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Victoria Hritonenko, Courtney Maloney, David J. Evans, Suzanne M. Fleiszig; Exos Mediates Survival Of P.aeruginosa Within Intracellular Vacuoles. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5867.
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© ARVO (1962-2015); The Authors (2016-present)
ExoS, a Type 3 Secretion System (T3SS) toxin enables P. aeruginosa to survive and replicate within plasma membrane bleb-niches. Mutants that cannot secrete T3SS effectors traffic to perinuclear vacuoles and do not thrive. Usually, T3SS toxins exert their effects after translocation across host cell membranes; indeed translocon (popB) mutants, like T3SS mutants, do not cause bleb-niche formation and traffic to perinuclear vacuoles. However, we have found that popB mutants still replicate intracellularly. Here we tested the hypothesis that ExoS (secreted, but not translocated by popB mutants) can mediate P. aeruginosa intracellular survival in the absence of the translocon.
Chromosomal gene knock-out of the P. aeruginosa T3SS translocon (popB-) was constructed in various T3SS effector mutant backgrounds to obtain the following: PAO1exoS-T-Y-popB- (negative control, triple effector and translocon mutant), PAO1exoT-Y-popB- (expresses ExoS without the translocon,) PAO1exoS-popB- (expresses ExoT and ExoY without the translocon). Intracellular replicative ability was compared to the popB- control expressing all known effectors (human corneal epithelial cells, ~106 CFU/ml bacteria, 3h intracellular survival assay). ANOVA was used for statistical analysis.
In the popB- translocon mutant background, intracellular survival by exoS- and triple effector mutants (neither secrete ExoS) was similar (p=0.65). exoT-Y-popB- mutants (secrete ExoS) showed increased capacity compared to either exoS-T-Y-popB- (p<0.001) or exoS-popB- mutants (neither secrete ExoS) (p<0.001). Similarly, exoT-Y-popB- mutants and popB- mutants (both secrete ExoS) replicated more efficiently intracellularly than exoS-popB- mutants (p<0.001 in each instance).
ExoS can promote intracellular survival of P. aeruginosa in the absence of the T3SS translocon and without plasma membrane bleb-niche formation. These data suggest ExoS secretion promotes bacterial survival locally in the perinculear vacuole (where exoS mutants instead die) without need for translocation across cell membranes. This may be the first documented example of a bacterial T3SS toxin impacting cell function without having to be translocated. It also provides evidence that intracellular P. aeruginosa can impact host cell function. Whether ADP-r activity of ExoS (needed for replication when the translocon is present, and shown to inhibit host cell endosomal maturation) is involved, is to be determined.
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