Induction of glutathione S-transferase in biofilms and germinating spores of Mucor hiemalis strain EH5 from cold sulfidic spring waters

Abstract

The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs ( approximately 27.8- and approximately 25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.

FIG. 1.

HPSEC elution profiles of purified GST fractions from M. hiemalis strain EH5. The thin solid line indicates the profile for the cytosolic GST fraction (A₂₆₀) with GST peaks 1 (major amount) and 2, the dotted line indicates the profile for the cytosolic GST fraction after treatment with a GST antibody (A₂₆₀), and the thick dashed line shows the corresponding fluorescence signal (fluorescence at 519 nm) for the cytosolic GST fraction after treatment with the GST antibody. 1, native GST peak 1 (A₂₆₀); 2, native GST peak 2 (A₂₆₀); 3, GST antibody (A₂₆₀); GAb, GST—anti-GST complex (fluorescence at 519 nm); mAU, milli absorbance units; a.u., arbitrary units; λ_ex, excitation wavelength; x, low-molecular-weight impurity (Alexa Fluor).

FIG. 2.

Phylogenetic tree based on 18S rRNA gene sequence data showing the position of aquatic M. hiemalis isolated from a sulfidic-sulfurous spring water biofilm. The topology of the phylogenetic tree was calculated by using the ClustalW and PAUP 4.0b software with 1,000 bootstrap trials and was visualized by using the TreeView software. Reference sequences were selected from the NCBI database to represent broad fungal diversity. Bar = 10% estimated difference in nucleotide sequences. Bootstrap values greater than 50% are indicated at the nodes.

FIG. 3.

Induction of GST activity in M. hiemalis strain EH5 by STS. The microsomal (a) and soluble cytosolic (b) GST activity with CDNB (○) and GST activity with fluorodifen (•) of M. hiemalis strain EH5 (ca. 4-week-old cultures) induced by different STS concentrations are shown. The responses of cytosolic GST activity with CDNB and microsomal GST activity with fluorodifen to STS concentrations were found to be linear and could be described by the following regression equations: y = 0.6278 + 161.3227x (r² = 0.99) and y = 26.0004 + 266.3944x (r² = 0.99), respectively. The responses of microsomal GST activity with CDNB and cytosolic GST activity with fluorodifen to STS could be represented by the following nonlinear three-parameter logarithmic functions showing slow saturation effects at STS concentrations higher than ∼1 mM: y = 58.9046 + 31.0094[ln(x + 0.1364)] (r² = 0.76) and y = 999.8441 + 213.1669[ln(x − 0.0306)] (r² = 0.94), respectively. The standard deviation (n = 3) at each data point was usually less than 10%.

FIG. 4.

Localization of in situ GST induction by STS employing Alexa Fluor-labeled GST antibody at various growth stages of germinating sporangiospores of M. hiemalis strain EH5. Strong GST-specific green fluorescence using anti-GST Alexa Fluor was observed in sporangiospores grown in the presence of 0.82 mM STS (a to c), in contrast to the fluorescence without STS (d to f). Panels a, b, d, and e were obtained by conventional fluorescence microscopy (bars = 10 μm), whereas panels c and f were obtained by using laser scanning confocal fluorescence microscopy (bars = 10 μm). The GST antibody immunofluorescence staining in an elongated hypha activated by 0.82 mM STS (c) is stronger than the fluorescence staining in the control (f), analogous to the activation of GST enzyme activity by STS (Fig. 3).