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 effectordependent bacterial survival and replication within intracellular perinuclear vacuoles.

Studies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., ␤-defensins, play a role in clearing bacteria from mouse corneas already infected with Pseudomonas aeruginosa. Less is known about the role of AMPs in allowing the cornea to resist infection when healthy. We previously reported that contact lens exposure, a major cause of P. aeruginosa keratitis, can inhibit the upregulation of human ␤-defensin 2 (hBD-2) by corneal epithelial cells in response to P. aeruginosa antigens in vitro. Here, we studied the role of AMPs in maintaining the corneal epithelial barrier to P. aeruginosa penetration using both in vitro (human) and in vivo (mouse) experiments. Results showed that preexposing human corneal epithelial multilayers to bacterial antigens in a culture supernatant (known to upregulate AMP expression) reduced epithelial susceptibility to P. aeruginosa traversal up to 6-fold (P < 0.001). Accordingly, small interfering RNA (siRNA) knockdown of any one of four AMPs expressed by human epithelia promoted P. aeruginosa traversal by more than 3-fold (P < 0.001). The combination knockdown of AMPs further enhanced susceptibility to bacterial traversal by ϳ8-fold (P < 0.001). In vivo experiments showed that the loss of murine ␤-defensin 3 (mBD-3), a murine ortholog of hBD-2, enhanced corneal susceptibility to P. aeruginosa. The uninjured ocular surface of mBD-3 ؊/؊ mice showed a reduced capacity to clear P. aeruginosa, and their corneal epithelia were more susceptible to bacterial colonization, even when inoculated ex vivo to exclude tear fluid effects. Together, these in vitro and in vivo data show functional roles for AMPs in normal corneal epithelial cell barrier function against P. aeruginosa.

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the