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Effects of Cryptococcus humicola killer toxin upon Cryptococcus terreus envelope: combined fluorometric and
E O Puchkov1, T V Yurkova, W I Golubev
1Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russian Federation. puchkov@ibpm.serpukhov.su
Abstract:
Killer toxin (microcin) produced by Cryptococcus humicola 9-6 induced interaction of the fluorogenic dyes, ethidium bromide, propidium iodide, and hemimagnesium 8-anilino-1-naphtalenesulfonate, with the sensitive strain of Cryptococcus terreus VKM Y-2253. The toxin also made the cells susceptible to cetyltrimethylammonium bromide and leaky for K+. When excited at 360 nm, cell-bound ethidium (propidium) fluorescence was enhanced by 8-anilino-1-naphtalensulfonate, and cell-bound 8-anilino-1-naphtalensulfonate fluorescence was quenched by ethidium (propidium), indicating energy transfer from 8-anilino-1-naphtalensulfonate to ethidium (propidium). These results suggest that at least a portion of the probe molecules had the same binding site, possibly the cytoplasmic membrane. The parameters of kinetics of microcin action were evaluated fluorometrically. They were found to be identical for all probes and depended on microcin concentration. The fluorescence increment of ethidium and 8-anilino-1-naphtalensulfonate upon binding to microcin-treated cells correlated with the fraction of stainable cells and viability.
Insights
Cryptococcus humicola killer toxin (microcin) alters Cryptococcus terreus cell membranes, increasing dye interaction and potassium leakage. This mechanism, studied via fluorometric probes, correlates with cell viability and stainability.
Area of Science:
- Microbiology
- Biochemistry
- Cell Biology
Background:
- Cryptococcus humicola produces a killer toxin, a microcin, with antimicrobial properties.
- The precise mechanism of microcin action on sensitive yeast strains is not fully understood.
- Investigating membrane interactions provides insights into microcin's cytotoxic effects.
Purpose of the Study:
- To elucidate the interaction of Cryptococcus humicola microcin with the cell membrane of Cryptococcus terreus.
- To characterize the biophysical changes induced by microcin using fluorogenic dyes.
- To evaluate the kinetics of microcin action and its correlation with cell viability.
Main Methods:
- Treatment of Cryptococcus terreus VKM Y-2253 with purified microcin from Cryptococcus humicola 9-6.
- Fluorometric analysis using ethidium bromide, propidium iodide, and 8-anilino-1-naphtalenesulfonate (ANS).
- Measurement of potassium (K+) leakage and susceptibility to cetyltrimethylammonium bromide.
- Kinetic analysis of microcin-cell interactions and fluorescence changes.
Main Results:
- Microcin induced enhanced interaction of fluorogenic dyes (ethidium, propidium iodide, ANS) with the sensitive strain.
- Evidence of energy transfer between ANS and ethidium/propidium iodide suggests shared binding sites, likely the cytoplasmic membrane.
- Microcin treatment increased cell susceptibility to cetyltrimethylammonium bromide and caused K+ leakage.
- Fluorometric parameters of microcin action were probe-independent but concentration-dependent.
- Increased fluorescence of bound dyes correlated with the fraction of stainable cells and overall viability.
Conclusions:
- Cryptococcus humicola microcin targets and perturbs the cytoplasmic membrane of sensitive Cryptococcus terreus cells.
- The observed dye interactions and K+ leakage are key indicators of microcin-induced membrane damage.
- Fluorometric methods provide a quantitative approach to study microcin kinetics and assess its cytotoxic impact on yeast viability.