Transcriptome and network analyses in Saccharomyces cerevisiae reveal that amphotericin B and lactoferrin synergy disrupt metal homeostasis and stress response

Chi Nam Ignatius Pang,Marc R Wilkins,Yu-Wen Lai,Leona T Campbell,Dee A Carter,Sharon C.-A Chen

doi:10.1038/srep40232

Chi Nam Ignatius Pang, Marc R Wilkins + Show 4 more

Open Access

https://doi.org/10.1038/srep40232

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Abstract

Invasive fungal infections are difficult to treat. The few available antifungal drugs have problems with toxicity or efficacy, and resistance is increasing. To overcome these challenges, existing therapies may be enhanced by synergistic combination with another agent. Previously, we found amphotericin B (AMB) and the iron chelator, lactoferrin (LF), were synergistic against a range of different fungal pathogens. This study investigates the mechanism of AMB-LF synergy, using RNA-seq and network analyses. AMB treatment resulted in increased expression of genes involved in iron homeostasis and ATP synthesis. Unexpectedly, AMB-LF treatment did not lead to increased expression of iron and zinc homeostasis genes. However, genes involved in adaptive response to zinc deficiency and oxidative stress had decreased expression. The clustering of co-expressed genes and network analysis revealed that many iron and zinc homeostasis genes are targets of transcription factors Aft1p and Zap1p. The aft1Δ and zap1Δ mutants were hypersensitive to AMB and H2O2, suggesting they are key regulators of the drug response. Mechanistically, AMB-LF synergy could involve AMB affecting the integrity of the cell wall and membrane, permitting LF to disrupt intracellular processes. We suggest that Zap1p- and Aft1p-binding molecules could be combined with existing antifungals to serve as synergistic treatments.

Highlights

Fungal infections, including invasive fungal disease, are on the rise, are often difficult to treat[1], and there is a sparse drug development pipeline for new antifungals[2]
Wild type S. cerevisiae cultures were treated with amphotericin B (AMB) or AMB-LF according to the Materials and Methods
We investigated the mechanistic basis of AMB-LF drug synergy in S. cerevisiae through RNA-seq and network analysis

Summary

Introduction

Fungal infections, including invasive fungal disease, are on the rise, are often difficult to treat[1], and there is a sparse drug development pipeline for new antifungals[2]. AMB and lactoferrin (LF), a multifunctional iron chelating and antimicrobial protein present in milk and tears[21], interacted synergistically against the model yeast Saccharomyces cerevisiae and the pathogen Cryptococcus This combination has been previously shown to be synergistic against C. albicans[22] and Aspergillus fumigatus[23], suggesting it may have broad spectrum of activity[3]. Saccharomyces cerevisiae has rich annotation resources[24] and the most comprehensively mapped intracellular networks of any eukaryotic organism[18,25] As it shares evolutionarily conserved genes, pathways and networks with pathogenic fungi including Cryptococcus, Candida and Aspergillus where genomic resources are relatively lacking[17], it is a useful model for analyzing cellular responses to antifungal agents. Knockout of AFT1 or ZAP1 caused increased susceptibility to AMB, H2O2 and other stressing agents, suggesting that they or their homologs may be suitable targets for adjunct therapies with AMB

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