The Benefits of Saccharomyces boulardii

2.1. Growth properties of S. boulardii

To further characterize this yeast, its growth properties at different temperatures are compared with other diploid yeast strains (all yeast strains used are summarized in Table 1). S. boulardii (I will keep this name in the text even though it is a S. cerevisiae strain) grows well on rich medium at 30°C as well as at 37°C but not at 40°C (Figure 1A). Growth at 37°C—though not ideal—is not uncommon to yeast strains such as the diploid strain BY4743 (a derivative of S288C) but as opposed to diploid strain RH2585/2586 (an Σ1278b derivative) which hardly grows at 37°C (Figure 1A). In that sense, growth at 37°C is not a particular and unique property of S. boulardii. It probably rather reflects its accommodation to hot climates such as those found often in Indochina.

A further investigated property is S. boulardii’s potential capability to form filaments. For this purpose, it was grown on SLAD plates which carry only limiting concentrations of ammonium sulfate (50 μM, about 1000× less than conventional SD minimal medium). As observed under the microscope, the diploid strain RH2585/2586 clearly shows filamentous properties under such ammonium-limiting conditions (Figure 1B) [9]. Deletion of the FLO8 gene encoding a transcriptional factor required for filamentation and adhesion completely abolished its filamentous properties. Flo8 is required to express, for example, Flo11, a cell-surface glycoprotein [10]. As opposed to RH2585/2586, S. boulardii hardly showed any filamentation properties. Interestingly, sequencing of the PCR-amplified FLO8 gene of S. boulardii indicated that it does not carry a premature stop codon (not shown) found in non-filamentous yeast strains as those derived from S288C [10]. So, other up- or downstream genes required for filamentation are likely dysfunctional in S. boulardii. Its lack of filamentation probably explains its low toxicity and its lack of establishment capacity in the gastrointestinal tract that otherwise could make it more persistent and thereby more problematic for medical applications.

Another interesting issue was to induce meiosis and sporulation in S. boulardii in order to obtain haploid progeny. For this purpose, diploid cells were incubated in a liquid medium with limiting nitrogen and very high potassium acetate concentration as a (poor) carbon source [11]. Despite several attempts, no tetrad formation was observed. In that respect, S. boulardii shows similar properties as RH2585/2586 (the filamentous diploid used in this work) that does not form tetrads upon treatment under the described conditions. In order to induce meiosis and sporulation, a double knockout of RME1 (Regulator of Meiosis 1) in S. boulardii was produced. RME1 is a negative regulator of meiosis that prevents the expression of meiosis-required proteins such as IME1 (Inducer of Meiosis 1) and promotes mitosis [12]. The S. boulardii ∆∆rme1 derivative did not show any phenotypical differences to the parental wild-type strain in terms of growth temperature (Figure 1A). Unfortunately, no tetrads were obtained from this knockout strain either. I conclude that S. boulardii does not undergo meiosis and haploid tetrad formation at least under laboratory conditions used here.

As shown in Figure 1C, S. boulardii does not carry any auxotrophic markers as it grows well on minimal medium (SD) devoid of amino acids or nucleic acid components such as adenine or uracil. Auxotrophic marker genes could be easily obtained by deleting the ADE2 (adenine biosynthesis) or HIS3 (histidine biosynthesis) genes (Figure 1C). These deletions were obtained by introducing dominant auxotrophic marker genes that provide resistance to the antibiotics kanamycin/G418 or nourseothricin. Deletion of a single gene copy of ADE2 or HIS3 still allowed for growth on minimal medium plates (not shown) indicating the clear diploid character of this yeast. Only double deletion of both HIS3- or ADE2-gene copies (which made it resistant against both kanamycin/G418 and nourseothricin; Figure 1C) made this yeast strain auxotrophic for histidine or for adenine. Newly gained histidine auxotrophy was used in a subsequent experiment to transform it with yeast plasmids carrying HIS3 as a selectable marker gene (see subsequent text).

2.2. Transformation of S. boulardii with conventional yeast plasmids

As an auxotrophic histidine-deficient yeast strain was now available, I decided to transform it with conventional yeast plasmids that complement for the lack of HIS3. For this purpose, S. boulardii ∆∆his3 (#1811) was grown in minimal SD medium supplemented with histidine. Cells were made competent by treating them with Li-acetate following a well-established yeast transformation protocol [13].

After 2–3 days of incubation at 30°C, his⁺-transformants were nicely observed (Figure 2; left panel), indicating that a simple yeast transformation protocol was sufficient to transform this yeast strain and to recover its prototrophy. Plasmids used for transformation (p301HIS3 GAL-p20-HA from S. cerevisiae and Candida albicans) allow for the expression of the protein p20 (a modulator of the activity of eIF4E, the cap-binding protein; see subsequent text) when growing cells in a medium containing galactose. As shown on a Western Blot obtained from yeast extracts, p20 from different sources was expressed in galactose but not in glucose-containing media (Figure 2; right panel). This confirms that, in S. boulardii, the GAL1/10 promoter is regulated in an identical manner as in conventional yeast strains used in the laboratory [14].

2.3. Sequencing and comparison of S. boulardii p20 gene

p20, a small acidic protein of 161 amino acids, is encoded by the non-essential gene CAF20 which only exists in a variety of yeast species (such as S. glabrata, Kluyveromyces lactis, C. albicans, S. cerevisiae). Its function is related to regulating the activity of the cap-binding protein eIF4E during translation in a yet not-well-understood manner [15]. A sequence alignment of different yeast species (see Figure 3; upper panel) shows a clear homology but not identity of the corresponding p20 proteins. Especially conserved are peptide motifs at the amino terminus (which are required for binding to eIF4E; the canonical motif YxxxxLL/I/F highlighted) and at the carboxy terminus (where precise function has still to be determined).

The CAF20 gene from S. boulardii was PCR-amplified using genomic DNA and oligonucleotides hybridizing at the 5′ and 3′ region of the S. cerevisiae CAF20 gene. Subsequent sequencing showed that CAF20 from S. cerevisiae and S. boulardii is nearly identical. The only difference detected is a conserved amino acid substitution (leucine to valine; highlighted) at position 16 (Figure 3; lower panel). Among those yeast species that carry the p20 gene conservation varies between 30 and 90% (not shown). The almost identity of both sequences shown here clearly confirms that S. boulardii is a variant of the species S. cerevisiae.