Invasion of human epithelial cells by Pseudomonas aeruginosa involves src-like tyrosine kinases p60Src and p59Fyn

Pseudomonas aeruginosa plays a major role in respiratory tract infections or sepsis in patients with cystic fibrosis or upon suppression of the immune system. Several P. aeruginosa strains have been shown to be internalized by human epithelial cells; however, the molecular mechanisms of the invasion process are poorly characterized. Here, we show that the internalization of P. aeruginosa into human epithelial cells results in and requires activation of the Src-like tyrosine kinases p59Fyn and p60Src and the consequent tyrosine phosphorylation of several eukaryotic proteins. The significance of Src-like tyrosine kinase activation is shown by an almost complete blockade of P. aeruginosa internalization, but not adhesion, upon inhibition of Src-like tyrosine kinases. Likewise, inhibition of P. aeruginosa binding to CFTR, which has been shown to block P. aeruginosa internalization, prevents Src and Fyn activation, supporting a pivotal role of Src-like tyrosine kinases for invasion by P. aeruginosa.     

Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity.

Intact tissues are relatively resistant to Pseudomonas aeruginosa-induced disease, and injury predisposes tissue to infection. Intact epithelia contain polarized cells that have distinct apical and basolateral membranes with unique lipids and proteins. In this study, the role of cell polarity in epithelial cell susceptibility to P. aeruginosa virulence mechanisms was tested. Madin-Darby canine kidney (MDCK) cells, human corneal epithelial cells, and primary cultures of two different types of airway epithelial cells were grown on Transwell filters or in plastic tissue culture wells. P. aeruginosa invasion of cells was quantified by gentamicin survival assays with two isolates that invade epithelial cells (6294 and PAO1). Cytotoxic activity was assessed by trypan blue exclusion assays with two cytotoxic strains (6206 and PA103). Basolateral surfaces of cells were exposed by one of two methods: EGTA pretreatment of epithelial cells or growth of cells in low-calcium medium. Both methods of exposing basolateral membranes increased epithelial cell susceptibility to P. aeruginosa invasion and cytotoxicity. Migrating cells were also found to be more susceptible to P. aeruginosa invasion than confluent monolayers that had established membrane polarity. Monolayers of MDCK cells that had been selected for resistance to killing by concanavalin A were resistant to both cytotoxicity and invasion by P. aeruginosa because they were more efficiently polarized for their susceptibility to P. aeruginosa virulence factors than regular MDCK cells and not because they were defective in glycosylation. These results suggest that there are factors on the basolateral surfaces of epithelial cells that promote interaction with P. aeruginosa or that there are inhibitory factors on the apical cell surface. Thus, cell polarity of intact epithelia is likely to contribute to defense against P. aeruginosa infection.     

Modification of Pseudomonas aeruginosa interactions with corneal epithelial cells by human tear fluid

Both cytotoxic and invasive strains of Pseudomonas aeruginosa can damage corneal epithelial cells in vitro, but neither can infect healthy corneas in vivo. We tested the hypothesis that whole human tear fluid can protect corneal epithelia against P. aeruginosa virulence mechanisms. Cultured corneal epithelial cells were inoculated with 10(6) CFU of one of 10 strains of P. aeruginosa (five cytotoxic, five invasive)/ml with or without reflex tear fluid collected from the conjunctival sacs of human volunteers. Cytotoxicity was assessed by observation of trypan blue staining and measurement of lactate dehydrogenase release; invasion was quantified by using gentamicin survival assays. Tear fluid retarded growth of only 50% of the P. aeruginosa strains (three of five invasive strains, two of five cytotoxic strains) yet protected corneal cells against invasion by or cytotoxicity of 9 of 10 strains. The only strain resistant to the tear cytoprotective effects was susceptible to tear bacteriostatic activity. Dilution of tear fluid threefold significantly reduced cytoprotection, while bacteriostatic activity prevailed with dilutions beyond 100-fold. Sulfacetamide (1 mg/ml) with bacteriostatic activity matching that of tear fluid was less cytoprotective than tear fluid (80% protection with tear fluid, 48% with sulfacetamide). Video microscopy revealed bacterial chain formation in both tear fluid and sulfacetamide, but tear fluid also blocked bacterial swimming motility. After prolonged tear contact, bacteria regained normal growth rates, swimming motility, and cytotoxic activity, suggesting a breakdown of protective tear factors. Boiled tear fluid lost bacteriostatic activity and effects on bacterial motility but retained cytoprotective function. These results suggest that human tear fluid can protect corneal epithelial cells against P. aeruginosa virulence mechanisms in a manner not dependent upon bacteriostatic activity or effects on bacterial motility. Whether overlapping tear film components are involved in these defense functions is to be determined.     

Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa.

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 Invasion and Cytotoxicity Are Independent Events, Both of Which Involve Protein Tyrosine Kinase Activity

Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.     

Adherence of Helicobacter pylori to cultured human gastric epithelial cells.

Experiments were performed to demonstrate that adherence of Helicobacter pylori to gastric epithelial cells causes alterations in the cell cytoskeleton. H. pylori intimately attached to cultured human gastric epithelial cells on small cellular projections, while there was no intimate association of H. pylori with cultured human esophageal epithelial cells. Fluorescein-conjugated phalloidin staining of gastric epithelial cells showed that H. pylori adherence stimulated actin polymerization; this stimulation was not observed with esophageal cells. Also, this organism's selectivity for gastric mucosa was supported by rare binding of bacteria to esophageal epithelial cells and gastric fibroblasts.     

Human tear fluid protects against Pseudomonas aeruginosa keratitis in a murine experimental model

Pseudomonas aeruginosa keratitis is an acute sight-threatening infection. We previously reported that human tear fluid could protect individual human corneal epithelial cells in vitro against invasion by and cytotoxicity due to clinical and laboratory isolates of P. aeruginosa and that the protective mechanism was independent of bacteriostatic activity. In the present study, we examined the effects of human tear fluid in vivo. Tears were collected from healthy human volunteers and were studied in vivo in mice. The effects on the virulence of both invasive and cytotoxic clinical isolates of P. aeruginosa were examined. Tear fluid was found to reduce the severity of disease when corneas were challenged with cytotoxic bacteria immediately after scratch injury, and it completely protected against susceptibility to infection by a cytotoxic strain in a model in which corneas were infected during the healing process 6 h after scratching. Visible protection correlated with the inhibition of bacterial colonization 1, 4, and 48 h postinoculation. Tear fluid also significantly reduced the severity of infections caused by invasive P. aeruginosa in the 6-h-healing model. This result also coincided with significantly reduced bacterial colonization at 48 h. In vitro, human tear fluid significantly reduced the ability of invasive and cytotoxic bacteria to translocate across corneal epithelia and increased transepithelial resistance with or without bacterial inoculation. These data show that human tear fluid can protect against P. aeruginosa corneal infection in vivo and that the mechanism likely involves enhanced epithelial barrier function in addition to protection of individual epithelial cells against bacterial internalization and cytotoxicity.     

Twitching motility contributes to the role of pili in corneal infection caused by Pseudomonas aeruginosa

Twitching motility is a form of surface-associated bacterial movement mediated by type IV pili of Pseudomonas aeruginosa. Others have shown that pilT and pilU mutants, which are piliated but defective in twitching motility, display reduced cytotoxic capacity towards epithelial cells in vitro. Although these mutants efficiently infected lungs in vivo, they were defective in dissemination to the liver. In this study the role of twitching motility in P. aeruginosa epithelial cell invasion and corneal disease pathogenesis was explored. pilU and pilT mutants of P. aeruginosa strain PAK were compared to a nonpiliated pilA mutant and to wild-type bacteria in their ability to associate with and to invade corneal epithelial cells in vitro and to cause disease in a murine model of corneal infection. As expected, the pilA mutant demonstrated reduced association and invasion of corneal epithelial cells (P < 0.05 in both cases). The pilT mutant, but not the pilU mutant, was less invasive than wild-type PAK was (P < 0.05 versus P = 0.43), while both pilU and pilT mutants exhibited association levels similar to those of the wild type (P = 0.31 and 0.52, respectively). In vivo, all mutants were markedly attenuated in virulence and showed reduced ability to colonize the cornea at 4 and 48 h (all P values < 0.02). Thus, twitching motility contributed to the role of pili in corneal disease but was not involved in the role of pili in adherence to or invasion of corneal epithelial cells.     

Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells

Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.     

Cytoskeleton and motor proteins are required for the transcytosis of Neisseria gonorrhoeae through polarized epithelial cells

Neisseria gonorrhoeae interact with polarized T84 epithelial cells by engaging carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) receptors. Adherent bacteria that are taken up by the cells are able to traverse the epithelial layer from the apical to the basal side. Herein, we demonstrate that the actin cytoskeleton of the cells is not required for the initial adherence of the bacteria, however, it is essential for invasion into and traversal through T84 cells. Furthermore, microtubule inhibitors blocked the traversal, but not the adherence and invasion of the bacteria. Inhibition of the motor activity of myosins reduced invasion and traversal, but not bacterial adherence. Immunofluorescence confocal laser scanning microscopy revealed the colocalization of the microtubule-based kinesin and dynein motors, and the actin-based motor myosin with adherent and intracellular gonococci. Transcytosis was reduced by blocking kinesin and myosin with specific antibodies. This underlines the importance of these motor proteins for the transcytosis of epithelial monolayers by N. gonorrhoeae.     

Role of heparan sulphate proteoglycans as potential receptors for non-piliated Pseudomonas aeruginosa adherence to non-polarised airway epithelial cells

Tight junctions seal polarised surface epithelial respiratory cells so as to prevent the passage of bacteria and toxins through the epithelial sheet. Disruption of tight junctions, which may occur during injury and repair processes of airway epithelium, favours potential bacterial interaction with receptors from cell basolateral membranes. Earlier studies reported that non-polarised and untight epithelial respiratory cells are highly susceptible to Pseudomonas aeruginosa adherence and internalisation. As heparan sulphate proteoglycans (HSP) from cell basolateral membranes in epithelial cells without tight junctions may become accessible to bacterial ligands, the present study investigated their role as potential receptors for non-piliate P. aeruginosa ligands. Treatment of cells with heparitinase I and II significantly reduced (51.2% and 51.7%, respectively) P. aeruginosa adherence to epithelial respiratory cells without tight junctions. The internalisation of bacteria was not affected by treatment with heparitinases. Treatment of the bacteria with heparin and heparan sulphate also significantly reduced their adherence to respiratory cells (34.3% and 43.7%, respectively). Treatment of cells with other enzymes (trypsin, lipase and chondroitinase ABC) or treatment of bacteria with chondroitin-4-sulphate did not modify the adherence to respiratory cells significantly. Both affinity chromatography and Western blotting assays showed the interaction of different P. aeruginosa outer-membrane proteins (OMPs) with heparin. Several bacterial strains showed differences in their profile of heparin-binding OMPs, but all exhibited low mol. wt (< 30 kDa) reactive proteins. Reactivity of whole bacterial cells with heparin was also observed by transmission electron microscopy. These results suggest that HSP are potential receptors for P. aeruginosa adherence to non-polarised and untight epithelial respiratory cells.     

Regulation of p38 mitogen-activated protein kinase during NK cell activation

The mitogen-activated protein kinase (MAPK) p38 modulates a variety of cellular functions, including proliferation, differentiation and cell death. However, we report here a novel function for p38, i.e. the regulation of cytotoxic lymphocyte-mediated cytotoxicity. Stimulation of NK cells by either cross-linking of their FcgammaRIII receptors or by binding to NK-sensitive target cells induces the phosphorylation and activation of p38, and also of its upstream regulators MKK3/MKK6. Pharmacologic analyses suggest that Src-family and Syk-family protein tyrosine kinases couple the NK cell surface receptors to p38 activation. The role of p38 in the cytotoxic function of NK cells was tested by treatment of NK cells with the cell-permeable, p38-specific inhibitor SB203580. Interestingly, exposure to the drug reduced both antibody-dependent cellular cytotoxicity and natural cytotoxicity, but maximal inhibitory concentrations resulted in only partial inhibition. Collectively, these results suggest that the p38 MAPK pathway is stimulated during the development of NK cell-mediated cytotoxicity and that efficient killing is influenced by both p38-dependent and p38-independent pathways. More broadly, this study identifies the regulation of cell-mediated killing as a novel role for p38 in cytotoxic lymphocytes.     

Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5alpha had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.     

Subinhibitory bismuth-thiols reduce virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common pathogen in mechanically ventilated patients and produces a wide array of virulence factors. Bismuth-thiols (BTs) are active in vitro against all bacterial lung pathogens, including P. aeruginosa. The objective of these studies was to examine the biochemical and morphologic effects of sublethal BT concentrations on P. aeruginosa and to evaluate virulence in cell culture. Bismuth-dimercaprol, at a fraction of the minimal inhibitory concentration, reduced alginate expression by 67% in P. aeruginosa, whereas subinhibitory bismuth-ethanedithiol (BisEDT) reduced alginate by 92% in P. syringae. BisEDT effects on lipopolysaccharide content and type III secreted cytoxins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subinhibitory BisEDT reduced cell-associated lipopolysaccharide, and inhibited processing of the secreted cytotoxic protein ExoU. BisEDT-induced outer membrane blebbing and aggregation of cytoplasmic material was noted in electron microscopy. Virulence of P. aeruginosa was assessed by adherence to epithelial cells and sensitivity to serum killing. BisEDT inhibited adherence of P. aeruginosa to 16HBE14o- cells by 28% and to a collagen matrix by 53%. BisEDT-treated bacteria were also 100-fold more sensitive to serum bactericidal activity. In summary, low BT concentrations affect P. aeruginosa in a variety of ways, the combination of which may help prevent or resolve respiratory tract infection.     

Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro.

Pseudomonas aeruginosa is usually considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have previously demonstrated that P. aeruginosa is able to invade corneal cells during infectious keratitis in mice. In vitro, P. aeruginosa was found to enter the following cells: human corneal cells removed by irrigation; epithelial cells in the cornea of rats, mice, and rabbits; and primary corneal epithelial cells cultured from rat and rabbit eyes. The level of invasion was related to the level of adherent or associated bacteria. In general, invasion was more efficient with cultured epithelial cells than with cells tested in situ. Invasion did not occur when assays were performed at 4 degrees C. Cytochalasin D but not colchicine inhibited bacterial invasion, suggesting that bacterial entry was an endocytic process dependent on actin microfilaments but not microtubules. Bacteria that invaded cultured corneal epithelial cells were found to multiply within cells. The ability of P. aeruginosa to invade and multiply within corneal epithelial cells may contribute to the virulence of this organism during infectious keratitis, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.     

Identification of a bacteriolysis-associated virulence factor against lung epithelial cells in Pseudomonas aeruginosa PAO-1 cell lysate

The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells.     

Interaction of Pseudomonas aeruginosa with human lung fibroblasts: role of bacterial elastase.

Colonization of cell surfaces by Pseudomonas aeruginosa is mediated by bacterial adherence, which, in turn, is influenced by both host and microbial factors. Previous studies with this organism suggest that elastase contributes to tissue invasion and necrosis. We studied the effects of Pseudomonas elastase (PE) on the adherence of P. aeruginosa to human lung fibroblast monolayers. Treatment of fibroblasts with PE (1 microgram/ml or 0.06 U/ml) increased adherence of 35S-labeled P. aeruginosa to cells, but heat-inactivated PE did not affect bacterial adhesion. Immunocytochemistry of cultured cells showed that PE (0.06 to 0.63 U/ml) decreased fibronectin (Fn) on the cell surface and extracellular matrix of cultured human lung fibroblasts. Data obtained by cytofluorography indicated that elastase also decreased Fn receptors on fibroblasts. Additional evidence for Fn degradation was provided by SDS-PAGE analysis of soluble Fn and proteins from surface iodinated cell monolayers treated with PE. We conclude that the increased bacterial adherence to fibroblasts may be due, in part, to elastase-induced proteolysis of Fn and its receptors on cell surfaces. Degradation of Fn could thus influence the extent and course of Pseudomonas infection in the lungs.     

Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells.

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence.     

Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin.

Infection of epithelial cells by two biovars of Chlamydia trachomatis results in the tyrosine phosphorylation of several host proteins. The most prominent change in host protein tyrosine phosphorylation involves a complex of proteins with molecular masses of 75 to 85 kDa (pp75/85) and 100 kDa (pp100). The C. trachomatis-induced tyrosine phosphorylation of pp75/85 and pp100 is observed in several cell lines, including epithelial cells, fibroblasts, and macrophages. Subcellular fractionation and detergent solubility properties of pp75/85 are consistent with its association with the cytoskeleton. Phosphoamino acid analysis demonstrates that the pp75/85 complex is phosphorylated on both tyrosine and serine residues. Immunofluorescence studies of chlamydia-infected cells by using fluorescein isothiocyanate-phalloidin and antibodies to phosphotyrosine and cortactin demonstrate that tyrosine-phosphorylated proteins, as well as cortactin, are localized to the chlamydial vacuole and that this process is facilitated by actin.