frakciojaval SDS-PAGE-t vegeztunk, es 3 protein savot talaltunk a 19 - 51 kDa tartomanyban. Tovabbi kiserletek szuksegesek a killer aktivitasu protein azonositasara. A F. capsuligenum sejtek minilizatainak agaroz gelelektroforezisevel kideritettuk, hogy az FC-1 proteint kromoszomalis gen kodolja. A toxin hatasmodjanak tanulmanyozasara a cellularis DNS mennyisegenek valtozasat a sejtciklus fuggvenyeben (DNS szintezis gatlas?) ill. a toxin kezelt sejtek FITC festődeset (sejtfal bioszintezis gatlas?) vizsgaltuk. Eredmenyeink szerint a killing mechanizmus sem a sejtciklustol, sem pedig a sejtfal bioszintezistől nem fugg. Sokkal inkabb ionofor proteinkent hat, amely a citoplazma membranfunkciojat rombolja. | The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the pathogen Cryptococcus neoformans. The goal of this project was to characterize the toxin, determine the genetic trait coding for it, and study its effect on C. neoformans cells. It was demonstrated that FC-1 toxin was specific for C. neoformans. The toxin had a cytocidal effect. FC-1 proved to be a protein: sensitive for heat and proteolytic enzymes. The optimal pH for the activity was within the range of pH 4-6. As receptor site for the killer protein beta-1,6-glucan in the cell wall of sensitive cells was determined. The specificity was used for purification of the toxin through affinity chromatography. The most active fraction from a pustulan-Sepharose 6B column was subjected to SDS-PAGE, and three protein bands, in molecular mass of 19 - 51 kDa, were detected. Additional investigations are necessary to identify the protein with killer property. Agarose gel electrophoresis of F. capsuligenum cell minilysate revealed that FC-1 protein is coded by the chromosomal DNA. Analysis of cellular DNA (inhibition of DNA synthesis?) by laser scanning cytometry and FITC staining (inhibition of cell wall biosynthesis?) of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it might act as an ionophoric protein that disrupts the cytoplasmic membrane function.
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Characterisation of the anticryptococcal effect of the FC-1 toxin produced by Filobasidium capsuligenum.
Mycoses, 2006Co-Authors: Andrea Keszthelyi, Kanji Takeo, M. Ohkusu, Ilona Pfeiffer, Judit Litter, Judit KucseraAbstract:The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the opportunistic fungal pathogen Cryptococcus neoformans. The aim of this work was to study the effect of the toxin on C. neoformans cells. The sensitivities of strains representing eight molecular subtypes (VNI-IV and VGI-IV) of the C. neoformans species complex, and of an additional 50 clinical and environmental isolates were determined. Analysis of cellular DNA by laser scanning cytometry and fluorescein isothiocyanate (FITC) staining of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it may act as an ionophoric protein that disrupts the cytoplasmic membrane function. The competition assay results suggest that beta-1,6-glucan in the cell wall may provide the binding site for the killer protein. This anticryptococcal toxin has the potential to be applied as a therapeutic agent for the treatment of cryptococcosis.
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Simple detection method for distinguishing dead and living yeast colonies
Journal of microbiological methods, 2000Co-Authors: Judit Kucsera, Kyoko Yarita, Kanji TakeoAbstract:A rapid and simple assay was developed for detection of yeast colonies containing dying or dead cells. Methylene blue, phloxin B, rose bengal and trypan blue at concentrations of 5-10 micromol l(-1) were shown to stain non-viable cells in colonies of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Filobasidium capsuligenum without staining or affecting the viability of living cells of the colonies.
Teun Boekhout - One of the best experts on this subject based on the ideXlab platform.
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Meningitis caused by Filobasidium uniguttulatum: case report and overview of the literature
Mycoses, 2011Co-Authors: Weihua Pan, Wanqing Liao, Ferry Hagen, Bart Theelen, Weimin Shi, Jacques F. Meis, Teun BoekhoutAbstract:Cryptococcal meningitis is mainly caused by Cryptococcus neoformans and Cryptococcus gattii, but occasionally other Cryptococcus species and phylogenetically related species are involved. Herein, we present a case of cryptococcal meningitis from China, which was caused by an azole and flucytosine resistant Filobasidium uniguttulatum. In addition, we present an overview of the literature of meningitis caused by Cryptococcus species other than C. neoformans and C. gattii. Eight cases were related to infections of the central nervous system. Leukaemia and cancer were important risk factors in HIV-negative patients. Molecular identification and susceptibility testing are important for proper management of patients because the species involved may differ in susceptibility to antifungal drugs. Keywords: Cryptococcus; Filobasidium uniguttulatum; identification; meningitis; treatmen
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Meningitis caused by Filobasidium uniguttulatum: case report and overview of the literature
Mycoses, 2011Co-Authors: Weihua Pan, Wanqing Liao, Ferry Hagen, Bart Theelen, Weimin Shi, Jacques F. Meis, Teun BoekhoutAbstract:Cryptococcal meningitis is mainly caused by Cryptococcus neoformans and Cryptococcus gattii, but occasionally other Cryptococcus species and phylogenetically related species are involved. Herein, we present a case of cryptococcal meningitis from China, which was caused by an azole and flucytosine resistant Filobasidium uniguttulatum. In addition, we present an overview of the literature of meningitis caused by Cryptococcus species other than C. neoformans and C. gattii. Eight cases were related to infections of the central nervous system. Leukaemia and cancer were important risk factors in HIV-negative patients. Molecular identification and susceptibility testing are important for proper management of patients because the species involved may differ in susceptibility to antifungal drugs.
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biodiversity and systematics of basidiomycetous yeasts as determined by large subunit rdna d1 d2 domain sequence analysis
International Journal of Systematic and Evolutionary Microbiology, 2000Co-Authors: Jack W Fell, Alvaro Fonseca, Gloria Scorzetti, Teun Boekhout, Adele StatzelltallmanAbstract:The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, CystoFilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.
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Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis.
International journal of systematic and evolutionary microbiology, 2000Co-Authors: Jack W Fell, Alvaro Fonseca, Gloria Scorzetti, Teun Boekhout, A Statzell-tallmanAbstract:The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, CystoFilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.
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Dimorphism in Itersonilia perplexans : yeast and hyphal phases differ in their sensitivity to mycocins produced by tremellaceous yeasts
Fems Microbiology Letters, 1992Co-Authors: Wladyslaw I. Golubev, Teun BoekhoutAbstract:The monokaryotic yeast phase of the heterobasidiomycete Itersonilia perplexans, unlike the hyphal phase, was found to be sensitive to mycocins produced by killer strains of Cryptococcus humicola, Cr. laurentii, CystoFilobasidium bisporidii and Rhodotorula fujisanense. Both the yeast and hyphal phases were resistant to mycocins of Cr. podzolicus, Filobasidium capsuligenum, Rhodotorula glutinis, Rh. mucilaginosa, Rh. pallida, Sporidiobolus johnsonii, Sb. pararoseus and Sporobolomyces alborubescens. The different sensitivity patterns of yeast and hyphal phases are probably caused by biochemical differences in the cell walls.
Andrea Keszthelyi - One of the best experts on this subject based on the ideXlab platform.
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Comparison of killer toxin-producing and non-producing strains of Filobasidium capsuligenum: proposal for two varieties.
Microbiological research, 2008Co-Authors: Andrea Keszthelyi, Ilona Pfeiffer, Zsuzsanna Hamari, Csaba Vágvölgyi, Judit KucseraAbstract:Summary The basidiomycetous yeast, Filobasidium capsuligenum, produces killer toxin against the opportunistic pathogen Cryptococcus neoformans. Not every strain isolated so far is able to produce the anti cryptococcal toxin. The aim of the present work was to study the relationship between the toxins and the toxin-producing and nonproducing isolates. The toxin was coded on chromosomal DNA in each producing strain as molecular analysis revealed. In addition, both the killing spectra and biochemical properties of the toxins proved to be identical, thus intraspecific variation in the toxin was not found. For molecular typing of the isolates, the D1/D2 region of 26S rDNA, partial sequences of internal transcribed spacer (ITS) regions, PCR fingerprinting RAPD and mtDNA-RFLP patterns were examinated. Phylogenetic analyses based on the different approaches showed that strains with the ability of killer-toxin production and those without it differ significantly and cluster into two distinct groups. The differences between the two groups and the similarity within them suggest the authority to separate the species into varieties. & 2008 Elsevier GmbH. All rights reserved.
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Characterisation of the anticryptococcal effect of the FC-1 toxin produced by Filobasidium capsuligenum.
Mycoses, 2006Co-Authors: Andrea Keszthelyi, Kanji Takeo, M. Ohkusu, Ilona Pfeiffer, Judit Litter, Judit KucseraAbstract:The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the opportunistic fungal pathogen Cryptococcus neoformans. The aim of this work was to study the effect of the toxin on C. neoformans cells. The sensitivities of strains representing eight molecular subtypes (VNI-IV and VGI-IV) of the C. neoformans species complex, and of an additional 50 clinical and environmental isolates were determined. Analysis of cellular DNA by laser scanning cytometry and fluorescein isothiocyanate (FITC) staining of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it may act as an ionophoric protein that disrupts the cytoplasmic membrane function. The competition assay results suggest that beta-1,6-glucan in the cell wall may provide the binding site for the killer protein. This anticryptococcal toxin has the potential to be applied as a therapeutic agent for the treatment of cryptococcosis.
Jack W Fell - One of the best experts on this subject based on the ideXlab platform.
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biodiversity and systematics of basidiomycetous yeasts as determined by large subunit rdna d1 d2 domain sequence analysis
International Journal of Systematic and Evolutionary Microbiology, 2000Co-Authors: Jack W Fell, Alvaro Fonseca, Gloria Scorzetti, Teun Boekhout, Adele StatzelltallmanAbstract:The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, CystoFilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.
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Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis.
International journal of systematic and evolutionary microbiology, 2000Co-Authors: Jack W Fell, Alvaro Fonseca, Gloria Scorzetti, Teun Boekhout, A Statzell-tallmanAbstract:The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, CystoFilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.
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diversity in the yeast cryptococcus albidus and related species as revealed by ribosomal dna sequence analysis
Canadian Journal of Microbiology, 1999Co-Authors: Alvaro Fonseca, Gloria Scorzetti, Jack W FellAbstract:Evidence accumulated from studies based on physiological, biochemical, and molecular characteristics has pointed to the heterogeneity of the ubiquitous anamorphic basidiomycetous yeast species Cryptococcus albidus (Saito) Skinner, with its current varieties and synonyms. The taxonomic status of this species has not been reappraised because different studies, mostly involving limited numbers of strains, have not been integrated. To assess species diversity within the clade containing Cryptococcus albidus and other phylogenetically related Cryptococcus and Filobasidium species, we determined ribosomal DNA (rDNA) sequences of 69 strains from the 5prime end of the 26S gene, D1/D2 region, and in some cases, the non-coding ITS2 region. Analysis of the sequence data together with available physiological, biochemical, and molecular characteristics, showed the segregation of C. albidus into at least 12 species, leading to the elevation of former varieties to the rank of species (C. aerius, C. diffluens), the reins...
Ilona Pfeiffer - One of the best experts on this subject based on the ideXlab platform.
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Comparison of killer toxin-producing and non-producing strains of Filobasidium capsuligenum: proposal for two varieties.
Microbiological research, 2008Co-Authors: Andrea Keszthelyi, Ilona Pfeiffer, Zsuzsanna Hamari, Csaba Vágvölgyi, Judit KucseraAbstract:Summary The basidiomycetous yeast, Filobasidium capsuligenum, produces killer toxin against the opportunistic pathogen Cryptococcus neoformans. Not every strain isolated so far is able to produce the anti cryptococcal toxin. The aim of the present work was to study the relationship between the toxins and the toxin-producing and nonproducing isolates. The toxin was coded on chromosomal DNA in each producing strain as molecular analysis revealed. In addition, both the killing spectra and biochemical properties of the toxins proved to be identical, thus intraspecific variation in the toxin was not found. For molecular typing of the isolates, the D1/D2 region of 26S rDNA, partial sequences of internal transcribed spacer (ITS) regions, PCR fingerprinting RAPD and mtDNA-RFLP patterns were examinated. Phylogenetic analyses based on the different approaches showed that strains with the ability of killer-toxin production and those without it differ significantly and cluster into two distinct groups. The differences between the two groups and the similarity within them suggest the authority to separate the species into varieties. & 2008 Elsevier GmbH. All rights reserved.
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Patogén Crytococcusok ellen hatásos killer toxin izolálása, jellemzése és a kódoló gén lokalizálása, klónozása Filobasidium capsulogenumban = Isolation and characterization of killer toxin active against pathogen Cryptococcus, localization and clonin
2007Co-Authors: Judit Kucsera, Ilona Pfeiffer, Zsuzsanna Hamari, Ferenc Kevei, Zsuzsanna Buzás, András Patthy, József Antal, Attila GácserAbstract:A basidiomycota Filobasidium capsuligenum egy olyan feherjet szekretal, amely elpusztitja a patogen Cryptococcus neoformanst. Palyazatunkban ezen killer toxin genetikai determinansat deritettuk fel, a toxin feherje izolalasat, jellemzeset valositottuk meg, ill a toxin hatasmechanizmusat is feltartuk. Az erzekenysegi tesztek alapjan az FC-1 toxin specifikusan, csak a C. neoformans-ra hatott. Bebizonyitottuk, hogy e hatas citocidikus. A toxin protein termeszetű: hő hatasara elbomlik, aktivitasanak optimuma pH 4-6. A toxin receptorakent az erzekeny sejtek sejtfalanak beta-1,6-glukanjat hataroztuk meg. E specificitast arra hasznaltuk fel, hogy a toxint affinitaskromatografiaval tisztitsuk. A pusztulan-Sepharose 6B oszlop legaktivabb frakciojaval SDS-PAGE-t vegeztunk, es 3 protein savot talaltunk a 19 - 51 kDa tartomanyban. Tovabbi kiserletek szuksegesek a killer aktivitasu protein azonositasara. A F. capsuligenum sejtek minilizatainak agaroz gelelektroforezisevel kideritettuk, hogy az FC-1 proteint kromoszomalis gen kodolja. A toxin hatasmodjanak tanulmanyozasara a cellularis DNS mennyisegenek valtozasat a sejtciklus fuggvenyeben (DNS szintezis gatlas?) ill. a toxin kezelt sejtek FITC festődeset (sejtfal bioszintezis gatlas?) vizsgaltuk. Eredmenyeink szerint a killing mechanizmus sem a sejtciklustol, sem pedig a sejtfal bioszintezistől nem fugg. Sokkal inkabb ionofor proteinkent hat, amely a citoplazma membranfunkciojat rombolja. | The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the pathogen Cryptococcus neoformans. The goal of this project was to characterize the toxin, determine the genetic trait coding for it, and study its effect on C. neoformans cells. It was demonstrated that FC-1 toxin was specific for C. neoformans. The toxin had a cytocidal effect. FC-1 proved to be a protein: sensitive for heat and proteolytic enzymes. The optimal pH for the activity was within the range of pH 4-6. As receptor site for the killer protein beta-1,6-glucan in the cell wall of sensitive cells was determined. The specificity was used for purification of the toxin through affinity chromatography. The most active fraction from a pustulan-Sepharose 6B column was subjected to SDS-PAGE, and three protein bands, in molecular mass of 19 - 51 kDa, were detected. Additional investigations are necessary to identify the protein with killer property. Agarose gel electrophoresis of F. capsuligenum cell minilysate revealed that FC-1 protein is coded by the chromosomal DNA. Analysis of cellular DNA (inhibition of DNA synthesis?) by laser scanning cytometry and FITC staining (inhibition of cell wall biosynthesis?) of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it might act as an ionophoric protein that disrupts the cytoplasmic membrane function.
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patogen crytococcusok ellen hatasos killer toxin izolalasa jellemzese es a kodolo gen lokalizalasa klonozasa Filobasidium capsulogenumban isolation and characterization of killer toxin active against pathogen cryptococcus localization and cloning of
2007Co-Authors: Judit Kucsera, Ilona Pfeiffer, Zsuzsanna Hamari, Ferenc Kevei, Zsuzsanna Buzás, András Patthy, József Antal, Attila GácserAbstract:A basidiomycota Filobasidium capsuligenum egy olyan feherjet szekretal, amely elpusztitja a patogen Cryptococcus neoformanst. Palyazatunkban ezen killer toxin genetikai determinansat deritettuk fel, a toxin feherje izolalasat, jellemzeset valositottuk meg, ill a toxin hatasmechanizmusat is feltartuk. Az erzekenysegi tesztek alapjan az FC-1 toxin specifikusan, csak a C. neoformans-ra hatott. Bebizonyitottuk, hogy e hatas citocidikus. A toxin protein termeszetű: hő hatasara elbomlik, aktivitasanak optimuma pH 4-6. A toxin receptorakent az erzekeny sejtek sejtfalanak beta-1,6-glukanjat hataroztuk meg. E specificitast arra hasznaltuk fel, hogy a toxint affinitaskromatografiaval tisztitsuk. A pusztulan-Sepharose 6B oszlop legaktivabb frakciojaval SDS-PAGE-t vegeztunk, es 3 protein savot talaltunk a 19 - 51 kDa tartomanyban. Tovabbi kiserletek szuksegesek a killer aktivitasu protein azonositasara. A F. capsuligenum sejtek minilizatainak agaroz gelelektroforezisevel kideritettuk, hogy az FC-1 proteint kromoszomalis gen kodolja. A toxin hatasmodjanak tanulmanyozasara a cellularis DNS mennyisegenek valtozasat a sejtciklus fuggvenyeben (DNS szintezis gatlas?) ill. a toxin kezelt sejtek FITC festődeset (sejtfal bioszintezis gatlas?) vizsgaltuk. Eredmenyeink szerint a killing mechanizmus sem a sejtciklustol, sem pedig a sejtfal bioszintezistől nem fugg. Sokkal inkabb ionofor proteinkent hat, amely a citoplazma membranfunkciojat rombolja. | The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the pathogen Cryptococcus neoformans. The goal of this project was to characterize the toxin, determine the genetic trait coding for it, and study its effect on C. neoformans cells. It was demonstrated that FC-1 toxin was specific for C. neoformans. The toxin had a cytocidal effect. FC-1 proved to be a protein: sensitive for heat and proteolytic enzymes. The optimal pH for the activity was within the range of pH 4-6. As receptor site for the killer protein beta-1,6-glucan in the cell wall of sensitive cells was determined. The specificity was used for purification of the toxin through affinity chromatography. The most active fraction from a pustulan-Sepharose 6B column was subjected to SDS-PAGE, and three protein bands, in molecular mass of 19 - 51 kDa, were detected. Additional investigations are necessary to identify the protein with killer property. Agarose gel electrophoresis of F. capsuligenum cell minilysate revealed that FC-1 protein is coded by the chromosomal DNA. Analysis of cellular DNA (inhibition of DNA synthesis?) by laser scanning cytometry and FITC staining (inhibition of cell wall biosynthesis?) of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it might act as an ionophoric protein that disrupts the cytoplasmic membrane function.
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Characterisation of the anticryptococcal effect of the FC-1 toxin produced by Filobasidium capsuligenum.
Mycoses, 2006Co-Authors: Andrea Keszthelyi, Kanji Takeo, M. Ohkusu, Ilona Pfeiffer, Judit Litter, Judit KucseraAbstract:The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the opportunistic fungal pathogen Cryptococcus neoformans. The aim of this work was to study the effect of the toxin on C. neoformans cells. The sensitivities of strains representing eight molecular subtypes (VNI-IV and VGI-IV) of the C. neoformans species complex, and of an additional 50 clinical and environmental isolates were determined. Analysis of cellular DNA by laser scanning cytometry and fluorescein isothiocyanate (FITC) staining of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it may act as an ionophoric protein that disrupts the cytoplasmic membrane function. The competition assay results suggest that beta-1,6-glucan in the cell wall may provide the binding site for the killer protein. This anticryptococcal toxin has the potential to be applied as a therapeutic agent for the treatment of cryptococcosis.