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I. Alarcon, S. M. J. Fleiszig; Bacterial Induced Apoptosis Is Involved in the Mechanism by Which Pseudomonas Aeruginosa Exits Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):501.
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© ARVO (1962-2015); The Authors (2016-present)
Cellular exit mechanisms used by most pathogens, including P. aeruginosa, are poorly understood. We previously showed that twitching motility, a pili-mediated form of surface associated movement, is required for bacterial virulence in a murine model of corneal infection, despite their competence for adherence to and invasion of corneal epithelial cells. To study the mechanism involved, twitching motility mutants were examined for their capacity to penetrate through corneal epithelial.
Rabbit corneal epithelial cell layers were grown on 24-well plates, cover slips or semi permeable filters in vitro. Wildtype P. aeruginosa invasive strain PAK was compared to pilU (twitching motility) or pilA (non-piliated, twitching motility) isogenic mutants in three different assays, each involving 2 - 6 h incubation with inocula of 106 CFU bacteria: 1. The number of bacteria crossing (traversing) multilayered corneal epithelium over time was quantified by viable counts of the basolateral chamber. 2. The number of viable intracellular bacteria persisting within cells was determined by extended gentamicin survival assays. 3. Bacterial exit from cells was monitored after gentamicin treatment by plating aliquots from the media bathing cells. The role of apoptosis in exit was determined using Caspase inhibitor I.
Twitching motility mutants were found defective in ability to translocate corneal epithelial cells as indicated by delayed initiation of traversal and also lower numbers observed at all time points ( > 2 log reduction at 6 h), when compared to wildtype PAK (p < 0.005). Twitching mutants were also found to accumulate within cells to numbers exceeding wildtype (p < 0.002) which corresponded with reduced ability to exit cells (p < 0.005). Wildtype, capable of efficient cell exit, was found to induce apoptosis in these assays (~4-fold increase as compared to unifected cells, p < 0.0002). The caspase inhibitor, which significantly reduced bacterial-induced apoptosis (~50% decrease p < 0.005) also inhibited bacterial exit (2-fold, p < 0.001).
Twitching motility enables P. aeruginosa to translocate across corneal epithelial layers and to contribute to epithelial cell exit. Exit from cells by wildtype PAK was reduced by Caspase inhibitor I, showing that apoptosis (previously reported to require twitching motility) was involved in the exit mechanism. The relationship between translocation and exit and their relevance to the demonstrated role of twitching motility in pathogenesis in vivo is to be determined.
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