Capric Acid Secreted by S. boulardii Inhibits C. albicans Filamentous Growth, Adhesion and Biofilm Formation

Candidiasis are life-threatening systemic fungal diseases, especially of gastro intestinal track, skin and mucous membranes lining various body cavities like the nostrils, the mouth, the lips, the eyelids, the ears or the genital area. Due to increasing resistance of candidiasis to existing drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. One promising strategy is the use of the probiotic microorganisms, which when administered in adequate amounts confer a health benefit. Such a probiotic microorganism is yeast Saccharomyces boulardii, a close relative of baker yeast. Saccharomyces boulardii cells and their extract affect the virulence factors of the important human fungal pathogen C. albicans, its hyphae formation, adhesion and biofilm development. Extract prepared from S. boulardii culture filtrate was fractionated and GC-MS analysis showed that the active fraction contained, apart from 2-phenylethanol, caproic, caprylic and capric acid whose presence was confirmed by ESI-MS analysis. Biological activity was tested on C. albicans using extract and pure identified compounds. Our study demonstrated that this probiotic yeast secretes into the medium active compounds reducing candidal virulence factors. The chief compound inhibiting filamentous C. albicans growth comparably to S. boulardii extract was capric acid, which is thus responsible for inhibition of hyphae formation. It also reduced candidal adhesion and biofilm formation, though three times less than the extract, which thus contains other factors suppressing C. albicans adherence. The expression profile of selected genes associated with C. albicans virulence by real-time PCR showed a reduced expression of HWP1, INO1 and CSH1 genes in C. albicans cells treated with capric acid and S. boulardii extract. Hence capric acid secreted by S. boulardii is responsible for inhibition of C. albicans filamentation and partially also adhesion and biofilm formation

The e¡ect of Saccharomyces boulardii on Candida albicans-infected human intestinal cell lines Caco-2 and Intestin 407

Abstract Saccharomyces boulardii is a probiotic strain that confers many benefits to human enterocolopathies and is used against a number of enteric pathogens. Candida albicans is an opportunistic pathogen that causes intestinal infections in immunocompromised patients, and after translocation into the bloodstream, is responsible for serious systemic candidiasis. In this study, we investigated the influence of S. boulardii cells and its culture extract on C. albicans adhesion to Caco-2 and Intestin 407 cell lines. We also tested the proinflammatory IL-1b, IL-6 and IL-8 cytokine expression by C. albicans-infected Caco-2 cells, using real-time RT-PCR. We found that both S. boulardii and its extract significantly inhibited C. albicans adhesion to epithelial cell lines. The IL-8 gene expression by C. albicans-infected Caco-2 cells was suppressed by the addition of S. boulardii extract. Our results indicate that S. boulardii affects C. albicans adhesion and reduces cytokinemediated inflammatory host response

Oxidative Stress and Replication-Independent DNA Breakage Induced by Arsenic in <i>Saccharomyces cerevisiae</i>

<div><p>Arsenic is a well-established human carcinogen of poorly understood mechanism of genotoxicity. It is generally accepted that arsenic acts indirectly by generating oxidative DNA damage that can be converted to replication-dependent DNA double-strand breaks (DSBs), as well as by interfering with DNA repair pathways and DNA methylation. Here we show that in budding yeast arsenic also causes replication and transcription-independent DSBs in all phases of the cell cycle, suggesting a direct genotoxic mode of arsenic action. This is accompanied by DNA damage checkpoint activation resulting in cell cycle delays in S and G2/M phases in wild type cells. In G1 phase, arsenic activates DNA damage response only in the absence of the Yku70–Yku80 complex which normally binds to DNA ends and inhibits resection of DSBs. This strongly indicates that DSBs are produced by arsenic in G1 but DNA ends are protected by Yku70–Yku80 and thus invisible for the checkpoint response. Arsenic-induced DSBs are processed by homologous recombination (HR), as shown by Rfa1 and Rad52 nuclear foci formation and requirement of HR proteins for cell survival during arsenic exposure. We show further that arsenic greatly sensitizes yeast to phleomycin as simultaneous treatment results in profound accumulation of DSBs. Importantly, we observed a similar response in fission yeast <i>Schizosaccharomyces pombe</i>, suggesting that the mechanisms of As(III) genotoxicity may be conserved in other organisms.</p></div

As(III) induces DNA damage checkpoint response in G1 phase in Yku70-deficient cells.

<p>(A) Histone H2A and Rad53 phosphorylation induction by As(III) in G1 cells in the absence of Yku70. The indicated strains were synchronized in G1 with α-factor and treated with 0.5 mM As(III) for 1 h followed by protein extraction and Western blot analysis. (B) Accumulation of Rfa1-YFP foci in the G1-synchronized <i>yku70</i>Δ strain reveals existence of As(III)-induced DSBs in G1 phase which undergo resection in the absence of Yku70. The representative image of <i>yku70</i>Δ cells in G1 phase containing As(III)-induced Rfa1 foci (arrows) is shown. Cell treatment was as in (A). Standard deviations are derived from three independent experiments (*<i>p</i><0.01; Student's <i>t</i>-test). DIC, differential interference contrast. (C) Analysis of DNA damage response activation in G1-synchronized cells devoid of BER (<i>apn1</i>Δ <i>apn2</i>Δ) or Yku70 reveals that As(III)-induced DNA lesions are different from those generated by H₂O₂ or MMS. The indicated strains were exposed to 0.5 mM As(III), 0.5 mM H₂O₂ or 0.03% methyl methanesulfonate (MMS) for 1 h and analyzed by western blot.</p

As(III) enhances cytotoxicity of phleomycin.

<p>(A) Serial dilutions of the indicated strains were spotted onto control YPD and As(III)-containing YPD plates or media containing phleomycin (PM) with or without As(III). (B) Alternatively, yeast cells were irradiated with various doses of ionizing radiation before plating in the presence or absence As(III). (C, D) Cells were also cultivated on media containing hydroxyurea (HU) (C) or methyl methanesulfonate (MMS) (D) with or without As(III). Cells were incubated at 30°C for 2 days.</p

Transcription-independent DNA damage induction by As(III).

<p>(A) To shut off transcription 3 µg/ml thiolutin, an inhibitor of RNA polymerases, was added to asynchronous and G2/M phase wild type cells and G1-arrested <i>yku70</i>Δ mutant for 1 h and then 0.5 mM As(III) was added to the cells for 1 h followed by protein extraction and western blotting analysis of histone H2A phosphorylation. (B) The <i>rpb1-1</i> cells bearing a temperature-sensitive mutation in the catalytic subunit of RNA polymerase II grown at permissive temperature (25°C) were shifted or not to non-permissive temperature (37°C) by adding YPD pre-warmed to 45°C to block transcription and exposed to 0.5 mM As(III) for 1 h. For a control wild type cells were treated in a similar way. Protein extracts were analyzed by western blotting to detect levels of phosphorylated H2A. Total histone H2A was used as a loading control.</p

As(III) and phleomycin co-treatment increases formation of DSBs.

<p>(A) Increased accumulation of Rad52-YFP nuclear foci in wild type cells after 1 h of 0.5 mM As(III) and 0.5 µg/ml phleomycin (PM) co-treatment. Standard deviations are derived from three independent experiments (*<i>p</i><0.05; Student's <i>t</i>-test). (B) Yeast chromosome breaks in asynchronously growing cells of indicated strains containing a circular chromosome III were measured using PFGE followed by Southern hybridization of the shown gel with a <i>LEU2</i>-probe to detect chromosome II and III. (C) DSB induction during As(III) and PM co-treatment in wild type cells synchronized and maintained in G1 or G2/M phase. (B, C) Cells were treated with 5 mM As(III) and 10 µg/ml PM in YPD medium for 4 h. (D) PFGE analysis of <i>S. cerevisiae</i> chromosomes isolated from wild type cells exposed to 5 mM As(III), 10 µg/ml PM or 4 mM copper sulfate [Cu(II)] in YPD medium for 4 h. PFGE experiments were repeated at least two times with similar results and representative images are shown.</p