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V. Hritonenko, A. A. Angus, D. J. Evans, S. M. J. Fleiszig; Role of Type III Secreted Effectors in P. Areuginosa-Induced Membrane Blebbing of Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):849.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that P. aeruginosa induces membrane blebs after invasion of corneal epithelial cells, and that the Type Three Secretion System (T3SS) was required. We subsequently showed that the effector ExoS was required for trafficking to membrane blebs, and intracellular survival. Here we tested the hypothesis that other effectors of P. aeruginosa participate in membrane blebbing.
Two invasive P. aeruginosa strains (PA01, PAK) and two cytotoxic strains (PA14, PA103) were used. Since ExoU is cytotoxic to cells before bacteria invade, non-cytotoxic exoU- mutants of PA14 and PA103 were used. Isogenic mutants in known effectors and T3SS needle mutants were used as controls. Corneal epithelial cells were inoculated with ~107 cfu of P. aeruginosa for 3 h followed by antibiotic treatment to kill extracellular bacteria. Live cell images and videos were recorded using phase-contrast microscopy from 6 to 12 h post-inoculation.
PA01exoS-exoT- (expresses only ExoY) and PAO1exoT-exoY- (expresses only ExoS) still induced membrane blebs, whereas PAO1exoS-exoY- (expresses only ExoT) and PAO1exoS-exoT-exoY- (expresses no known effectors) did not, showing that ExoY could also induce membrane blebbing in the absence of ExoS. Interestingly, none of the PA14 mutants (PA14exoU-, PA14exoU-exoT-exoY-, and needle mutant PA14pscD-) were observed to cause membrane blebbing, but instead localized to intracellular vacuoles. In contrast, blebbing was observed in cells exposed to mutants in all known effectors of both PAKexoS-exoT-exoY- and PA103exoU-exoT-. Blebbing associated with PAKexoS-exoT-exoY- was observed 1-2 h later than wild type. T3SS needle mutants did not cause blebbing for any strain.
These data show that ExoY can also induce membrane blebbing (at least in PA01), and confirmed the known role for ExoS, but not ExoT, in this effect. ExoS and ExoY do not share known enzymatic activities, suggesting that they induce blebbing through different mechanisms. The lack of ExoY-induced blebbing in PA14 requires further study. Results from unsequenced strains PAK (invasive) and PA103 (cytotoxic), suggest that unidentified effectors can also induce blebbing and that they are absent in sequenced strains.
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