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.

We have reported that some strains of Pseudomonas aeruginosa can enter corneal epithelial cells during experimental murine eye infection and when the cells are cultured in vitro. Following invasion, both the host cell and the intracellular bacteria can remain viable for up to 24 h. Others have reported that toxin-mediated damage of epithelial cells contributes to the pathogenesis of P. aeruginosa keratitis. To clarify the relationship between cell invasion and cytotoxicity, fourteen P. aeruginosa isolates were compared for their capacity to enter epithelial cells and for their ability to induce cytotoxicity. Bacterial invasion was quantified by gentamicin survival assays both in vivo and in vitro. Cytotoxicity was examined qualitatively by trypan blue exclusion assays and quantitatively by chromium release assays in vitro. A significant inverse correlation was found between the ability to induce cytotoxicity and epithelial cell invasion as measured by gentamicin survival assays. Both cytotoxic and noncytotoxic strains were identified among corneal and noncorneal isolates; all isolates that were not cytotoxic were capable of epithelial cell invasion. Efficient host cell invasion could not be demonstrated for cytotoxic strains; however, the gentamicin survival assay relies upon host cells retaining viability in order to yield useful results, and this may limit the effectiveness of this assay for testing epithelial cell invasion by cytotoxic strains. Since all of the corneal isolates that were tested were virulent in vivo, the results show that there are at least two different types of P. aeruginosa-induced disease, one caused by strains that are cytotoxic and the other involving bacteria that can enter epithelial cells and survive intracellularly without killing the host cell.

Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion of Salmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in trans restored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, an flhA mutant containing the plasmid vector control, or an fliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC and flhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhA mutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by ϳ80% for the flhA mutant and ϳ50% for the fliC mutant, showing that only part of the role of flhA in invasion involves fliC. Invasion was restored by complementing the flhA mutant with flhA in trans but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhA and fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosa invasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacterial internalization.Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that can cause sight-threatening corneal infection in contact lens wearers and life-threatening infections among neutropenic patients, individuals with severe burns, and those with cystic fibrosis (3, 21). Although P. aeruginosa is traditionally thought to be an extracellular pathogen, evidence is accumulating to implicate corneal epithelial cell invasion in the pathogenesis of corneal disease caused by this bacterium (9, 10). Blocking the cystic fibrosis transmembrane conductance regulator, which has been shown to be a corneal cell factor involved in P. aeruginosa invasion, reduces corneal pa...

Pseudomonas aeruginosa can establish a niche within the plasma membrane of epithelial cells (bleb niches) within which bacteria can survive, replicate, and swim at speeds detectable by real-time phase-contrast imaging. This novel virulence strategy is dependent on the bacterial type three secretion system (T3SS), since mutants lacking the T3SS needle or known T3SS effectors localize to perinuclear vacuoles and fail to replicate. Here, we determined which of the three effectors (ExoS, ExoT, or ExoY) were required for bleb niche formation and intracellular replication. PAO1 strains with mutations in exoS, exoT, exoY, or combinations thereof were compared to wild-type and complemented strains. P. aeruginosa exoS mutants, but not exoT or exoY mutants, lost the capacity for bleb niche formation and intracellular replication. Complementation with exoS rescued both phenotypes, either in the background of an exoS mutant or in a mutant lacking all three known effectors. Complementation with activity domain mutants of exoS revealed that the ADP-ribosyltransferase (ADP-r) activity of ExoS, but not the Rho-GAP activity nor the membrane localization domain (MLD) of ExoS, was required to elicit this phenotype. Membrane bleb niches that contained P. aeruginosa also bound annexin V-enhanced green fluorescent protein (EGFP), a marker of early apoptosis. These data show that P. aeruginosa bleb niches and intracellular survival involve ExoS ADP-r activity and implicate a connection between bleb niche formation and the known role(s) of ExoS-mediated apoptosis and/or Rab GTPase inactivation.

Epithelial cells express antimicrobial proteins in response to invading pathogens, although little is known regarding epithelial defense mechanisms during healthy conditions. Here we report that epithelial cytokeratins have innate defense properties because they constitutively produce cytoprotective antimicrobial peptides. Glycine-rich C-terminal fragments derived from human cytokeratin 6A were identified in bactericidal lysate fractions of human corneal epithelial cells. Structural analysis revealed that these keratin-derived antimicrobial peptides (KDAMPs) exhibited coil structures with low α-helical content. Synthetic analogs of these KDAMPS showed rapid bactericidal activity against multiple pathogens and protected epithelial cells against bacterial virulence mechanisms, while a scrambled peptide showed no bactericidal activity. However, the bactericidal activity of a specific KDAMP was somewhat reduced by glycine-alanine substitutions. KDAMP activity involved bacterial binding and permeabilization, but the activity was unaffected by peptide charge or physiological salt concentration. Knockdown of cytokeratin 6A markedly reduced the bactericidal activity of epithelial cell lysates in vitro and increased the susceptibility of murine corneas to bacterial adherence in vivo. These data suggest that epithelial cytokeratins function as endogenous antimicrobial peptides in the host defense against infection and that keratin-derived antimicrobials may serve as effective therapeutic agents.