Research ArticleHEALTH AND MEDICINE

A modular yeast biosensor for low-cost point-of-care pathogen detection

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Science Advances  28 Jun 2017:
Vol. 3, no. 6, e1603221
DOI: 10.1126/sciadv.1603221
  • Fig. 1 S. cerevisiae biosensor for detection of fungal pathogens.

    (A) Overview of biosensor components. Highly specific fungal receptors provide sensitive response to mating peptides secreted by pathogenic fungi. Activation of the downstream mating signaling pathway induces transcriptional activation of biosynthetic genes for production of red lycopene pigment visible to the naked eye. FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. (B) Color signal as shown in paper-based dipstick assay. Scale bars, 0.5 cm.

  • Fig. 2 Biosensor functionality and lycopene optimization.

    (A) Activation of C. albicans mating receptor (Ca.Ste2) in S. cerevisiae by its cognate mating peptide. Fluorescence (black) and lycopene absorbance (red) were used as a transcriptional readout for receptor activation. (B) Specificity of Ca.Ste2 and Sc.Ste2 receptors. Fluorescence was determined after 9 hours using 5 μM synthetic fungal peptides. (C) Optimization of lycopene production. Maximal lycopene yield was measured after induction with 10 μM synthetic S. cerevisiae mating peptide. Null, parental strain (no lycopene genes); Lyco-1, parental strain with single-copy CrtE, CrtB, and CrtI; 2xCrtI, Lyco-1 with additional plasmid-borne copy of CrtI; Fad1, Lyco-1 with additional plasmid-borne copy of Fad1; Lyco-2, all genes genomically integrated into Lyco-1. (D) Time course of lycopene production in lycopene-producing strains. Induction as in (C). (E) Representative photos of cell pellets (5 × 107 cells) corresponding to strains in (D). Lycopene per cell was determined by spectroscopy (see Supplementary Methods).

  • Fig. 3 Yeast biosensor for multiple fungal peptides.

    (A) Activation of fungal mating receptors in S. cerevisiae by the corresponding cognate synthetic mating peptides (40 μM) (see also fig. S4). Dotted line denotes the effective visible threshold from Fig. 2A. (B) EC50 values calculated for fungal receptors in S. cerevisiae using cognate ligands. (C) Specificity of heterologous fungal receptors. Receptors were activated by 5 μM synthetic peptides. Response was measured by fluorescence and normalized for each receptor (see fig. S5). (D) Comparative scoring of all biosensors. (E) Lycopene production induced by culture supernatant from clinically isolated fungal pathogens. Lycopene per cell measured by spectroscopy at 9 hours (see Supplementary Methods). **P ≤ 0.01, ***P ≤ 0.001; n = 3.

  • Fig. 4 Paper-based dipstick assay for detection of fungal pathogens.

    (A) Dipstick device. Inset: “+,” positive biosensor strain; “−,” negative control strain. (B) Quantitative analysis of lycopene production using dipstick assay, as scored by time-lapse photography (see Supplementary Methods) for detection of 1 μM synthetic P. brasiliensis mating peptide. Individual runs are shown in light color, and average response is shown in dark color. Shading indicates visible threshold. (C) P. brasiliensis and C. albicans mating peptides were reproducibly detected using the dipstick assay. Maximal response was achieved by 12 hours after exposure to the respective peptides (1 μM). (D) Detection of P. brasiliensis mating peptide in complex samples. Liquid samples were supplemented with synthetic P. brasiliensis mating peptide (blue) or water (gray), and scored as in (B). YPD, medium only; soil, standard potting soil; urine, 50% pooled human urine; serum, 50% human serum; blood, 2% whole blood. All experiments were performed using 1 μM peptide and supplemented with YPD medium. AU, arbitrary units.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1603221/DC1

    Supplementary Methods

    fig. S1. Optimization of peptide-induced lycopene production.

    fig. S2. C. albicans biosensor robustness in liquid culture.

    fig. S3. Sequence analysis of fungal mating receptors.

    fig. S4. Dose-response curves for fungal mating receptors.

    fig. S5. Specificity of fungal mating receptors.

    fig. S6. P. brasiliensis biosensor characterization in liquid culture.

    fig. S7. Comparison of mating receptors from human pathogens P. brasiliensis and H. capsulatum.

    fig. S8. Paper-based dipstick assay.

    fig. S9. Detection of C. albicans using dipstick assay.

    fig. S10. Long-term stability of paper-based dipsticks stored at room temperature.

    table S1. Fungal pathogen peptides and receptor genes used in this study.

    table S2. Strains used in this study.

    table S3. Plasmids used in this study.

    table S4. List of expression modules constructed in this study.

    table S5. Primers for cloning of fungal receptors and for genotyping of C. albicans isolates.

    table S6. DNA sequence for fungal receptor ORFs used in this study.

    movie S1. Yeast dipstick assay with plastic holder.

    movie S2. Yeast dipstick assay in soil.

    movie S3. Yeast dipstick assay in urine.

    movie S4. Yeast dipstick assay in serum.

    movie S5. Yeast dipstick assay in blood.

    References (5255)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • fig. S1. Optimization of peptide-induced lycopene production.
    • fig. S2. C. albicans biosensor robustness in liquid culture.
    • fig. S3. Sequence analysis of fungal mating receptors.
    • fig. S4. Dose-response curves for fungal mating receptors.
    • fig. S5. Specificity of fungal mating receptors.
    • fig. S6. P. brasiliensis biosensor characterization in liquid culture.
    • fig. S7. Comparison of mating receptors from human pathogens P. brasiliensis and H. capsulatum.
    • fig. S8. Paper-based dipstick assay.
    • fig. S9. Detection of C. albicans using dipstick assay.
    • fig. S10. Long-term stability of paper-based dipsticks stored at room temperature.
    • table S1. Fungal pathogen peptides and receptor genes used in this study.
    • table S2. Strains used in this study.
    • table S3. Plasmids used in this study.
    • table S4. List of expression modules constructed in this study.
    • table S5. Primers for cloning of fungal receptors and for genotyping of C. albicans isolates.
    • table S6. DNA sequence for fungal receptor ORFs used in this study.
    • Legends for movies S1 to S5
    • References (52–55)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Yeast dipstick assay with plastic holder.
    • movie S2 (.mp4 format). Yeast dipstick assay in soil.
    • movie S3 (.mp4 format). Yeast dipstick assay in urine.
    • movie S4 (.mp4 format). Yeast dipstick assay in serum.
    • movie S5 (.mp4 format). Yeast dipstick assay in blood.

    Download Movies S1 to S5

    Files in this Data Supplement:

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