Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy
Correspondence
Maurizio Ciani
m.ciani{at}univpm.it
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ABSTRACT |
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INTRODUCTION |
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Recently, it has been shown that Kluyveromyces phaffii (since reclassified as Tetrapisispora phaffii) (Ueda-Nishimura & Mikata, 1999) produces a killer toxin (KpKt) that is active on wine spoilage yeasts (Ciani & Fatichenti, 2001
). KpKt has an extensive anti-Hansenispora/Kloeckera activity under winemaking conditions and, therefore, is of particular interest for its potential application as an antimicrobial agent in the wine industry (Ciani & Fatichenti, 2001
). At present, the inhibition of wild spoilage yeast at the pre-fermentative stage is achieved by the addition of SO2 to freshly pressed must. This antiseptic agent, which has been shown to have a toxic action on humans, is also re-added at the end of fermentation for its antioxidant properties. The use of KpKt as a substitute for SO2 during the pre-fermentative stage would limit SO2 use to only the post-fermentative stage, thus reducing the total amount of this antimicrobial in the final product. Moreover, as KpKt is also active against yeasts belonging to the species Saccharomycodes ludwigii, Zygosaccharomyces bailii and Zygosaccharomyces rouxii, the possible use of this toxin for the control of spoilage yeasts in sweet beverages may also be promising (Palpacelli et al., 1991
).
In order to gain information on the nature and mode of action of KpKt in view of its possible use in the wine and beverage industries, the purpose of this study was the characterization of this K. phaffii killer toxin. The active protein was purified and assayed for its molecular mass, glycosylation and enzymic activity. The KpKt NH2-terminal sequencing, primary binding sites within the cell wall of sensitive yeasts, and mode of action were also investigated.
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METHODS |
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Killer activity assay and measurement.
KpKt activity was determined according to Somers & Bevan (1969). Briefly, 70 µl toxin samples were filter-sterilized through 0·45 µm pore-size membrane filters (Millipore) and put into wells (7 mm diameter) cut in the malt agar plates that had previously been seeded with 105 cells ml1 of a sensitive indicator strain. The killing activity was measured as the diameter of the inhibition halo around the well after incubation for 72 h at 20 °C, and is defined as the mean zone of inhibition of replicate wells. The linear relationship observed between the logarithm of killer toxin concentration and the diameter of the inhibition halo assayed by this well-test method was used to define KpKt activity in arbitrary units (aU). One aU is defined as the toxin concentrationc that results in an inhibition halo of 15 mm (actual diameter 8 mm, considering the 7 mm diameter of the well) (Ciani & Fatichenti, 2001
). One aU corresponds to about 1·0 µg killer protein.
KpKt production.
K. phaffii (DBVPG 6076) was cultivated in SM in a 2 l bench-top fermenter (Biostat C, B. Braun Biotech) with a 1·8 l working volume. The oxygen concentration was maintained at 20 % by automatically regulating the stirrer. The temperature was maintained at 25 °C. After 40 h, the culture was centrifuged (5000 g for 10 min, 4 °C) and the supernatant was filter-sterilized through 0·45 µm pore-size membrane filters (Millipore).
Purification of KpKt.
The filter-sterilized supernatant was concentrated with Amicon YM10 (10 kDa cut-off membrane; Pharmacia) to a final volume of 12 ml, which was then dialysed with 10 mM citrate/phosphate buffer, pH 4·5, using dialysis membrane (1214 kDa, Medicell), and applied to a pre-equilibrated (10 mM citrate/phosphate buffer, pH 4·5) Q-Sepharose Fast Flow IEX column (50 ml bed volume; 3 ml min1 flow rate; Amersham Biosciences). After application of the sample (34 mg protein in 12 ml) to the column, the bound protein was eluted with the following step-wise increases in the NaCl concentration in the elution buffer (10 mM citrate/phosphate, pH 4·5): 0, 100, 125, 150, 175, 200, 300, 400, 500 mM.
Electrophoresis and mass spectrometry.
SDS-PAGE was performed according to Laemmli (1970). The protein was stained with Coomassie blue R-250 (Sigma) and the molecular mass determined by comparison with known marker proteins (Pre-stained Protein Ladder, MBI Fermentas; LMW-SDS, Pharmacia Biotech). The molecular mass of the purified killer toxin was confirmed by mass spectrometry (MALDI-TOF-MS; Waters Corp.), using the following modalities: instrument, TofSpec-E; source voltage, 20 000 V; mode, linear; ionization mode, LD+. The mass accuracy and precision of the technique are consistently within 0·04 %.
Endoglycosidase H treatment of KpKt.
KpKt was treated with endoglycosidase H [45 IU (mg protein)1]; ICN Biomedicals). The assay was performed following the procedure described by Elgersma et al. (1997). In brief, 5 µl endoglycosidase H (0·01 IU µl1) was added to 25 µl KpKt (25 aU) and 70 µl buffer (150 mM sodium citrate, pH 5·5, 1 mM PMSF, 10 µM pepstatin, 5 mM sodium azide, 643 µl H2O). Samples were incubated at 37 °C for 48 h, with gentle agitation, and subjected to SDS-PAGE, as described above.
NH2-terminal amino acid sequencing.
After electrophoresis, the purified KpKt was transferred to a PVDF membrane and stained with Coomassie blue, as described by Steinberg et al. (2001). The relevant band was cut out and subjected to 15 cycles of sequence analysis in a protein sequencer (Applied Biosystems).
Enzymic activity.
-Glucanase activity was determined as described by Notario (1982)
by using as enzymic substrates laminarin and glucan. The units of
-glucanase activity are defined as µmol glucose liberated per mg protein per min. Glucose was determined by using the enzymic kit no. 716251 (Boehringer Mannheim). Laminarinase (Sigma-Aldrich) was used as positive control of enzymic activity.
Binding of KpKt by cell-wall polysaccharides.
The sensitive H. uvarum strain (DBVPG 3037; 105 cells ml1) was treated with the killer toxin (70 aU ml1) in the absence or presence of 9 mg ml1 of the following cell-wall polysaccharides: laminarin (Sigma), mannan (Sigma), glucan (Sigma) and pustulan (Calbiochem). After an incubation at 25 °C for 24 h, the cell samples were subjected to viable cell counts to assess the binding activities of the polysaccharides.
Evaluation of KpKt mode of action.
The sensitive strains (106 cells ml1) were incubated with 46 aU killer toxins (KpKt and K1) at 25 °C in a final volume of 1·5 ml. At each sampling time, 100 µl cell suspension was pelleted by centrifugation (2 min at 400 g), resuspended in 500 µl PBS buffer (8 mM Na2HPO4, 1·47 mM KH2PO4, 137 mM NaCl, 2·7 mM KCl) to which 50 µl propidium iodide (PI) solution (1 mg ml1, in the same buffer) was added, and finally processed on a Coulter Epics XL (Beckman Coulter). Forward scatter (FS) and side scatter (SS) were recorded using a linear scale. The intensity of fluorescence at FL3 (red fluorescence, 620 nm) was measured on a logarithmic scale and displayed in a single-parameter histogram. At the time defined for the flow cytometry assay, aliquots of the cell suspensions were subjected to a viable plate count in triplicate in YPD agar plates. The plates were incubated for a minimum of 48 h at 25 °C, the colonies were counted, and the results were expressed as percentage reduction in c.f.u. ml1.
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RESULTS |
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According to the viable plate counts, when S. cerevisiae cells were treated with KpKt (46 aU with 106 cells ml1) the great majority of the sensitive cells were killed after 8 h (5x104 cells ml1 survival) and after 24 h almost the entire population died (3x102 cells ml1 survival) (Fig. 6a). Flow cytometry analysis, performed at the corresponding times, shows that treatments of either 8 h or 24 h with KpKt were not sufficient to cause PI staining of the cells (Fig. 6c, d
). Similar results were obtained when H. uvarum DBVPG 3037 was utilized as the sensitive strain (data not shown).
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DISCUSSION |
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As with the killer toxins secreted by S. cerevisiae (K2 and K28) (Pfeiffer & Radler, 1984), Kluyveromyces lactis (Stark et al., 1990
), Pichia kluyveri (Middelbeek et al., 1979
), Candida sp. SW-55 (Yokomori et al., 1988
) and Hansenula anomala (Kagiyama et al., 1988
), KpKt is a glycosylated protein. The estimated mass of the carbohydrate part of KpKt is similar to that exhibited by K28 killer toxin of S. cerevisiae (Pfeiffer & Radler, 1982
). However, its molecular mass and NH2-terminal sequence do not show any similarities with those of other known killer toxins (Shimizu, 1993
; Magliani et al., 1997
).
According to our BLAST analysis of the 15 amino acids of the N-terminal sequence, KpKt is strikingly similar to -1,3-glucanase of S. cerevisiae and
-1,3-glucan transferase of C. albicans. These two proteins are involved in connecting newly synthesized
-1,3-glucan chains to existing chains, and in linking them through the
-1,6-linkage (Goldman et al., 1995
; Mr
a et al., 1993
; Smits et al., 2001
), and they have been shown to be homologous to a
-1,3-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus (Mouyna et al., 1998
). As we show for KpKt, these proteins have low glycosylation, and have molecular masses of about 30 kDa (Mouyna et al., 1998
). Therefore, KpKt was further characterized to assess whether it shows similar biochemical properties to these enzymes, in terms of enzymic activity and ability to bind cell-wall components.
KpKt -glucanase activity toward laminarin and glucan is in accordance with its sequence similarity with
-1,3-glucanase of S. cerevisiae and
-1,3-glucan transferase of C. albicans.
The competitive inhibition of this killer toxin activity by cell-wall polysaccharides shows that the cytocidal action of KpKt is prevented by glucan (-1,3- and
-1,6-branched glucans) and not by laminarin (mainly
-1,3-, with a few
-1,6-linked glucans) or pustulan (
-1,6-glucan). Thus, KpKt is different from S. cerevisiae
-1,3-glucanase, which binds glucan, laminarin and chitin (Klebl & Tanner, 1989
; Mr
a et al., 1993
). This different behaviour probably arises from the steric conformation of
-1,3 and
-1,6 branched glucans (Kopecka & Kreger, 1986
; Saito et al., 1990
), which may play an important role in the binding process of KpKt. Moreover, the observed KpKt specificity of the binding site is not accompanied by a similar specificity of the catalytic site.
Indeed, the inhibitory effect of glucan and the enzymic activity of the purified protein strongly suggest that -glucanase activity is involved in KpKt killing action, but a definitive confirmation of that would require the development of molecular tools suitable for the genetic manipulation of this unconventional yeast and necessary for the achievement of a K. phaffii BGL2 knock-out strain.
However, our results seem to be, at least in part, in agreement with those regarding the killer toxin secreted by Williopsis saturnus var. mrakii MUCL 41968 (WmKT). This toxin binds sensitive micro-organisms through the recognition of -glucans and its killing action appears to be mediated by a
-glucanase activity that results in cell-wall permeabilization and subsequent cell lysis. The results obtained by Guyard et al. (2002)
indicated that sensitive cells are quickly permeabilized and hence are prone to PI staining upon WmKT treatment, as expected with a toxin that is able to cause cell lysis. Similarly, the S. cerevisiae K1 toxin, which can also generate pores on the cell membrane, has been documented by several authors (Bussey & Sherman, 1973
; Skipper & Bussey, 1977
; Martinac et al., 1990
; Ahmed et al., 1999
), and we see here that it is able to permeabilize sensitive cells and cause PI staining within the first 8 h of treatment.
By contrast, our investigations into the mode of action of KpKt under the same experimental conditions have highlighted that cell death induced by this K. phaffi killer toxin is slower than that caused by K1, and it is not accompanied by prompt PI staining of the dead cells. Thus KpKt killing action seems to take place through a different mode of action when compared to K1 and WmKT.
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ACKNOWLEDGEMENTS |
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Received 8 March 2004;
revised 29 April 2004;
accepted 20 May 2004.
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