P. aeruginosa uses twitching motility to disperse within human corneal epithelial cells and multiple phospholipases for subsequent exit

Abstract

Purpose : Pseudomonas aeruginosa invasion into corneal epithelial cells contributes to the pathogenesis of microbial keratitis. After cell entry, P. aeruginosa replicates, spreads intracellularly, then exits the cell. Previously, we showed that twitching motility, a form of movement dependent on bacterial appendages called pili, is required for P. aeruginosa corneal virulence and its capacity to exit cells. Here, we explored mechanisms by which twitching contributes to cellular escape.

Methods : Human corneal epithelial cells (hTCEPi) were infected with P. aeruginosa strain PAO1 or mutants in pilin (pilA), twitching (pilT), and other mutants, including some lacking secretion systems and phosphoplipases. After 3 h to allow internalization, extracellular bacteria were killed using aminoglycoside antibiotics. Infected cells were imaged for 6 h by fluorescence microscopy, and velocity of intracellular bacteria quantified by ImageJ. Transmission electron microscopy (TEM) was used to study localization of intracellular bacteria in more detail. The impact of nocodazole (100 ng/mL) or latrunculin A (0.5 µM) to depolymerise microtubules and actin respectively was also explored. Bacterial exit was quantified hourly for 6 h by viable counts, and gene expression by qPCR.

Results : PAO1 dispersed throughout the cytosol of human corneal epithelial cells with a mean (+/- SD) velocity of 0.04 (± 0.03) µm/s. Intracellular pilA and pilT mutants were stationary and formed intracellular aggregates. TEM confirmed that the mutants were not inside vacuoles, but surrounded by conjoined electron-lucent halos that also encompassed individual bacteria. Inhibitors of actin or microtubules did not impact PAO1 intracellular motility. Mutants in the phospholipase plcB and PA2155 (a putative phospholipase) were defective in exit (~34 % and 25% of PAO1 respectively after 6 h, p < 0.05, ANOVA), but both retained capacity to disperse in the cytosol. Conversely, exit defective twitching pilA and pilT mutants expressed plcB and PA2155 similarly to PAO1.

Conclusions : Internalized P. aeruginosa use twitching motility to move throughout the cytosol of human corneal epithelial cells without requiring actin or microtubules, a novel strategy for intracellular motility by pathogens. Cellular exit following twitching-dependent dispersal within the cell involves phospholipases via mechanisms yet to be determined.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.