Transcriptome Analysis of Pseudogymnoascus destructans Under Copper Stress Conditions Reveals Adaptive Responses and Putative Virulence Factors

Copper (Cu) is an essential metal micronutrient, and a fungal pathogens' ability to thrive in diverse niches across a broad range of bioavailable copper levels is vital for host-colonization and fungal-propagation. Recent transcriptomic studies have imple-mented that trace metal acquisition is important for the propagation of the white nose syndrome (WNS) causing fungus, Pseudogymnoascus destructans, on bat hosts. This report characterizes the P. destructans transcriptional response to Cu-withholding and Cu-over-load stress. We identify 583 differently expressed genes (DEGs) that respond to Cu-with-holding stress and 667 DEGs that respond to Cu-overload stress. We find that P. de-structans the Cu-transporter genes CTR1a and CTR1b, as well as two homologs to Crypto-coccus neoformans Cbi1/BIM1 VC83_03095 (BLP2) and VC83_07867 (BLP3) are highly reg-ulated by Cu-withholding stress. We identify a cluster of genes, VC83_01834 – VC83_01837, that are regulated by Cu bioavailability, which we identify as the Cu Re-sponsive gene Cluster (CRC). We find that chronic exposure to elevated Cu levels leads to an increase in genes associated with DNA repair and DNA replication fidelity. A com-parison of our transcriptomic data sets with P. destructans at WNS fungal infection sites reveals several putative fungal virulence factors that respond to environmental copper stress. The Pseudogymnoascus destructans (Pd) strain (MYA4855) was purchased from the American tissue culture collection (ATCC strain number 20631-21) and used for all experiments. The long-term propagation of Pd was carried out using solid Yeast extract-peptone and glucose (YPD) plates at 15ºC and consisted of 10 g/L of yeast extract, 20 g/L of peptone, 20 g/L glucose, and 15 g/L of agar. Metal growth studies were performed using chemically defined growth plates (SC-Ura) which were made using Milli-Q water and 1.8 g/L of yeast nitrogen base, 5g/L ammonium sulfate (NH4SO4), 20 g/L glucose, 100 mg/L histidine, 1.7 g/L of the SC-Ura-His amino acid supplement mixture, and 15 g/L agar. Prior to autoclaving 1 pellet (~200 mg) of NaOH was added to the SC-Ura media prior to autoclave sterilization. The addition of sterile Cu-sulfate and Bathocuproine sulfonic acid sodium salt (BCS) was added to the SC-URA growth media using a 125 mM stock solution after autoclave sterilization at a temperature of approximately 50 ºC. All samples were subsequently used for gene expression profiling