Ellen J. Lee

Pseudomonas aeruginosa Induces Membrane Blebs in Epithelial Cells, Which Are Utilized as a Niche for Intracellular Replication and Motility

ABSTRACT Pseudomonas aeruginosa is known to invade epithelial cells during infection and in vitro. However, little is known of bacterial or epithelial factors modulating P. aeruginosa intracellular survival or replication after invasion, except that it requires a complete lipopolysaccharide core. In this study, real-time video microscopy revealed that invasive P. aeruginosa isolates induced the formation of membrane blebs in multiple epithelial cell types and that these were then exploited for intracellular replication and rapid real-time motility. Further studies revealed that the type three secretion system (T3SS) of P. aeruginosa was required for blebbing. Mutants lacking either the entire T3SS or specific T3SS components were instead localized to intracellular perinuclear vacuoles. Most T3SS mutants that trafficked to perinuclear vacuoles gradually lost intracellular viability, and vacuoles containing those bacteria were labeled by the late endosomal marker lysosome-associated marker protein 3 (LAMP-3). Interestingly, mutants deficient only in the T3SS translocon structure survived and replicated within the vacuoles that did not label with LAMP-3. Taken together, these data suggest two novel roles of the P. aeruginosa T3SS in enabling bacterial intracellular survival: translocon-dependent formation of membrane blebs, which form a host cell niche for bacterial growth and motility, and effector-dependent bacterial survival and replication within intracellular perinuclear vacuoles.

A comparison of invasive and cytotoxic Pseudomonas aeruginosa strain-induced corneal disease responses to therapeutics.

Purpose. During corneal infection, cytotoxic Pseudomonas aeruginosa strains remain mostly extracellular, while invasive strains can enter corneal cells and replicate within them. We tested the hypothesis that ofloxacin, which easily penetrates host cell membranes, would be more effective than the less cell-permeable antibiotic tobramycin, for treatment of corneal infection by an invasive P. aeruginosa strain. Methods. A murine model of P. aeruginosa keratitis was used to compare the response to ofloxacin, tobramycin, prednisolone acetate, and non-preserved saline treatment, as well as combination antibiotic-corticosteroid therapy for infection caused by a cytotoxic strain (6206) and an invasive strain (PAO1). Treatment involved hourly eye drop administration for 12 hours. Results. As expected, tobramycin was less effective at eradicating viable bacteria from corneas infected with the invasive strain. Despite rapid sterilization of corneas in other antibiotic treated groups, disease progression occurred during the 12 hour treatment period. Both antibiotics hastened disease resolution over the next 7 days for infections caused by either strain. Corticosteroid use during the 12 hour treatment period was of little added benefit. Conclusions. Differences between invasive and cytotoxic strain infections in their early response to the different therapeutic regimens did not translate to notable differences after 7 days, but the effects of antibiotics in halting disease progression were delayed for both strain types. These results suggest that successful management might be improved by addressing factors contributing to disease progression during sterilization of the cornea by antibiotics.