Transcriptional adaptations of Pseudomonas aeruginosa upon exposure to human tear fluid

Abstract

Purpose : The healthy cornea is usually resistant to infection. While the tear film covering the ocular surface contains a multitude of antimicrobial factors, the common corneal pathogen Pseudomonas aeruginosa can survive and replicate in human tear fluid in vitro. RNA-sequencing studies performed in our laboratory have shown numerous alterations to gene expression in P. aeruginosa when exposed to human tear fluid, and many suggest changes to the bacterial outer membrane. Here, we sought to identify P. aeruginosa genes required for survival in tear fluid by characterizing genes whose lack of function resulted in bacterial cell death in tear fluid.

Methods : A Tn-Sequencing experiment was designed, in which a pool of transposon mutants with mutations in non-essential genes, was sequentially passaged in tear fluid for 96 h at 24 h intervals. 701 genes were identified to be conditionally essential for survival in tear fluid. Those genes were overlayed onto the RNA-sequencing dataset containing 331 differentially-expressed genes to identify only those genes that were important for survival in tear fluid and differentially-expressed (p < 0.05, Log₂ fold-change > 8). Enrichment analysis was performed to furhter identify groups of similarly acting bacterial genes required for survival in tear fluid.

Results : This approach yielded 75 candidate genes with potential involvement in the mechanisms underlying P. aeruginosasurvival in tear fluid. These conditionally-essential genes were associated with cell wall remodeling (arn operon, 14-fold enrichment), glycerol uptake (glp operon, 30-fold enrichment), dicarboxylate transport (dct operon, 31-fold enrichment), 5 two-component systems that regulate bacterial motility (cheY), cell wall biosynthesis and biofilm formation, and include a novel putative two-component sensor histidine kinase.

Conclusions : These data suggest that P. aeruginosaovercomes the antimicrobial activity of tear fluid by altering the expression of genes involved in the remodeling of the bacterial cell wall, overcoming nutritional limitation via uptake of alternative metabolites, and likely other adaptive responses.Targeting these adaptive changes could facilitate development of novel strategies to manage P. aeruginosa infections at the ocular surface and beyond.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.