Characterization of a Williopsis saturnus var. mrakii high molecular weight secreted killer toxin with broad-spectrum antimicrobial activity

Cyril Guyard1,2,*, Nathalie Séguy3, Jean-Charles Cailliez1,2,4, Hervé Drobecq5, Luciano Polonelli6, Eduardo Dei-Cas1,2,7, Annick Mercenier1,2 and Franco D. Menozzi2,8

1Département de Microbiologie des Ecosystèmes and 2IFR17, Institut Pasteur de Lille, 1 rue Calmette, BP 245, 59019 Lille; 3Laboratoire de Mycologie, Université de Bourgogne, Faculté de Pharmacie et de Médecine, 7 boulevard Jeanne d’Arc, BP 87900, 21079 Dijon Cedex; 4Faculté Libre des Sciences, Université Catholique, Lille; 5UMR 8525, Université de Lille II, Institut de Biologie de Lille, 1 rue Calmette, 59019 Lille, France; 6Dipartimento di Patologia e Medicina di Laboratorio, Sezione di Microbiologia, Università degli Studi di Parma, Parma, Italy; 7Centre Hospitalier et Faculté de Médecine, Lille; 8INSERM U447, Mécanismes Moléculaires de la Pathogénie Microbienne, Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue Calmette, 59019 Lille, France

Received 3 August 2001; returned 14 December 2001; revised 25 January 2002; accepted 19 February 2002.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Williopsis saturnus var. mrakii MUCL 41968 secretes a killer toxin (WmKT), which is active against a wide range of pathogens. From the W. saturnus var. mrakii MUCL 41968 culture supernatant a protein of 85 kDa with killer activity was purified to homogeneity. The purified protein was demonstrated to be a killer toxin since it displays the toxin activity and cross-reacts with mAbKT4, a monoclonal antibody that blocks WmKT activity. Its partial amino acid sequencing revealed that WmKT might be related to yeast SUN proteins, but not to other killer toxins described. Immunofluorescence studies using polyclonal antibodies raised against purified WmKT revealed that it acts by binding to the cell surface of sensitive strains. We showed that WmKT is inactive against mutant strains of Saccharomyces cerevisiae deficient in the synthesis of ß-glucans, indicating that these polysaccharides constitute the target of the toxin. WmKT was demonstrated to induce rapid lethal cell permeation, since strong propidium iodide labelling was shown for sensitive strains treated with the killer toxin. These findings indicate that WmKT is a novel killer toxin whose molecular characterization may lead to the development of new wide-spectrum antimicrobial compounds.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The emergence and rapid dissemination of infectious agents resistant to antibiotic treatments have emphasized the need for alternative chemotherapies.1 Among the new antimicrobial molecules under investigation, specific yeast killer toxins represent promising candidates because they display wide spectra of activity.2,3 The yeast killer phenomenon was first described in Saccharomyces cerevisiae strains producing toxins that inhibit the growth of, or exert lethal effects on, closely related yeast strains.4 It has since been demonstrated that this phenomenon is widespread among yeast species.2,5 Due to a mechanism called self-immunity, yeast killer strains are resistant to the activity of their own killer toxins. In the past decade, numerous killer toxins have been described and shown to differ in their mode of action, molecular structure or maturation process.2 Although killer toxins secreted by Williopsis spp. generally have low molecular weights (5–19 kDa),68 a killer toxin with an apparent molecular weight of 85 kDa was described recently in Williopsis saturnus var. mrakii MUCL 41968 (WmKT),9 an environmental yeast strain that is characterized by the saturniform shape of its ascospores. WmKT is characterized by a particularly wide spectrum of activity: it is active against Candida albicans, Pichia anomala, Pneumocystis carinii and S. cerevisiae.10,11 The high sensitivity of mouse- and rat-derived P. carinii strains to WmKT has been demonstrated both in vitro and in animal models.11 WmKT is encoded by a nuclear gene and its microbicidal activity is neutralized by mAbKT4, an anti-WmKT monoclonal antibody.12 Preliminary characterization studies have also shown that WmKT is active in acidic conditions (around pH 4.6), and at temperatures between 25 and 28°C.11

Anti-idiotypic antibodies (KTIdAbs) obtained after immunization of mice with mAbKT413 were shown to exert a lethal effect against WmKT-sensitive yeast strains, as well as multidrug-resistant isolates of Mycobacterium tuberculosis and antibiotic-resistant Gram-positive cocci.14,15 Since the antimicrobial activity of KTIdAbs is neutralized by mAbKT4, it has been proposed that KTIdAbs mimic the activity of WmKT. Moreover, single chain fragment anti-idiotypic antibodies (ScFv KTIdAbs) produced from splenic lymphocytes of mice immunized with mAbKT4 were shown to display wide antimicrobial activity.16 Recently, recombinant strains of Streptococcus gordonii expressing ScFv KTIdAbs were used to treat rats suffering from experimental vaginitis caused by C. albicans.17 In this experiment, S. gordonii secreting ScFv KTIdAbs displayed therapeutic effects equivalent to those of fluconazole.

Taken together, these results indicate that WmKT or derivatives thereof could be used as novel therapeutic agents against microbial infections. However, previous studies of the WmKT activity spectrum were performed with KTIdAbs or crude W. saturnus var. mrakii MUCL 41968 culture supernatant and there was virtually no information on the protein itself and the corresponding gene. Therefore, as reported here, we purified WmKT and demonstrated for the first time that this killer toxin is a cell surface 85 kDa glycoprotein that exhibits some relatedness to the yeast SUN proteins and causes cell permeation after contact with target cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Yeast strains

The killer yeast strains used were W. saturnus var. mrakii MUCL 419685 from Mycothèque de l’Université de Louvain and P. anomala ATCC 96603.18 P. anomala strain MUCL 41969 and S. cerevisiae W303 were used as killer toxin-sensitive strains. S. cerevisiae ZH 401-1-d knr4 disruption mutant19 and its parental JY102 strain were used to study the interaction of WmKT with cell surface 1,3-ß-glucan. S. cerevisiae SEY6210 and its derivative carrying a disruption of the KRE1 gene20 were used to study the interaction of WmKT with cell surface 1,6-ß-glucan.

Monoclonal antibody

mAbKT4 is a neutralizing monoclonal antibody raised against the killer toxin of P. anomala ATCC 9660318 that cross-reacts with WmKT and blocks its activity.21,22

Killer toxin purification

A colony of W. saturnus var. mrakii MUCL 41968 was used to inoculate a 200 mL preculture in yeast minimal medium (YMM),23 buffered at pH 3.5 with 25 mM citric acid. After 48 h of growth at 26°C and 70 rpm, four flasks (2 L volume) containing 450 mL of YMM were each seeded with 50 mL of this preculture. After 15 h of growth at 26°C and 70 rpm, yeast cells were harvested by centrifugation (5000g for 10 min), and the clarified supernatant was collected. After addition of 10 tablets of EDTA-free protease inhibitor cocktail (Complete; Roche, Mannheim, Germany), the 2 L of spent growth medium was applied to a Macro-Prep High S (Bio-Rad, Hercules, CA, USA) ion exchange chromatography column (10 x 2.5 cm) equilibrated with 25 mM sodium acetate pH 3.0. After extensive washing with 25 mM sodium acetate buffer pH 3.5, elution was achieved by passing 25 mM sodium acetate buffer pH 4.5 containing 1 M NaCl. Eluted fractions (5 mL) were screened for killer activity as described below. The active fractions were pooled, lyophilized and resuspended in 25 mM sodium acetate pH 3.0. After dialysis against 25 mM sodium acetate pH 3.0, the sample was analysed by SDS–PAGE and immunoblotting.24

Killer toxin activity assay

A 50 µL aliquot of spent growth medium or chromatographic fraction was mixed with 200 µL of liquid Sabouraud broth (AES Laboratory, Combourg, France) containing 5 x 104 cells of the killer toxin-sensitive P. anomala strain MUCL 41969 seeded into the wells of 96-well microdilution plates (Costar, Brumath, France). The plates were incubated at 26°C for 18–20 h and growth of the indicator strain was followed by measuring the optical density at 630 nm (OD630) using an automatic plate recorder (Bio-tek Instruments, Inc., Winooski, VT, USA). Killer toxin activity was detected by growth inhibition of the sensitive strain, using a toxin-free microculture as control.9 Assays were performed in triplicate and the results expressed as the mean of OD630 ± S.D.

SDS–PAGE and immunoblotting

SDS–PAGE was performed using a 4–15% linear acrylamide gel.24 After SDS–PAGE, proteins were transferred on to nitrocellulose membranes (Bio-Rad SA, Ivry-sur-Seine, France) as described by Towbin et al.25 Killer toxins were detected with mAbKT4 or WmKTAbs (see below) at a concentration of 50 or 10 µg/mL, respectively. Immune complexes were revealed with alkaline phosphatase-linked goat anti-mouse IgG (Roche Diagnostics SA, Meylan, France) or alkaline phosphatase-linked goat anti-rabbit IgG (Promega, Madison, WI, USA).

Amino acid sequencing

To determine their N-terminal sequence, the protein bands recognized by WmKTAbs were excised from the PVDF membrane (Millipore SA, Saint Quentin en Yvelines, France), and submitted directly to Edman degradation using an automated microsequencer (Procise 492; Applied Biosystems, Palo Alto, CA, USA). Internal amino acid sequences were obtained by a procedure described previously.26 Briefly, purified WmKT was submitted to SDS–PAGE followed by Coomassie blue staining. The band corresponding to the killer toxin was excised and treated with acetonitrile and ammonium carbonate to remove impurities. After dehydration, the protein was digested with endoproteinase Lys-C (Roche) for 18 h at 37°C. The resulting peptides were then purified by C18 reverse-phase HPLC (Microgradient System 140C; Applied Biosystems) and submitted to N-terminal microsequencing.

Polyclonal antibody production and purification

The polyacrylamide band containing the 85 kDa protein recognized by mAbKT4 and corresponding to WmKT, was excised from the SDS–polyacrylamide gel, minced and resuspended in 4 mL of phosphate-buffered saline (PBS) to obtain a final WmKT concentration of 400 µg/mL, and used to immunize rabbits by the subcutaneous route. The first injection contained 0.5 mL of WmKT suspension (200 µg of WmKT) and 0.5 mL of complete Freund’s adjuvant (Sigma-Aldrich, Saint Quentin Fallavier, France). Three boosts were carried out at 2 week intervals with 0.5 mL of WmKT suspension mixed with 0.5 mL of incomplete Freund’s adjuvant (Sigma-Aldrich). Two weeks after the last boost, the rabbits were bled and sera were collected. All animal experiments were carried out according to the Institut Pasteur de Lille guidelines for laboratory husbandry. WmKTAbs were then purified using protein A–Sepharose CL-4B (Amersham Pharmacia Biotech, Uppsala, Sweden). The concentration of purified polyclonal antibodies was determined using the BCA protein assay (Pierce, Rockford, IL, USA). Neutralization of killer toxin activity was assayed by adding 10 µL of WmKTAbs at a concentration of 10 mg/mL to serially diluted culture supernatant of W. saturnus var. mrakii MUCL 41968. After overnight incubation at 4°C, the WmKT activity was measured as described above. Protein A-purified rabbit antibodies from pre-immune serum were used as negative control at a concentration of 10 mg/mL. Assays were performed in triplicate and results expressed as the mean of OD630 ± S.D.

Glycosylation studies

Concanavalin A conjugated to digoxigenin was purchased from Roche Diagnostics. The specific affinity of digoxigenin-labelled concanavalin A for the carbohydrate moiety of WmKT was determined according to the DIG glycan differentiation kit protocol (Roche Diagnostics). Deglycosylation of WmKT was performed by treating the toxin with NANase II, O-glycosidase DS and PNGase F cocktail (Bio-Rad, USA) for 24 h at 37°C, without prior denaturation of the protein. After deglycosylation, WmKT was analysed by SDS–PAGE followed by western blotting.

Immunofluorescence assays

Yeast cells from an overnight culture were centrifuged for 4 min at 10 000g and resuspended in PBS to a final concentration of c. 5 x 106 cells/mL.

For immunofluorescence microscopy, 20 µL of yeast suspension was pipetted into the wells of an immunofluorescence assay (IFA) slide and left to dry before fixing for 45 min at 50°C. Twenty-five microlitres of WmKTAbs at 100 µg/mL in PBS was then added to each well and the slide was incubated for 35 min at 37°C in a humid chamber. Following three washes with PBS, 25 µL of fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit IgG (1.6 µg/mL) (Dako, Mississauga, Canada) was pipetted into the wells. After a 90 min incubation at 37°C followed by three washes with PBS, the slides were mounted under a coverslide with mounting medium containing glycerol/mowiol/Dabco (Sigma-Aldrich). Epifluorescence was observed with an Axiophot 2 microscope (Zeiss SA, Le Pecq, France).

For flow cytometry analysis, 106 yeast cells were incubated for 90 min at 37°C in 100 µL PBS containing WmKTAbs or control rabbit antibodies at a concentration of 10 µg/mL. The cells were then washed three times with 25 µL of PBS before incubation for 30 min at 37°C with 100 µL of FITC–swine anti-rabbit IgG at 1.6 µg/mL in PBS. After three final washes with PBS, the cells were resuspended in 1 mL PBS and analysed by flow cytometry. Cells exhibiting a FITC signal greater than that of control cells were considered positive. Assays were performed in triplicate and results expressed as the mean of labelled fluorescent cells ± S.D.

For analysis of the binding of WmKT to sensitive yeast cells, 106 yeast cells were incubated for 1 h at 26°C in 1 mL of W. saturnus var. mrakii MUCL 41968 spent growth medium. The IFA was performed as described above except that all incubation and washing steps were done in 25 mM sodium acetate buffer pH 4.6.

Flow cytometry assays

Flow cytometry analyses were performed using a Coulter Epics XL flow cytometer (Coultronics, Margency, France). FITC signal was collected through a 525 nm dichroic band-pass filter after being reflected by a 550 nm dichroic long pass filter. Propidium iodide (PI) fluorescence was collected through a 645 nm dichroic band-pass filter after being reflected by a 620 nm dichroic long pass filter. Before each analysis, 3 and 6 µm green latex beads (Coultronics) were used to calibrate the light scatter and fluorescence parameters. Killer toxin sensitivity was measured by flow cytometry analysis of 10 000 cells. Overnight-grown yeast cells were numerated using a Thoma cell count chamber, and 107 cells were mixed with spent growth medium or purified WmKT at a final concentration of 500 µg/mL. After 3 h of incubation at 26°C, the cells were harvested by centrifugation (10 000g, 10 min at 4°C), washed twice with PBS, then resuspended in 1 mL of PBS containing 10 µg/mL of PI (Sigma-Aldrich) and PI fluorescence was determined.

Zymolyase resistance assay

Yeast cells were grown to mid-log phase in 50 mL Sabouraud broth buffered at pH 4.6 with 0.1 M citric acid and 0.2 M sodium phosphate. After harvesting by low-speed centrifugation, the cells were diluted to an OD600 of 0.25 using 10 mM Tris–HCl pH 7.5 containing zymolyase (Sigma-Aldrich) at 20 µg/mL, and incubated at 37°C. Zymolyase sensitivity was monitored by measuring the OD600 of the cell suspensions every 20 min for 160 min.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
W. saturnus var. mrakii MUCL 41968 killer toxin purification

Spent growth medium (see Materials and methods) from a W. saturnus var. mrakii MUCL 41968 culture performed in YMM buffered at pH 3.5 was filter sterilized and applied directly to a Macro-Prep High S ion exchange chromatography column. Elution of adsorbed proteins was performed with a stepwise NaCl gradient ranging from 50 mM to 1 M. Eluted fractions were screened for the presence of killer toxin activity and the highest activity was recovered in fractions eluted at 1 M NaCl. Killer toxin assay performed with these fractions showed that the specific activity of the purified sample was increased by a factor of 20. SDS–PAGE analysis of killer fractions showed three major proteins of 85, 73 and 60 kDa (Figure 1, lane A). In immunoblots, only the 85 kDa protein was recognized by mAbKT4, which neutralizes the W. saturnus var. mrakii MUCL 41968 killer toxin activity, indicating that this protein displays killer activity (Figure 1, lane B). This was further supported by demonstrating that the 85 kDa protein purified to homogeneity by gel filtration chromatography retains the killer toxin activity (data not shown). Nevertheless, due to the low yield of purified 85 kDa protein obtained after gel filtration, the fractions resulting from the ion exchange chromatography purification were retained for further analysis. This result, indicating a high molecular weight for WmKT, is in agreement with previous results showing that killer activity might be associated with a protein of 85 kDa present in the crude culture supernatant of W. saturnus var. mrakii MUCL 41968.21



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Figure 1. Ion exchange purification, immunoblotting, concanavalin A–DIG binding and enzymic deglycosylation of WmKT. W. saturnus var. mrakii MUCL 41968 conditioned growth medium was subjected to cation exchange chromatography and elution was performed using a stepwise NaCl gradient. The fraction eluted at 1 M NaCl and containing killer toxin activity was analysed by SDS–PAGE (lane A) and immunoblotting using mAbKT4 after enzymic deglycosylation (lane F). W. saturnus var. mrakii MUCL 41968 conditioned growth medium was also analysed by western blotting using mAbKT4 (lane B); anti-WmKT serum (lane C), pre-immune serum (lane D) and concanavalin A–DIG (lane E). The arrows in the right-hand margin indicate the position of the 85, 73 and 60 kDa proteins present in the 1 M NaCl elution peak.

 
Amino acid sequencing

Analysis of the N-terminal amino acid sequence of the 85 kDa protein gave the following sequence: GVVVVLEXQVAY. A search in the protein databases failed to detect significant homologies with known proteins. To establish whether the 73 and 60 kDa proteins were related to the 85 kDa one, their N-terminal amino acid sequences were also determined. The three proteins turned out to possess identical N-terminal amino acid sequences, suggesting that they may represent three isoforms of the same protein. Alternatively, the 73 and 60 kDa proteins may represent degradation products of the 85 kDa protein.

To characterize the 85 kDa protein further, its partial internal amino acid sequence was determined using six peptides derived from endoproteinase Lys-C treatment of the protein followed by reverse-phase HPLC separation. The sequences obtained and respectively labelled peptide sequences 1–6 were: KGYLYRSNTAK, KXPMSVVDNSDDYYN, KGITYSPYTSSGT, KTTPNYNIK, KATANFVFY and KQQTANIK. For peptides 1–5, a search in databases revealed similarities with proteins belonging to the SUN family (Figure 2). Peptide 1 showed a strong similarity, since eight of 11 invariant amino acids were identical to SUN proteins. For peptides 2–5, more limited similarities were observed; however, identities ranging from 27 to 78% were determined with each protein of the SUN family. The SUN protein family has been defined on the basis of a common 258-amino-acid C-terminal domain2730 and is composed of seven proteins that are involved in different yeast cellular functions such as regulation of mitochondria biogenesis and cell septation. The similarities observed between the internal sequences of the 85 kDa protein and the SUN proteins are all located within the SUN common C-terminal domain, indicating that the W. saturnus var. mrakii MUCL 41968 85 kDa protein could share molecular properties with the yeast SUN protein family. For peptide 6, no significant similarity with known proteins was found.



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Figure 2. Sequence alignment of SUN proteins with WmKT peptides. UTH1p, SUN4p, SIMp and NCA3p are SUN proteins from S. cerevisiae. BglA is a ß-glucosidase of C. wickerhamii (Cw). PSU1 and c2G2 are two proteins of Schizosaccharomyces pombe (Sp). Alignment of SUN proteins was performed using the Clustal software. Alignment of peptides with SUN proteins was deduced from a similarity search in the protein sequence database with the BLAST network. GenBank accession nos: PSU1, 5822708; NCA3, 1008306; UTH1, 486485; SUN4, 1301945; SIM1, 322376.

 
Immunological characterization

To characterize functionally the killer activity associated with the 85 kDa protein, rabbit polyclonal antibodies (WmKTAbs) were raised against the purified protein. Immunoblotting analysis of spent growth medium of W. saturnus var. mrakii MUCL 41968 showed that WmKTAbs and mAbKT4 cross-react strongly with an 85 kDa protein (Figure 1, lane C). Interestingly, WmKTAbs also react with the 73 and 60 kDa proteins associated with the 85 kDa protein, which corroborates the hypothesis that these three polypeptides are related.

Immunofluorescence studies were then performed using WmKTAbs to localize the killer toxin on the surface of yeast cells. As shown in Figure 3a, strong and homogeneous binding of WmKTAbs was observed at the surface of the W. saturnus var. mrakii MUCL 41968 killer strain, indicating that the toxin accumulates in the cell wall during its secretion process. To check the specificity of WmKTAbs, two killer strains and two killer toxin-sensitive strains were studied by IFA, and labelling signals were monitored by flow cytometry. As shown in Figure 3b, labelling by WmKTAbs was of 85.3% and 54.9% of the two killer strains W. saturnus var. mrakii MUCL 41968 and P. anomala ATCC 96603, respectively. Under the same conditions, S. cerevisiae W303 and P. anomala MUCL 41969, both sensitive to the killer toxin, exhibited labelling of 0.1% and 27.0%, respectively. Control experiments using pre-immune rabbit serum or secondary antibody alone failed to induce significant yeast labelling, demonstrating that the binding of WmKTAbs is much higher for killer yeast strains than for sensitive ones.



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Figure 3. Immunodetection of WmKT with anti-WmKT polyclonal antibodies (WmKTAbs). (a) Fluorescence microscopy analysis of W. saturnus var. mrakii MUCL 41968 immunolabelled with WmKTAbs. (b) Flow cytometry analysis of yeasts immunolabelled with WmKTAbs (horizontal bars). As negative controls, the assays were carried out using pre-immune serum instead of WmKTAbs (cross-hatching), and to check non-specific immunolabelling with secondary antibody, IFAs were performed using PBS instead of WmKTAbs (filled diamonds). The results are expressed as the mean of the percentage of labelled cells ± S.D.

 
To investigate the specificity of WmKTAbs further, neutralization assays of WmKT activity were performed by mixing the polyclonal antibodies with serial dilutions of spent growth medium of W. saturnus var. mrakii MUCL 41968. Figure 4 shows that a 1/8 dilution of the WmKT-containing growth medium was totally inhibited (no drop in the OD630 of the WmKT-sensitive culture) by WmKTAbs at the concentration 1.6 mg/mL, a concentration that still significantly reduced the KT activity contained in 1/4 dilution. Pre-incubation with control antibodies had no impact on WmKT activity. WmKT was thus specifically neutralized by WmKTAbs as described for mAbKT4.22



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Figure 4. Neutralization of killer toxin activity using WmKTAbs. One hundred micrograms of anti-killer toxin WmKTAbs and serial dilutions of W. saturnus var. mrakii MUCL 41968 conditioned growth medium were mixed. The mixtures were incubated overnight at 4°C and the killer toxin activity assays were performed as described in Materials and methods. Means and standard deviations were calculated from three independent experiments: (triangles) toxin-free control; (diamonds) WmKTAbs mixed with diluted W. saturnus var. mrakii MUCL 41968 conditioned growth medium; (squares) control antibodies mixed with dilution of conditioned supernatant.

 
Evidence for glycosylation of WmKT

Preliminary observations suggested that WmKT may bear a polysaccharide moiety. This hypothesis was first investigated in a western blot probed with concanavalin A, a lectin that interacts with terminal {alpha}-D-mannosyl and {alpha}-D-glucosyl residues. As shown in Figure 1 (lane E), concanavalin A conjugated to digoxigenin bound to a c. 85 kDa protein, which is also recognized by mAbKT4 and WmKTAbs, supporting the hypothesis that WmKT is a glycoprotein. In addition, and based on their strong reactivity with concanavalin A, the cross-reactive 73 and 60 kDa proteins also appeared glycosylated.

To prove glycosylation, WmKT was submitted to enzymic deglycosylation. Following treatment of purified WmKT with combined NANase II, O-glycosidase DS and PNGase F, mAbKT4 was shown to react in immunoblotting with a protein band of c. 55 kDa (Figure 1, lane F), confirming that WmKT carries a polysaccharide moiety that accounts for c. 35% of the protein by weight.

Effect of WmKT on PI staining of sensitive yeasts

According to Green et al.,31 antifungal effects can be monitored by flow cytometry analysis, since treatment with antifungal agents leads to increased PI staining of sensitive yeasts. The PI staining pattern of the WmKT-sensitive P. anomala MUCL 41969 strain after incubation with WmKT was therefore analysed. Untreated P. anomala MUCL 41969 stained with PI exhibited only low auto-fluorescence (Figure 5a). The same analysis performed on P. anomala MUCL 41969 cells killed by heating at 100°C for 10 min led to intense PI staining of the entire cell population (Figure 5b). A similar PI staining pattern was observed when P. anomala MUCL 41969 cells were incubated for 3 h at 26°C with W. saturnus var. mrakii MUCL 41968 spent growth medium (Figure 5c), indicating an alteration of cell integrity as a result of the WmKT activity. To test this hypothesis, P. anomala MUCL 41969 cells were incubated for 3 h at 26°C with purified WmKT at a concentration of 500 µg/mL, followed by flow cytometry analysis of PI staining. The control experiment was carried out with purified WmKT that had been heated previously to 100°C for 10 min. While heat-treated WmKT failed to induce significant PI staining (data not shown), purified WmKT led to results similar to those obtained with W. saturnus var. mrakii MUCL 41968 spent growth medium (Figure 5d). A time-course study over 80 min showed that after 10 min of incubation at 26°C with WmKT, 90.5% of the sensitive cells were stained.



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Figure 5. Flow cytometry analysis of a susceptible yeast strain treated with WmKT and stained with PI. A total of 106 P. anomala MUCL 41969 cells was mixed with conditioned growth medium of W. saturnus var. mrakii MUCL 41968 or purified WmKT. After 3 h of incubation cells were resuspended and stained in a PBS solution containing 10 µg/mL PI. (a) PI fluorescence histogram of cells mixed with unconditioned growth medium. (b) Fluorescence histogram of heat-killed cells stained with PI. (c) PI fluorescence histogram of cells mixed with conditioned growth medium. (d) PI fluorescence histogram of cells mixed with purified WmKT.

 
Molecular targets of WmKT

To determine whether WmKT acts by direct binding to the cell surface of sensitive cells, IFA tests with WmKTAbs were used to localize WmKT after incubation with P. anomala MUCL 41969-sensitive cells. An increased fluorescence signal was detected by flow cytometry (Figure 6a) compared with the control incubated in the absence of WmKT. As shown in Figure 6b, 51.5% of cell labelling was detected when the sensitive strain was incubated with WmKT whereas only 27% of cell labelling was observed in the control assay. This increased fluorescence suggests that the toxin indeed acts by binding to the cell surface. To study the influence of pH on the WmKT cell binding properties, IFA tests were repeated using PBS pH 7.5 instead of sodium acetate buffer pH 4.6. In these conditions, no increase in fluorescence was detected when P. anomala MUCL 41969 was treated with WmKT (data not shown). The WmKT binding properties thus seem to be affected directly by pH.



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Figure 6. Flow cytometry analysis of immunolabelled killer toxin bound to sensitive yeast cells. Sensitive yeast cells were incubated with conditioned growth medium of W. saturnus var. mrakii MUCL 41968. After this incubation, IFAs were carried out using WmKTAbs. (a) FITC fluorescence histograms: 1, untreated cells; 2, IFA performed without prior incubation with WmKT; 3, IFA performed after incubation with WmKT. (b) FITC fluorescence intensities greater than that obtained with the untreated control cells were defined as a labelling signal and the proportion of fluorescently labelled cells was determined. All IFA experiments were performed in triplicate and results are expressed as the mean of the percentage of labelled cells ± S.D.

 
To characterize the molecular targets of WmKT at the cell surface of sensitive strains, we studied the WmKT sensitivity of S. cerevisiae mutants defective in the production of cell wall ß-glucan. The S. cerevisiae PM359-1d strain, which is deficient in 1,3-ß-glucan synthesis due to the knr4 mutation,19 was resistant to WmKT, whereas its parental JY102 strain (KNR4) was demonstrated to be sensitive (Figure 7a). Similar experiments were performed with S. cerevisiae SEY6210 strain, bearing the kre1 mutation,20 which is associated with a defect in cell wall 1,6-ß-glucan production. Again, the mutant strain displayed a resistant phenotype, whereas the parental strain (KRE1) was highly sensitive to WmKT (Figure 7a). Flow cytometry was used to quantify the PI staining of these mutant strains after treatment with WmKT (Figure 7b). For a yeast strain bearing the kre1 mutation, only 1.6% ± 0.1 were stained, whereas under the same conditions 93.1% ± 1.3 of parental cells were stained by PI. Analysis of the staining phenotype linked to the knr4 mutation indicated that 31.8% ± 3.3 of the mutant cells and 95.6% ± 0.1 of the parental cells were PI labelled after contact with WmKT. This result indicates that the kre1 mutation induces total resistance to WmKT, while only partial resistance is conferred by the knr4 mutation.



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Figure 7. Study of WmKT activity against cell wall ß-glucan mutants of S. cerevisiae. The susceptibility to WmKT of S. cerevisiae SEY6210 kre1, S. cerevisiae PM359-1d knr4 and corresponding wild-type strains was compared. As positive controls, killer toxin assays were performed against P. anomala MUCL 41969. (a) Killer toxin assays in liquid medium. (b) PI staining of cells treated with WmKT supernatant. Hatched bars, cells treated with heat-inactivated WmKT; dotted bars, cells treated with WmKT.

 
Zymolyase sensitivity and WmKT activity

Yeast killer strains are immune to their own toxin.2 Since WmKT activity is decreased or abolished in S. cerevisiae strains with mutations in the ß-glucan synthesis pathway, we hypothesized that these cell wall compounds could be involved in the self-immunity mechanisms of W. saturnus var. mrakii MUCL 41968 to its toxin. To test this hypothesis, we compared the sensitivity of four yeast strains, i.e. two WmKT-sensitive and two KT-producing ones, to the ß-glucan-degrading enzyme zymolyase. Sensitivity was monitored by a time-course decrease in the OD600 of a standardized yeast suspension containing zymolyase at a concentration of 20 µg/mL. The two WmKT-sensitive strains, S. cerevisiae W303 and P. anomala MUCL 41969, turned out to be highly sensitive to zymolyase, since a 40 min incubation led to a 70% decrease in OD600. In contrast, in the same conditions, only a 0–20% decrease in the OD600 was observed for the KT-producing strains W. saturnus var. mrakii MUCL 41968 and P. anomala ATCC 96603. These results point to a correlation between sensitivity to zymolyase and to WmKT.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
W. saturnus var. mrakii MUCL 41968 has been found to secrete a killer toxin exhibiting a lethal effect against a broad range of pathogenic microorganisms, including P. carinii, C. albicans and M. tuberculosis.11,32,33 Because of its broad spectrum of activity, WmKT has been proposed as the basis for novel therapeutic strategies to treat insidious infectious diseases.34 However, no biochemical characterization of WmKT had been achieved so far and its mode of action remained poorly explored.35

In this study, we have fractionated by ion exchange chromatography the spent growth medium of W. saturnus var. mrakii MUCL 41968 and isolated fractions exhibiting high killer toxin activity. SDS–PAGE analysis of these fractions revealed the presence of three proteins with apparent molecular weights of 85, 73 and 60 kDa. Immunoblot analysis showed that only the 85 kDa protein was recognized by mAbKT4, which blocks WmKT activity.18 Such a high molecular weight of WmKT7,8 indicated that it might belong to a new class of killer toxins. This hypothesis is supported by the N-terminal amino acid sequence determination of purified WmKT, which revealed no significant similarity with sequences available in databases including those from killer toxins. Interestingly, the N-terminal amino acid sequencing of internal peptides demonstrated that this toxin exhibits similarities with proteins belonging to the yeast SUN family. Although the SUN proteins are involved in multiple cellular processes, so far no killer activity has been associated with any of them.27,28

To establish that the 85 kDa protein is WmKT, immunoblots and inhibition assays were performed using monospecific antisera directed against the purified 85 kDa protein. Since both mAbKT4 and WmKTAbs reacted specifically in western blot experiments with the same 85 kDa protein, and also abolished the killer activity of culture supernatants of W. saturnus var. mrakii MUCL 41968,12 it indicates that the 85 kDa protein is the WmKT. Immunofluorescence assays performed on various yeast strains showed that WmKTAbs have higher affinity for killer strains, such as W. saturnus var. mrakii MUCL 41968 and P. anomala ATCC 96603, than for killer toxin-sensitive strains. This correlates well with our previous results,21 which indicated that these killer yeast strains secrete toxins bearing common epitopes.

In contrast to mAbKT4, antisera raised against the 85 kDa protein also react with the 73 and 60 kDa proteins that co-elute in the ion-exchange fractionation with WmKT, indicating the presence of common epitopes. Amino acid sequencing of these three proteins revealed that they share an identical 12-amino-acid N-terminal sequence. These findings suggest that the 73 and 60 kDa proteins are either isoforms or degradation products of WmKT.

Preliminary observations indicated that WmKT may be glycosylated.9 In the present study, we have proven the glycosidic nature of WmKT by both concanavalin A binding assay and enzymic deglycosylation of purified WmKT. Interestingly, treatment of WmKT with combined NANase II, O-glycosidase DS and PNGase F generated a peptide of 55 kDa that is still recognized by mAbKT4, indicating that this monoclonal antibody recognizes a peptidic epitope. Assuming that complete deglycosylation of WmKT is achieved by such an enzymic treatment, a peptidic moiety of 55 kDa is in agreement with the hypothesis that it belongs to the SUN family proteins, since the average molecular weight of SUN proteins is c. 50 kDa, and some of them appear to be glycosylated.27

To elucidate the action of WmKT at the cellular level, WmKTAbs were incubated with sensitive strains exposed to WmKT, and antibody binding was monitored by flow cytometry analysis. Whereas an intense fluorescence signal was observed at pH 4.6, it disappeared at neutral pH, suggesting a pH-dependent binding of the killer toxin to the surface of target cells. This hypothesis is further supported by the lability of the WmKT activity in neutral or basic conditions.11

WmKT-sensitive yeast strains exhibited high PI fluorescence when exposed to WmKT. Since a recent study indicated that flow cytometry measurement of PI fluorescence can discriminate fungicidal activity from a fungistatic effect,30 it appears that WmKT exerts a microbicidal activity, leading to increased membrane permeability. A kinetic study demonstrated that WmKT acts rapidly on target cells since increased PI staining was already observed after 10 min incubation of yeasts with WmKT. Similar results have been reported for a 1.8–5.0 kDa killer toxin (K-500) produced by W. mrakii NCYC 500. K-500 is active at pH 4 and at 30°C, conditions that are analogous to those of WmKT, and induces rapid membrane permeability after contact with target cells.7 It has been suggested that K-500 killer toxin acts in a similar manner to the K1 S. cerevisiae killer toxin36 and the Pichia kluyveri toxin.37

Using microcultures and PI staining assay, we showed that a null mutant of S. cerevisiae for the KNR4 gene involved in 1,3-ß-glucan synthesis19 becomes partially resistant to WmKT. Moreover, a S. cerevisiae strain with a mutation in the KRE1 gene, phenotypically characterized by an altered cell wall 1,6-ß-glucan, is totally resistant to WmKT.20 Since S. cerevisiae strains bearing mutations in the ß-glucan synthesis pathway are more resistant to WmKT, it suggests that ß-glucans may be the cell wall target of the killer toxin, which could therefore act as a glucanase. This is further supported by the finding that two members of the SUN family, Sun4p from S. cerevisiae and BglAp proteins from Candida wickerhamii are cell wall proteins that have been postulated to display glucanase activity.27,38 The hypothetical glucanase activity of WmKT is also corroborated by the observation that killer strains W. mrakii MUCL 41968 and P. anomala 96603 are resistant to zymolyase, an enzyme with 1,3-ß-glucanase activity. Resistance to zymolyase degradation may be explained by either an increased amount of 1,3-ß-glucan in the cell wall or a modified cross-linkage between 1,3-ß-glucan motifs and other wall components such that carbohydrate linkages become inaccessible to the enzyme.19 In accordance with the fact that ß-glucans may be potential targets for WmKT on the yeast surface, the self-immunity mechanism of W. saturnus var. mrakii MUCL 41968 to its own toxin could be explained by increased ß-glucan synthesis or by the inaccessibility of these ß-glucans at the cell surface of the killer yeast.

The results reported here represent the first information concerning the mode of action and biochemical properties of WmKT. The potential ß-glucanase activity of WmKT may explain the wide spectrum of activity reported for WmKT, since ß-glucans are supposed to be present at the cell surface of most WmKT-sensitive pathogens. Actually, ß-glucans are major structural components of yeast cell walls39 and recent studies have revealed that the cell surface of P. carinii is also composed largely of ß-glucans.40 Moreover, M. tuberculosis, which is also sensitive to killer toxin, is surrounded by a capsule containing glucans.41 These capsular glucans are potential virulence factors since they are involved in interaction between M. tuberculosis and the CR3 ß-glucan receptor present at the surface of host cells.42 If the action of WmKT is based on the degradation of capsular glucans, WmKT might reduce mycobacterial virulence and might be useful in treating multidrug-resistant tuberculosis. Investigations into the potential ß-glucanase activity of WmKT are currently in progress in our laboratory using biochemical assays. We are also cloning the WmKT-encoding gene to produce recombinant WmKT in Escherichia coli and yeast strains. These recombinant forms will be useful in further characterizing WmKT activity and stability, and in specifying its mechanism of action, a prerequisite in the development of new anti-infectious strategies based on WmKT.


    Acknowledgements
 
We acknowledge Beth Didomenico at Schering-Plough Research Institute (Kenilworth, NJ, USA) for providing S. cerevisiae ZH 401-1-d and M. Schmitt at Universität des Saarlandes (Germany) for providing S. cerevisiae SEY6210 kre1. We are grateful to Christian Sergheraert for his support during this work. C.G. is supported by a PhD grant from the Région Nord Pas-de-Calais (France) and the Institut Pasteur de Lille (France). This work was funded in part by grants from the Fondation pour la Recherche Médicale and FEDER.


    Footnotes
 
* Corresponding author. Tel: +33-3-20-87-71-57; Fax: +33-3-20-87-79-08; E-mail: Cyril.Guyard{at}pasteur-lille.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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