Isolation and characterization of killer toxin active against pathogen Cryptococcus, localization and cloning of the toxin coding gene(s) in Filobasidium capsulogenum.
Patogén Crytococcusok ellen hatásos killer toxin izolálása, jellemzése és a kódoló gén lokalizálása, klónozása Filoba-sidium capsulogenumban
Title in English
Isolation and characterization of killer toxin active against pathogen Cryptococcus, localization and cloning of the toxin coding gene(s) in Filobasidium capsulogenum.
Panel
Immunity, Cancer and Microbiology
Department or equivalent
Department of Microbiology (University of Szeged)
Participants
Antal, József Buzás, Zsuzsanna Gácser, Attila Hamari, Zsuzsanna Kevei, Ferenc Patthy, András Pfeiffer, Ilona Anikó
Starting date
2001-01-01
Closing date
2005-12-31
Funding (in million HUF)
8.664
FTE (full time equivalent)
0.00
state
closed project
Final report
Results in Hungarian
A basidiomycota Filobasidium capsuligenum egy olyan fehérjét szekretál, amely elpusztítja a patogén Cryptococcus neoformanst. Pályázatunkban ezen killer toxin genetikai determinánsát derítettük fel, a toxin fehérje izolálását, jellemzését valósítottuk meg, ill a toxin hatásmechanizmusát is feltártuk.
Az érzékenységi tesztek alapján az FC-1 toxin specifikusan, csak a C. neoformans-ra hatott. Bebizonyítottuk, hogy e hatás citocidikus. A toxin protein természetű: hő hatására elbomlik, aktivitásának optimuma pH 4-6. A toxin receptoraként az érzékeny sejtek sejtfalának béta-1,6-glükánját határoztuk meg. E specificitást arra használtuk fel, hogy a toxint affinitáskromatográfiával tisztítsuk. A pusztulán-Sepharose 6B oszlop legaktívabb frakciójával SDS-PAGE-t végeztünk, és 3 protein sávot találtunk a 19 – 51 kDa tartományban. További kísérletek szükségesek a killer aktivitású protein azonosítására. A F. capsuligenum sejtek minilizátainak agaróz gélelektroforézisével kiderítettük, hogy az FC-1 proteint kromoszómális gén kódolja. A toxin hatásmódjának tanulmányozására a celluláris DNS mennyiségének változását a sejtciklus függvényében (DNS szintézis gátlás?) ill. a toxin kezelt sejtek FITC festődését (sejtfal bioszintézis gátlás?) vizsgáltuk. Eredményeink szerint a killing mechanizmus sem a sejtciklustól, sem pedig a sejtfal bioszintézistől nem függ. Sokkal inkább ionofor proteinként hat, amely a citoplazma membránfunkcióját rombolja.
Results in English
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.
Kucsera J; Keszthelyi A; Kovács M; Palatinus I; Pfeiffer I: Molecular characterisation of the anti-cryptococcal toxin producing Filobasidium capsuligenum, In: Book of Abstract 11th International Congress on Yeast, Rio de Janeiro, Brazília, pp. 173, 2004
Litter J, Keszthelyi A, Hamari Zs, Pfeiffer I, Kucsera J: Differences in the mitochondrial genome organization of Cryptococcus neroformans strains, Antonie van Leeuwenhoek 88: 249-255, 2005
Kucsera J; Ohkusu M; Takeo K; Pfeiffer I; Litter J; Kevei F: Filobasidium capsuligenum IFM 40078 produces a toxin with anti-cryptococcal activity, Mycoses 45 : 33, 2002
Litter J; Hamari Zs; Pfeiffer I; Kucsera J: Study of the mitochondrial genomes of Cryptococcus neoformans, Mycoses 45: 35, 2002
Pfeiffer I; Kucsera J; Gácser A; Litter J; Golubev WI: Diversity of extrachromosomal elements in yeasts, Acta Microbiol Immunol Hung 49: 315-319., 2002
Litter J; Hamari Zs; Pfeiffer I; Kucsera J: Comparison of the mitochondrial DNA in two strains of Cryptococcus neoformans, FEMS Microbiol Letters 222: 469-470., 2003
Kucsera J; Ohkusu M; Pfeiffer I; Litter J; Takeo K: Partial characterization of FC-1 killer toxin active against Cryptococcus neoformans, FEMS Microbiol Letters 222: 440, 2003
Kucsera J; Ohkusu M, Pfeiffer I; Litter J; Takeo K: The effect of mycocin FC-1 on Cryptococcus neoformans cells, In: 23rd International Specialized Symposium on Yeasts “Interaction between Yeasts and other Organisms”, pp. 130, 2003
Litter J; Hamari Zs; Pfeiffer I; Kucsera J: Analysis of mitochondrial genome variations in Cryptococcus neoformans, In: 23rd International Specialized Symposium on Yeasts “Interaction between Yeasts and other Organisms”, pp. 131, 2003
Kucsera J; Keszthelyi A; Kovács M; Palatinus I; Pfeiffer I: Molecular characterisation of the anti-cryptococcal toxin producing Filobasidium capsuligenum, In: Book of Abstract 11th International Congress on Yeast, Rio de Janeiro, Brazília, pp. 173, 2004
A. Keszthelyi, M. Ohkusu, K. Takeo, I. Pfeiffer, J. Litter and J. Kucsera: Characterization of the anti-cryptococcal effect of the FC-1 toxin produced by Filobasidium capsuligenum, Mycoses 49/3:, 2006
Keszthelyi A, Kovács M, Palatinus I, Buzas Zs, Kucsera J: Isolation and purification of the killer toxin of Filobasidium capsuligenum IFM 40078 strain, Acat Microbiol Immunol Hung, 2005
