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Desire Takawira, Olufolarin Oke, David J. Evans, Suzanne M. Fleiszig; Formation of Pseudomonas aeruginosa-induced Bleb-Niches in Corneal Epithelial Cells Involves Myosin II and ERMs. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4200.
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© ARVO (1962-2015); The Authors (2016-present)
Pseudomonas aeruginosa (PA), a leading cause of microbial keratitis, can invade and survive within corneal epithelial cells. Intracellular PA occupies membrane-bound vacuoles and cell membrane protrusions (bleb-niches). Both intracellular survival and bleb-niche formation require ADP-ribosyltransferase (ADPr) activity of ExoS, a type three secreted toxin. Previously, we observed that bleb-niches have disrupted cell membrane-actin cortex interactions. Since myosin II is the major motor responsible for actin organization, and ezrin/radixin/moesin (ERMs) regulate linkage between the cell membrane and actin, we hypothesized that these proteins contribute to bleb-niche formation.
Human corneal epithelial cells (telomerase immortalized) were grown to 70% confluence on glass coverslips, and infected with ~106 cfu/mL of PA or isogenic mutants lacking ExoS ADPr activity. Blebbistatin, a myosin II inhibitor, or ML-7, a myosin light chain kinase inhibitor were added 30 min pre-infection, at infection, or 30 min, 1 h, and 2 h post-infection. Controls verified that drugs were not toxic to cells or bacteria. Protein extracts of infected cells were collected hourly up to 6 h post-infection and analyzed for phosphorylated ERMs or myosin light chain by Western immunoblot. Bacterial survival was determined from cultures of cell extracts.
Phosphorylation of ERMs and myosin light chain were all reduced within 3 h of infection by PA with ExoS ADPr activity, but not with the ADPr mutant bacteria. PA-induced bleb-niche formation was inhibited by either blebbistatin or ML-7, but only if drugs were added within 2 h of infection. However, inhibition of bleb-niche formation did not alter levels of bacterial internalization or survival within the epithelial cells.
Together, these data suggest that both myosin II and ERMs are involved in the initiation of PA-induced bleb-niches, and that targeting of these proteins by ExoS ADPr activity is involved. The data also confirmed our previous findings that PA can survive intracellularly without bleb-niches. Further studies are needed to determine the relationship between ERMs, myosin II, and ExoS ADPr activity, and if bleb-niches represent a novel host defense to expel bacteria and/or a novel strategy for bacterial dissemination.
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