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

Abstract

The availability of simple, specific, and inexpensive on-site detection methods is of key importance for deployment of pathogen surveillance networks. We developed a nontechnical and highly specific colorimetric assay for detection of pathogen-derived peptides based on Saccharomyces cerevisiae-a genetically tractable model organism and household product. Integrating G protein-coupled receptors with a visible, reagent-free lycopene readout, we demonstrate differential detection of major human, plant, and food fungal pathogens with nanomolar sensitivity. We further optimized a one-step rapid dipstick prototype that can be used in complex samples, including blood, urine, and soil. This modular biosensor can be economically produced at large scale, is not reliant on cold-chain storage, can be detected without additional equipment, and is thus a compelling platform scalable to global surveillance of pathogens.

Keywords: G-Protein coupled receptor; Yeast; biosensor; fungal pathogens; invasive fungal infections; low-cost diagnostic; lycopene; peptide biomarker; point-of-care; synthetic biology.

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.