Spatial transcriptomics: P. aeruginosa corneal infection

To holistically unravel the complexity of pathogen-host interactions within infected tissues we leverage a dual spatial transcriptomic approach that, for the first time, simultaneously captures the expression of Pseudomonas aeruginosa genes alongside the entire host transcriptome in a model of ocular infection. This innovative method reveals differential pathogen and host-specific gene expression patterns across specific anatomical regions generating a unified transcriptional map of infection. By integrating these data, we developed a predictive ridge regression model trained on images from infected tissues. Overall design: Overnight cultures of P. aeruginosa were grown to an OD600 of 0.20 in LB broth, and cultures were then washed and resuspended in 1x sterile PBS (Corning). C57BL/6J mice were anesthetized with ketamine/xylazine (MWI Animal Health) and corneal epithelium was abraded with three, parallel 10mm scratches, performed with a 26 ½ -gauge needle (BD). Pseudomonas was then applied to the abraded cornea topically with 2µl containing 5x105 CFU in PBS in the PA14 infection studies and with 5ml of 1x106 CFU in the PAO1 infection studies. For use in the infection experiments, 100µl of overnight culture was added to 10ml of fresh LB and was subcultured for 2.5-3h to reach OD600=0.20. 1ml of culture was spun down at 3,000 x g (rotor, centrifuge) for 10 min and diluted to a desired inoculum. Experiments were carried out predominantly with male 7-8 week old C57Bl6/J mice. After 24h or 48h, mice were euthanized, and corneas were imaged by brightfield microscopy to observe opacification. Bacterial CFU was quantified at 24h and 48h by serial dilutions of whole eye homogenates onto LB plates and were counted manually.