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Abby Kroken, Vincent Nieto, Amber Jolly, David J Evans, Suzanne M J Fleiszig; Pseudomonas aeruginosa T3SS effectors delay epithelial cell death to favor intracellular survival. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6411.
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© ARVO (1962-2015); The Authors (2016-present)
Pseudomonas aeruginosa uses a type three secretion system (T3SS) and T3SS effectors to promote bacterial virulence in the cornea. Since T3SS effectors are generally thought cytotoxic, and T3SS needle proteins can trigger programmed cell death when in host cell cytoplasm, it is unexpected that the T3SS is required for P. aeruginosa to thrive in the cytosol of corneal epithelial cells. We studied the relationship between intracellular persistence and cell death to reconcile how the T3SS and its effectors influence P. aeruginosa-corneal epithelial cell interactions.
Telomerase immortalized corneal epithelial (hTCEpi) or HeLa cells were infected with bacteria expressing T3SS-driven GFP. Invasive strain PAO1 was compared to mutants lacking T3SS effectors or all T3SS components. Extracellular bacteria were eliminated using amikacin at 3 h, and intracellular bacteria studied from 4 to 20 h. Cell death was detected by nuclear stains and transcriptional responses examined using Qiagen pathway arrays.
Mutants lacking effectors failed to replicate in hTCEpi cells (CFU, 4 v 8 h, P=0.48), but thrived in HeLa cells dividing rapidly in the cytosol (P<0.0001). The limiting factor for corneal cells is more rapid death. Both cell types survived longer when infected with wild type expressing effectors (HeLa P<0.05 from 6-20 h, with invaded cells surviving 13.4 v 10.5 h; hTCEpi P<0.05 from 5-8 h, with invaded cells surviving 12.2 v 5.3 h), correlating with above noted differences in bacterial intracellular persistence. Surprisingly, mutants lacking the entire T3SS (harmless to HeLa cells), caused corneal cell death, correlating with upregulated BCL2A1 and TNF; HeLa cells required the T3SS for this. Wild type bacteria expressing T3SS effectors suppressed this phenotype in both cell lines.
P. aeruginosa can use T3SS effectors to prolong viability of corneal and HeLa epithelial cells. This protects their intracellular niche, which otherwise deliberately dies when T3SS apparatus proteins are detected in the cytoplasm. Corneal, but not HeLa cells, can also die in response to mutants lacking the T3SS, correlating with upregulation of host factors known to modulate programmed death. These results show that the fate of both intracellular bacteria and the invaded host cell depends on complex countermeasures on both sides of the equation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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