The Type VI secretion system regulates Pseudomonas aeruginosa internalization in human corneal epithelial cells

Abstract

Purpose : The Type VI secretion system (T6SS) has been shown in gram-negative bacteria to function as a major virulence factor. In P. aeruginosa (PA), three T6SS loci, H1- to H3-T6SS have been identified. Of these, the H2-T6SS has been shown in non-ocular tissues to promote bacterial internalization into epithelial cells. The purpose of this study was to test whether loss of a functional H2-T6SS in PA impacts corneal epithelial cell internalization in vitro compared to the parental wild type strain PAO1.

Methods : The standard invasive test strain PA01 and the wild type mutant that lacks a functional H2-T6SS (PAO1ΔclpV2) were used in this study. Each strain was grown for variable time points corresponding to log, transition, and stationary phases before use. Experiments to confirm the timing of the different growth phases were performed for both strains. At the indicated time points, bacteria were re-suspended to a concentration of 1 x 10⁸ CFU/mL. To test the effect on corneal epithelial cell internalization, telomerase-immortalized human corneal epithelial (hTCEpi) cells were grown overnight in serum-free keratinocyte growth media. hTCEpi cells were infected with 1 x 10⁸ CFU/mL of either PAO1 or PAO1ΔclpV2. Non-infected cells were used as controls. hTCEpi cells were incubated for 30 minutes, washed, and allowed to incubate an additional 30 minutes. External PA was killed by treatment with 400 μg/mL gentamicin. hTCEpi cells were then lysed, serially diluted in PBS and plated in triplicate for colony counts analysis.

Results : PAO1ΔclpV2 had 4.8 fold greater internalization than the wild type strain when cultured for 10 hours prior to infection (late log phase, p<0.0001). Both strains were internalized more at 11 and 12.5 hours compared to 10 hours (transition phase); however, PAO1ΔclpV2 internalization was 2.0 fold greater than PAO1 at 11 hours (p<0.01). There was no significant difference between the two groups at 12.5 hours (stationary phase) or later time points (p=0.58).

Conclusions : During log to transition growth phases, PAO1ΔclpV2 internalized greater than the wild type strain PAO1. This difference was lost upon reaching stationary phase. We posit that this is due to the corresponding upregulation of the H3-T6SS in stationary phase. Further studies to investigate the reciprocal relationship between the H2- and H3-T6SS are underway.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.