Transcriptomics analysis of amphotericin B and lactoferrin drug synergy in Cryptococcus neoformans H99

Invasive fungal infections (IFIs) are difficult to treat. Few effective antifungal drugs are available and many have problems with toxicity, efficacy and drug-resistance. To overcome these challenges, existing therapies may be enhanced using more than one agent acting in synergy. Previously, we have found amphotericin B (AMB) and the iron chelator, lactoferrin (LF), were synergistic against Cryptococcus neoformans and Saccharomyces cerevisiae. This study investigates the mechanism of AMB+LF synergy using RNA-seq in Cryptococcus neoformans H99. Overall design: We extracted mRNA from C. neoformans treated with i) AMB only (0.06 µg/mL), ii) LF only (2 µg/mL), iii) a combination of AMB + LF (0.06 µg/mL and 2 µg/mL, respectively) or iv) corresponding matching untreated controls. ID20 (inhibition of cell growth by 20%) was chosen as the time point for RNA isolation from all treatments, as at this point cells are stressed but there should be few confounding effects of cell death, allowing a direct comparison among treatments. The ID20 for AMB of 60 minutes, and AMB-LF of 50 minutes. The same incubation times were applied to each of the matched control samples. RNA-seq data were generated using Illumina HiSeq 2000 and Illumina NextSeq 500 with biological triplicates multiplexed and randomized across two sequencing lanes. Using EdgeR, we identified differentially expressed genes by comparing AMB treatment with the matched control. We also compared AMB + LF treatment with its matched control.