Abstract
The cell wall is an essential, unique and highly conserved structure in fungi, thus representing an ideal set of targets for antifungal drugs. In the model yeast S. cerevisiae, the Pkc1-mediated cell integrity signalling pathway is essential for maintenance of the cell wall. Adaptation to cell wall stress involves the transcriptional activation of genes functionally relevant for cell wall remodelling. One of these activated genes, namely MLP1/ YKL161c, is an ideal indicator of cell wall perturbations, Mlp1p, being almost undetectable under normal growth conditions, accumulated in large amounts when cell wall integrity was compromised. We have developed a reporter system based on the expression of the nourseothricin resistance gene under the control of the regulatory sequences of MLP1. Yeast cells transformed with this reporter construct, subjected to a cell wall stress, by chemical agents present in the culture medium, attained a high level of nourseothricin-resistance with respect to non-stressed cells, as a consequence of increased MLP1 expression. A genetically modified S. cerevisiae strain (AT-1) including the reporter system integrated into the native MLP1 chromosomal locus was also developed. This strain was tested against several compounds, grouping different mechanisms of yeast growth inhibition, responding specifically to cell wall-perturbing agents. Our results demonstrate the usefulness and feasibility of the AT-1 strain as a biosensor to perform high-throughput antifungal screenings for the identification of antifungal compounds active on the cell wall.
Published Version
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