The Epithelial adhesin 1 tandem repeat region mediates protein display through multiple mechanisms

Abstract

The pathogenic yeast Candida glabrata is reliant on a suite of cell surface adhesins that play a variety of roles necessary for transmission, establishment and proliferation during infection. One particular adhesin, Epithelial Adhesin 1 [Epa1p], is responsible for binding to host tissue, a process which is essential for fungal propagation. Epa1p structure consists of three domains: an N-terminal intercellular binding domain responsible for epithelial cell binding, a C-terminal GPI anchor for cell wall linkage and a serine/threonine-rich linker domain connecting these terminal domains. The linker domain contains a 40-amino acid tandem repeat region, which we have found to be variable in repeat copy number between isolates from clinical sources. We hypothesized that natural variation in Epa1p repeat copy may modulate protein function. To test this, we recombinantly expressed Epa1p with various repeat copy numbers in S. cerevisiae to determine how differences in repeat copy number affect Epa1p expression, surface display and binding to human epithelial cells. Our data suggest that repeat copy number variation has pleiotropic effects, influencing gene expression, protein surface display and shedding from the cell surface of the Epa1p adhesin. This study serves to demonstrate repeat copy number variation can modulate protein function through a number of mechanisms in order to contribute to pathogenicity of C. glabrata.

Keywords: antimicrobial; killer yeasts; mycocins.

Figure 1.

EPA1 codes for an adhesin with variable repeat copy number. (A)EPA1 repeat region amplified from gDNA of 24 C. glabrata clinical isolates (1–24) and BG14 reference strain (R) through PCR and visualized on an agarose gel. (B)EPA1 repeat copy number frequency from the 24 clinical isolates. (C) pSPHA-EPA1 expression vector cloned into S. cerevisiae allows for constitutive expression of HA-tagged Eap1p (A domain—purple, B domain—light blue, GPI anchor—dark blue) with a 3x HA-tag (green) directed to the surface by the Epa1p signal sequence (pink) under the control of the ADH promoter (grey arrow) and CYC1 terminator (grey box). Five repeat copy number variants of EPA1 were cloned; the black arrows represent each individual tandem repeat. pADH is the empty vector backbone.

Figure 2.

Epa1p repeat copy number affects surface display. HA-Epa1p with 0, 3, 4 and 5 repeats was expressed in S. cerevisiae under constitutive expression under the ADH promoter, and surface display was measured via ɑHA flow cytometry. (A) Flow cytometry gating strategy. Single cells were selected from debris and doublets, and the negative control (pADH) was utilized to set the gate for the HA + population detected by positive AlexaFluor 488 fluorescence. (B) Surface display of HA-Epa1p with various repeat copy numbers. Y-axis counts are normalized by division by the count of the mode fluorescent intensity of each sample. Plot is representative of multiple experiments. (C) Percentage of cells stained positively for HA-Epa1p. (D) Mean fluorescence intensity (arbitrary units, AU) of HA + stained cells. Mean + SEM is shown. n = 3. Representative experiment from multiple independent experiments. ** = P < 0.01, **** = P < 0.0001 as determined by paired Bonferroni corrected T-tests between all experimental groups.

Figure 3.

EPA1 mRNA expression levels decrease with increased repeat copy number. (A) mRNA expression levels of EPA1 in S. cerevisiae carrying the empty pADH vector or pSPHA-EPA1 (3 rep) or BG14, a lab strain of C. glabrata measured by rt-qPCR. Expression is expressed as fold change over Actin (ACTI). (B) RT-qPCR-quantified expression levels of HA-EPA1 with various repeat copy numbers (0, 3, 4, 5, 10) of the EPA1 repeat region expressed through the pSPHA-EPA1 plasmid system. n.d. = no detection. Expression is expressed as fold-change over expression of Actin (3 rep). Mean + SEM is shown. n = 2. Representative from multiple independent experiments.

Figure 4.

Shedding of HA-Epa1p fragments increases with reduced repeat copy number. (A) HA-Epa1p shedding assay. Briefly, cells were grown to log phase and their growth media was collected by centrifugation. Media was then concentrated by lyophilization and resuspended at 40x in Tris-EDTA. (B) ɑHA western blot of concentrated media from cells expressing HA-Epa1p with various repeat copy numbers (0, 3, 4, 5 rep) or the empty pSPHA vector. Media were collected from an equivalent number of cells and concentrated via lyophilization before western blot analysis. Blot is representative of three replicates.

Figure 5.

Quantifying HA-Epa1p mediated epithelial cell adherence. (A) Binding of S. cerevisiae expressing HA-Epa1p repeat copy number variants (0, 3, 5 repeats) to epithelial cells was measured via an epithelial cell adherence assay. Unbound yeast were then removed via washing in PBS, and HeLa cells were chemically lysed. Bound yeast were quantified by counting colony forming units (CFUs) and comparing them to the CFUs on input control plates. (B) Binding normalized to HA-Epa1p (3 rep). Values are mean + SEM deviation. n = 5–6. Data pooled from multiple experiments.