Abstract
Over the last few decades, the emergence of resistance to commonly used antifungal molecules has become a major barrier to effective treatment of recurrent life-threatening fungal diseases. Resistance combined with the increased incidence of fungal diseases has created the need for new antifungals, such as the plant defensin NaD1, with different mechanisms of action to broaden treatment options. Antimicrobial peptides produced in plants and animals are promising new molecules in the arsenal of antifungal agents because they have different mechanisms of action to current antifungals and are often targeted specifically to fungal pathogens (van der Weerden et al., 2013). A key step in the development of novel antifungals is an understanding of the potential for the fungus to develop resistance. Here, we have used the prototypic plant defensin NaD1 in serial passages with the model fungus Saccharomyces cerevisiae to examine the evolution of resistance to plant antifungal peptides. The yeast strains did develop tolerance to NaD1, but it occurred more slowly than to the clinically used antifungal caspofungin. Sequencing the genomes of the strains with increased tolerance failed to identify any ‘hotspot’ mutations associated with increased tolerance to NaD1 and led to the identification of 12 genes that are involved in resistance. Characterization of the strains with increased tolerance to NaD1 also revealed changes in tolerance to abiotic stressors. Resistance developed slowly via an accumulation of single nucleotide mutations and had a fitness penalty associated with it. One of the genes identified FPS1, revealed that there is a common mechanism of resistance to NaD1 that involves the osmotic stress response pathway. These data indicate that it is more difficult to generate resistance to antimicrobial peptides such as NaD1 compared to small molecule antifungals.
Highlights
Pathogenic fungi have become a serious threat to both agriculture and human health (Murray and Brennan, 2009)
The NaD1-resistant isolates were 10-fold more resistant to NaD1 than the no-treatment control lines that had been passaged at the same time, with an minimum inhibitory concentration (MIC) of 40 μM compared to an MIC of 4 μM (Figure 1B and Table 1)
We described the development of S. cerevisiae tolerance to an antifungal plant protein, the defensin NaD1
Summary
Pathogenic fungi have become a serious threat to both agriculture and human health (Murray and Brennan, 2009). Fungicide resistance occurs when a fungal pathogen becomes less susceptible to an antifungal agent. Resistance is broadly characterized by the mechanism by which it occurs These mechanisms include; alteration of the target site in a protein, detoxification of the fungicide, overexpression of the target site, and the use of efflux pumps to expel the fungicide (Leroux et al, 2002). The increased use of the small molecule antifungal drugs that are currently in the clinic as well as related molecules used in agriculture has led to reports of fungal pathogens resistant to almost all common antifungals (Verweij et al, 2009). There is a need for new antifungal agents to battle the phenomenon of fungal resistance; antifungal proteins are one attractive option for development (Sanglard et al, 1996; van der Weerden et al, 2013)
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