Pseudomonas aeruginosa gene regulation by Staphylococcus aureus supernatant or added secreted products

Pseudomonas aeruginosa and Staphylococcus aureus are often co-isolated in persistent infections. The goal of this study was to determine how secreted products that were identified in S. aureus supernatant affect gene expression in P. aeruginosa. Therefore, media control, the indicated products in media, or S. aureus supernatant was added to P. aeruginosa cultures at 25% total volume and gene expression was measured at 20 min and 2 h using RNA-seq. The individual products induced distinct pathways in P. aeruginosa. The products in combination recapitulated much of the differential gene expression seen in P. aeruginosa in response to S. aureus supernatant. We prepared RNA-seq samples from P. aeruginosa cultures at the time of treatment, and 20 minutes and 2 hours after the addition of 25% (v/v) media control, S. aureus supernatant, or the following concentrations of additives either together or separately in media: 80 µM staphylopine, mix of 1000 µM diethylene triamine pentaacetic acid (DTPA) with 500 µM deferoxamine, 150 µM citrate, or 150 µM acetoin. RNAprotect (Qiagen) was added to each sample before purification using the Total RNA Purification Plus Kit (Norgen). Genomic DNA was removed using Turbo DNase (Invitrogen) and rRNA was removed using riboPOOLs (siTOOLs Biotech). The cDNA libraries were generated using NEBNext Ultra II Directional Library Kit and amplified with NEBNext index primers (New England Biolabs). Sequencing was performed at the National Cancer Institute Center for Cancer Research Genomics Core.