Neither fluorescein staining nor bacterial adhesion necessarily predict or enable corneal susceptibility to bacterial penetration or disease. Corneal epithelial defenses limiting traversal by adherent bacteria include EGTA-sensitive factors and SP-D. Understanding mechanisms modulating epithelial traversal by microbes could improve our understanding of susceptibility to infection and may indicate new strategies for preventing disease.

Purpose The authors have shown that twitching motility, a pilus-mediated form of bacterial surface movement, is required for Pseudomonas aeruginosa virulence in a murine model of keratitis. To study the role of twitching motility in virulence, Pseudomonas traversal of multilayered corneal epithelia in vitro was investigated. Methods Translocation of multilayered corneal epithelia was investigated with the invasive strain PAK and isogenic twitching motility mutants. Rabbit corneal epithelial cells were grown to multilayers with filters and inoculated on their apical surfaces with 106 colony-forming unit bacteria, and translocating bacteria were quantified by viable counts of the basal chamber. Transepithelial resistance (TER) was recorded. Cellular exit of P. aeruginosa after invasion was quantified with modified gentamicin survival assays, and the role of apoptosis in exit was explored. Results PAK translocated the epithelia as early as 1 hour after infection, and by 8 hours apical and basal numbers of bacteria were similar. Bacterial translocation did not reduce TER. Each twitching motility mutant (pilU, pilT with pili, pilA lacking pili) was defective in translocation (>2 log reduction vs. PAK; P < 0.005). All twitching mutants were competent for cell invasion but defective in cellular exit, accumulating intracellularly to numbers exceeding those of PAK. Inhibiting apoptosis reduced the cellular exit of PAK. Conclusions These results show that twitching motility enables P. aeruginosa to translocate corneal epithelial layers and suggest that it contributes to epithelial cell exit by a mechanism involving apoptosis. The relationship between these in vitro findings and the role of twitching motility in P. aeruginosa virulence in vivo remains to be determined.

Pseudomonas aeruginosa can invade corneal epithelial cells and translocates multilayered corneal epithelia in vitro, but it does not penetrate the intact corneal epithelium in vivo. In healthy corneas, the epithelium is separated from the underlying stroma by a basement membrane containing extracellular matrix proteins and pores smaller than bacteria. Here we used in vivo and in vitro models to investigate the potential of the basement membrane to defend against P. aeruginosa. Transmission electron microscopy of infected mouse corneas in vivo showed penetration of the stroma by P. aeruginosa only where the basement membrane was visibly disrupted by scratch injury, suggesting that the intact basement membrane prevented penetration. This hypothesis was explored using an in vitro Matrigel Transwell model to mimic the corneal basement membrane. P. aeruginosa translocation of multilayered corneal epithelia grown on Matrigel was ϳ100-fold lower than that of cells grown without Matrigel (P < 0.005, t test). Matrigel did not increase transepithelial resistance. Matrigel-grown cells blocked translocation by a P. aeruginosa protease mutant. Without cells, Matrigel also reduced traversal of P. aeruginosa and the protease mutant. Fluorescence microscopy revealed a relative accumulation of bacteria at the superficial epithelium of cells grown on Matrigel at 3 h compared to cells grown on uncoated filters. By 5 h, bacteria accumulated beneath the cells, suggesting direct trapping by the Matrigel. These findings suggest that the basement membrane helps defend the cornea against infection via physical barrier effects and influences on the epithelium and that these roles could be compromised by P. aeruginosa proteases.Pseudomonas aeruginosa is an important opportunistic pathogen, commonly affecting burn victims, individuals with cystic fibrosis, patients in hospital intensive care units, and contact lens wearers (9-11). In the absence of contact lens wear, the cornea is remarkably resistant to infection, with P. aeruginosa effectively colonizing this tissue only if it is injured or otherwise compromised (41). To initiate clinically significant corneal pathology, P. aeruginosa (and almost all other microbes) must first access the corneal stroma, which is normally protected by a multilayered epithelium and associated basement membrane (1).The corneal epithelial basement membrane is secreted by the overlying epithelia and is comprised of sheets of extracellular matrix constituents, including type IV collagen, heparan sulfate proteoglycan, and various glycoproteins (laminin, entactin, nidogen, and fibronectin) and growth factors that mediate cellular function (1). Quantitative imaging of the corneal epithelial basal membrane in the rhesus macaque has shown a complex cross-linking of fibers and proteins intermingled with pores ranging from 30 to 400 nm in size (2). As shown for other basement membranes in the body (17,19,33), the corneal basement membrane anchors epithelial cells and provides positional information for healing, tissu...

To catalog factors that may contribute to the completion of myogenesis, we have been looking for molecular differences between BC3H1 and C2C12 cells. Cells of the BC3H1 tumor line, though myogenic, are nonfusing, and withdraw from the cell cycle only reversibly, whereas cells of the C2C12 line fuse, differentiate terminally, and express several muscle-specific gene products that BC3H1 cells do not. Relative to C2C12 cells, BC3H1 cells underaccumulated cyclin-dependent kinase inhibitor p21 and underaccumulated transcripts for p21, GADD45, CDO, decorin, osteopontin, H19, fibronectin, and thrombospondin-1 (tsp-1). Levels of accumulation of H19, tsp-1, and larger isoforms of fibronectin messenger ribonucleic acid (mRNA) were found to increase in response to expression of myogenic regulatory factors as shown by their accumulation in differentiated myogenically converted 10T1/2 cells but not in 10T1/2 fibroblasts. BC3H1s accumulated a temperature-insensitive, geldanamycin-sensitive, misfolded form of p53 incapable of transactivating a p53 responsive reporter, consistent with underexpression of p21, GADD45, and tsp-1. BC3H1 and C2C12 cells were similar with respect to upregulation of p27 protein, downregulation of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein, upregulation of retinoblastoma (Rb) mRNA, and nuclear localization of hypophosphorylated Rb. Cells of both lines expressed the muscle-specific 1b isoform of MEF2D. Although nonfusing in the short term, after more than 18 d in differentiation medium, some cultures of BC3H1 cells formed viable multinucleated cells in which the nuclei did not reinitiate synthesis of DNA in response to serum. Our findings suggest participation of tsp-1 and specific isoforms of fibronectin in myogenesis and suggest additional avenues of research in myogenesis and oncogenesis.