Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Abstract

Cell culture systems have greatly expanded our understanding of how bacterial pathogens target signaling pathways to manipulate the host and cause infection. Advances in genetic engineering have allowed for the creation of fluorescent protein readouts within signaling path-ways, but these techniques have been underutilized in pathogen biology. Here, we genetically engineered a lung cell line with fluorescent reporters for extracellular signal-related kinase (ERK) and the downstream transcription factor FOS-related antigen 1 (Fra1) and evaluated signaling after inoculation with pathogenic and non-pathogenic bacteria. Cells were inoculated with 100 colony-forming units of Acinetobacter baylyi, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus aga-lactiae, or Staphylococcus epidermidis and imaged in a multi-mode reader. The alamarBlue cell viability assay was used as a reference test and showed that pathogenic P. aeruginosa induced significant (p < 0.05) cell death after 8 h in both wild-type and engineered cell lines compared to non-pathogenic S. epidermidis. In engineered cells, we found that Fra1 signaling was disrupted in as little as 4 h after inoculation with bacterial pathogens compared to delayed disruption in signaling by non-patho-genic S. epidermidis. Overall, we demonstrate that low levels of pathogenic versus non-pathogenic bacteria can be rapidly and sensitively screened based on ERK-Fra1 signaling.