Purpose: In excess of 1.5 million people die from fungal diseases each year. More than one third are caused by invasive as well as chronic Aspergillus infections, predominantly by the major human mould pathogen Aspergillus fumigatus. The current therapeutic arsenal for the treatment of invasive fungal infections only comprises four classes of antifungal agents - azoles, polyenes, candins and nucleobase analogs. 5-flucytosine (5FC), the only member of the nucleobase-analog drug class is rarely used as a monotherapeutic agent to treat Aspergillus infections, as resistance develops rapidly. Furthermore, at neutral pH (pH 7.0) A. fumigatus displays intrinsic resistance to 5FC. At low pH (pH 5.0), however, 5FC is highly active against this mould pathogen.
Methods: To elucidate genetic factors and the mechanism causing 5FC resistance in A. fumigatus, we applied a combination of microbiological, genetic and molecular tools. Genetically modified strains were generated that underwent EUCAST and modified plate-assay based drug susceptibility testing (pH 5.0 and pH 7.0, low/rich nutrient medium, blood agar). RNA-sequencing based whole transcriptome analysis of transcription factor mutants, showing increased 5FC susceptibility, was carried out. To study 5FC efficacy during infection with wild-type and selected mutants, we established a Galleria mellonella larvae based 5FC treatment model.
Results: fcyB, encoding the major 5FC uptake protein, is repressed at pH 7.0 and transcriptionally activated at pH 5.0 in A. fumigatus. We identify the CCAAT binding complex (CBC) and the pH regulatory protein PacC as repressors of the gene. Genetic inactivation of the CBC or PacC leads to elevated fcyB transcript levels and results in increased 5FC susceptibility. Upregulating fcyB using a xylose-inducible expression system to mimic CBC and PacC mutation, we can dramatically increase 5FC susceptibility at pH 7.0. A similar outcome was observed by downregulating pacC.
Plate assay based susceptibility testing reveals a 5FC concentration level (>100mg/L) that causes severe growth inhibition of A. fumigatus, independent of the nutrient availability and the pH. Furthermore, we find that at this concentration level A. fumigatus' growth is inhibited even in the absence of FcyB mediated uptake. A similar finding was observed during 5FC treatment modelling. Survival of larvae infected with wild-type or an fcyB deletion mutant was significantly increased at when treated with 100 or 200mg/kg 5FC.
Conclusion: In this study we demonstrate CBC and PacC mediated repression of fcyB to be the major cause of 5FC resistance at neutral pH, the standard pH used for EUCAST and CLSI based susceptibility testing. Hence, bypassing this repression might illustrate a strategy to increase activity of 5FC against A. fumigatus.
Plate assay based susceptibility testing and G. melonella basedtreatment modelling suggest FcyB independent 5FC uptake routes playing a crucial role in 5FC import at high concentration levels.
Full conference title:
- AAA 8th (2018)