Fungicide-tolerant persister formation during cryptococcal pulmonary infection
Bacterial persisters, a subpopulation of genetically susceptible cells that are normally dormant and tolerant to bactericides, have been studied extensively because of their clinical importance. In comparison, much less is known about the determinants underlying fungicide-tolerant fungal persister formation in vivo. Here, we report that during mouse lung infection, Cryptococcus neoformans forms persisters that are highly tolerant to amphotericin B (AmB), the standard of care for treating cryptococcosis. By exploring stationary-phase indicator molecules and developing single-cell tracking strategies, we show that in the lung, AmB persisters are enriched in cryptococcal cells that abundantly produce stationary-phase molecules. The antioxidant ergothioneine plays a specific and key role in AmB persistence, which is conserved in phylogenetically distant fungi. Furthermore, the antidepressant sertraline shows potent activity specifically against cryptococcal AmB persisters. Our results provide evidence for and the determinant of AmB-tolerant persister formation in pulmonary cryptococcosis, which has potential clinical significance. Overall design: Cryptococcus cells were cultured for 12 h at 30°C with shaking at 220 rpm in YPD liquid medium and then transferred to fresh YPD liquid medium and incubated for 6, 12, 18, 24, 36, 48, 72, 96 and 120 h. Cells were harvested for RNA extraction at each time point. The concentration and integrity of RNA in each sample were assessed by a Qubit RNA Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. RNA purity was determined using a Nano Photometer spectrophotometer (IMPLEN, CA, USA). The transcriptome libraries were constructed using the VAHTS mRNA-seq v2 Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) according to the manufacturer's instructions. RNA-seq was performed by Annoroad Gene Technology Co., Ltd (Beijing, China). The samples were clustered using VAHRS RNA adapters set1/set2 and sequenced on an Illumina platform.