RNA interference-dependent epimutations as a mechanism of drug resistance in Mucor circinelloides

Z Chang1, RB Billmyre2, SC Lee3, J Heitman1

Author address: 

1Department of Molecular Genetics and Microbiology, Duke University, Durham, USA 2Stowers Institute for Medical Research, Kansas City, USA 3Department of Biology, University of Texas, San Antonio, USA

Abstract: 

Introduction: The opportunistic fungal infection mucormycosis is notable for high mortality as well as increasing incidence. Treatment is complicated by the fact that Mucor circinelloides, a major cause of mucormycosis, demonstrates high intrinsic resistance to most antifungal agents. However, the mechanisms driving this extensive resistance remain poorly understood.

Purpose: Previous work demonstrated that Mucor is capable of developing transient resistance to the antifungal FK506 through a novel, RNA interference (RNAi) dependent mechanism known as epimutation. Epimutants silence the drug target gene and can be selected by exposure to FK506; the target gene is re-expressed in these strains following passage without selective pressure. This silencing process involves the generation of small RNAs (sRNA) against the target gene via core RNAi pathway proteins. We investigated whether this novel mechanism plays a role in Mucor’s intrinsic resistance to additional antifungal agents.

Methods: We studied the development of resistance to the antifungal agent 5-fluoroorotic acid (5-FOA). 5-FOA resistant isolates were generated from multiple strains of Mucor. sRNA hybridization was then used to identify epimutant strains which induce resistance through selective silencing of either of the target genes, pyrF or pyrG. sRNA expression was further characterized via analysis of sRNA libraries. Transience of epimutant-derived resistance was determined by passages on nonselective media followed by repeat sRNA hybridization and library generation.

Results: We have identified epimutants that exhibit resistance to 5-FOA without mutations in either pyrF or pyrG. sRNA hybridization documented the presence of sRNA against pyrF or pyrG in these epimutants, which is lost after reversion to drug sensitivity. Analysis of sRNA libraries generated from these epimutants demonstrated expression of sRNA against the pyrF and pyrG loci, respectively, without evidence of increased expression in other loci.

Conclusion: From this work, we conclude that epimutation serves as a general mechanism capable of targeting multiple genes, and thus enables Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis, as well as of fungal pathogenesis and drug resistance more generally.

2018

Full conference title: 

The 8th Advances Against Aspergillus, Lisbon Conference Center, Lisbon, Portugal
    • AAA 8th (2018)