Abf1 negatively regulates the expression of EPA1 and affects adhesion in Candida glabrata

<b>Introduction</b>. Adherence is a major virulence trait in <i>Candida glabrata</i> that, in many strains, depends on the <i>EPA</i> (epithelial adhesin) genes, which confer the ability to adhere to epithelial and endothelial cells of the host. The <i>EPA</i> genes are generally found at subtelomeric regions, which makes them subject to subtelomeric silencing. In <i>C. glabrata</i>, subtelomeric silencing depends on different protein complexes, such as silent information regulator and yKu complexes, and other proteins, such as Repressor/activator protein 1 (Rap1) and Abf1. At the <i>EPA1</i> locus, which encodes the main adhesin Epa1, we previously found at least two <i>cis</i>-acting elements, the protosilencer Sil2126 and the negative element, that contribute to the propagation of silencing from the telomere to the subtelomeric region.<b>Hypothesis</b>. Abf1 binds to the regulatory regions of <i>EPA1</i> and other regions at the telomere E-R, thereby negatively regulating <i>EPA1</i> transcription.<b>Aim</b>. To determine whether Abf1 and Rap1 silencing proteins bind to previously identified <i>cis-</i>acting elements on the right telomere of chromosome E (E-R subtelomeric region), resulting in negative regulation of <i>EPA1</i> transcription and infer Abf1 and Rap1 recognition sites in <i>C. glabrata</i>.<b>Methodology</b>. We used chromatin immunoprecipitation (ChIP) followed by quantitative PCR to determine the binding sites for Abf1 and Rap1 in the intergenic regions between <i>EPA1</i> and <i>EPA2</i> and <i>HYR1</i> and <i>EPA1</i>, and mutants were used to determine the silencing level of the <i>EPA1</i> promoter region.<b>Results</b>. We found that Abf1 predominantly binds to the <i>EPA1</i> promoter region, leading to negative regulation of <i>EPA1</i> expression. Furthermore, the mutant <i>abf1-43</i>, which lacks the last 43 amino acids at its C-terminal end and is defective for subtelomeric silencing, exhibits hyperadherence to epithelial cells <i>in vitro</i> compared to the parental strain, suggesting that <i>EPA1</i> is derepressed. We also determined the motif-binding sequences for Abf1 and Rap1 in <i>C. glabrata</i> using data from the ChIP assays.<b>Conclusion</b>. Together these data indicate that Abf1 negatively regulates <i>EPA1</i> expression, leading to decreased adhesion of <i>C. glabrata</i> to epithelial cells.