Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: cornea: epithelium • bacterial disease • pseudomonas