Peptide Detection of Fungal Functional Amyloids in Infected Tissue

Many fungal cell adhesion proteins form functional amyloid patches on the surface of adhering cells. The Candida albicans Agglutinin-like sequence (Als) adhesins are exemplars for this phenomenon, and have amyloid forming sequences that are conserved between family members. The Als5p amyloid sequence mediates amyloid fibril formation and is critical for cell adhesion and biofilm formation, and is also present in the related adhesins Als1p and Als3p. We have developed a fluorescent peptide probe containing the conserved Als amyloid-forming sequence. This peptide bound specifically to yeast expressing Als5p, but not to cells lacking the adhesin. The probe bound to both yeast and hyphal forms of C. albicans. Δals1/Δals3 single and double deletion strains exhibited reduced fluorescence, indicating that probe binding required expression of these proteins. Additionally, the Als peptide specifically stained fungal cells in abscesses in autopsy sections. Counterstaining with calcofluor white showed colocalization with the amyloid peptide. In addition, fungi in autopsy sections derived from the gastrointestinal tract showed colocalization of the amyloid-specific dye thioflavin T and the fluorescent peptide. Collectively, our data demonstrate that we can exploit amyloid sequence specificity for detection of functional amyloids in situ

Als5p expressing S. cerevisiae cells stained with fluorescent peptides.

<p><i>S. cerevisiae</i> harboring an empty vector (A,C) or expressing Als5p (B, D) were stained with amyloid peptide (200 µg/ml; A, B) or a scrambled-sequence peptide of identical composition (200 µg/ml; C, D). Lower micrographs are bright field images. All scale bars are 30 µm.</p

Autopsy sections stained with Als5p amyloid peptide and calcofluor white.

<p>A-C) Confocal microscopy of gut section stained with A) amyloid peptide (200 µg/ml), B) calcofluor white, C) merged image. D-F) Spleen section stained with D) amyloid peptide (200 µg/ml), E) calcofluor white, F) merged image.</p

Confocal micrographs of <i>C. albicans</i> yeast and hyphae binding Als5p peptide.

<p><i>C. albicans</i> cultured in spider medium, with 20 µg/ml (A, C) amyloid or (B, D) scrambled sequence peptide. Fields were imaged containing (A, B) yeast or (C, D) hyphae at a gain of 6.5. Inset images in A and B were acquired at a higher gain of 7.0.</p

Autopsy section stained with Als5p amyloid peptide and thioflavin T.

<p>Confocal images of A-C) gut section stained with A) amyloid peptide (200 µg/ml), B) thioflavin T (100 nM), C) merged image, D) brightfield. All scale bars are 40 µm.</p

Gomori’s Methenamine Silver stained autopsy section

<p>. <i>Candida</i> yeast and filamentous forms (hyphae and pseudohyphae) invading human gastrointestinal epithelium at the luminal border <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086067#pone.0086067-Gilchrist1" target="_blank">[8]</a>. Tissue is a green-blue and the fungi are black.</p

Peptide staining of <i>als1/als1 als3/als3</i> deletion strains.

<p>All strains were induced to form hyphae by incubating overnight in spider media at 37^°C. A) CAI4 and B) <i>als1/als1 als3/als3</i> double deletion strains were probed with 20 µg/ml amyloid peptide with their corresponding brightfield images (right). All scale bars are 40 µm. C) Quantification of the average intensity of images A and B. Error bars represent standard error of the mean (n = 4; see text for details).</p

Aggregation and amyloid peptide staining of <i>C. albicans</i>.

<p>A) Aggregates of wild type (CAI4) and double deletion strains binding with heat denatured BSA-coated magnetic beads. B) The yeast form of <i>C. albicans</i> strains, SC5314, CAI4 and <i>als1/als1 als3/als3</i> double deletion were probed with 20 µg/ml amyloid peptide. All scale bars are 20 µm.</p