Internalization of<i>Pseudomonas aeruginosa</i>Is Mediated by Lipid Rafts in Contact Lens–Wearing Rabbit and Cultured Human Corneal Epithelial Cells

Yamamoto, Naoka; Yamamoto, Norio; Petroll, Matthew; Cavanagh, Harrison D; Jester, James V.

doi:10.1167/iovs.04-0542

Cited by 62 publications

(57 citation statements)

References 39 publications

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“…This finding was not anticipated, in view of previous reports that had suggested that P. aeruginosa is internalized at advanced stages of pulmonary infection, possibly via a receptor located in the basolateral membrane (16). While this putative receptor remains to be identified, a possible role for CFTR has been suggested (22,26,43), even though this chloride channel is normally located within the apical membranes of epithelial cells (32,55). In this situation, access to the basolateral membrane is not needed for P. aeruginosa to interact with CFTR, in contrast to the finding of such an early access documented in this and previous studies (19).…”

Section: Discussionmentioning

confidence: 81%

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia byPseudomonas aeruginosa

Zulianello¹,

Canard²,

et al. 2006

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The opportunistic bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis and immunocompromised individuals. Bacterial adherence to the basolateral domain of the host cells and internalization are thought to participate in P. aeruginosa pathogenicity. However, the mechanism by which the pathogen initially modulates the paracellular permeability of polarized respiratory epithelia remains to be understood. To investigate this mechanism, we have searched for virulence factors secreted by P. aeruginosa that affect the structure of human airway epithelium in the early stages of infection. We have found that only bacterial strains secreting rhamnolipids were efficient in modulating the barrier function of an in vitroreconstituted human respiratory epithelium, irrespective of their release of elastase and lipopolysaccharide. In contrast to previous reports, we document that P. aeruginosa was not internalized by epithelial cells. We further report that purified rhamnolipids, applied on the surfaces of the epithelia, were sufficient to functionally disrupt the epithelia and to promote the paracellular invasion of rhamnolipid-deficient P. aeruginosa. The mechanism involves the incorporation of rhamnolipids within the host cell membrane, leading to tight-junction alterations. The study provides direct evidence for a hitherto unknown mechanism whereby the junctiondependent barrier of the respiratory epithelium is selectively altered by rhamnolipids.The airway mucosa is an efficient barrier to protect the host from infection by pathogens. An essential component of this barrier is the polarity of surface epithelial cells, which allows them to segregate the receptors that trigger the adherence and internalization of several pathogens within the basolateral domain of the membrane (51). Normally, this domain is not accessible to microbial organisms, due to the presence of tight junctions (TJ) that separate the apical from the basolateral membrane components and seal the paracellular space (37). To overcome this protective barrier, several microorganisms have developed strategies to alter either the apical membrane of epithelial cells (39) or the TJ barrier (53). Understanding the mechanism underlying these alterations is a prerequisite for development of novel therapeutic strategies targeted to specific molecular and cellular events. This is particularly necessary in the context of infections, such as those caused by Pseudomonas aeruginosa, which is difficult to eradicate by conventional antibiotic treatments (45).P. aeruginosa is an opportunistic gram negative bacterium that does not invade normal mucosae but causes serious nosocomial infections in immunocompromised individuals and in cystic fibrosis patients (7). The pathogenic mechanism accounting for these infections is not fully clarified and has been variously attributed to the production of different cell-associated and secreted virulence factors (41). The finding that P. aeruginosa is internalized more readily by dispersed and migrating ...

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Section: Discussionmentioning

confidence: 81%

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia byPseudomonas aeruginosa

Zulianello¹,

Canard²,

et al. 2006

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“…The images in panel C show representative cells from three separate experiments that were performed. Bars ϭ 10 m. (21,49). Internalization of LFA-1 could occur either independent of or in association with lipid rafts (14).…”

Section: Discussionmentioning

confidence: 99%

Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and Clathrin-Dependent Manner

Atapattu

Czuprynski

2007

Infect Immun

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Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex. Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the ␤ 2 -integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demonstrated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3) death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly, expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation, reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internalization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the cytoskeletal network.Mannheimia haemolytica is the principal bacterial agent responsible for the bovine respiratory disease complex (50). M. haemolytica produces a leukotoxin (LKT) that is considered its most important virulence factor. The LKT belongs to the RTX (repeats in toxin) family of toxins that contain glycine-rich repeats in the primary structure and are produced by a variety of gram-negative bacteria (47). LKT binds to the ␤ 2 -integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in apoptosis or cytolysis of bovine cells (2,25,32). This response is contrary to the usual outcome of cell signaling via CD11a/ CD18, which generally leads to cell activation and survival rather than cell death (11,24).It has been shown that upon stimulation, LFA-1 receptors in human neutrophils associate in detergent-resistant membrane microdomains called lipid rafts (14). Lipid rafts are cholesterol-and sphingolipid-enriched membrane structures that are approximately 50 to 100 nm in diameter (39). Coalescence of lipid rafts can result in membrane domains up to 400 nm in diameter that involve up to 30 to 50% of the cell surface, depending on the cell type (22, 42). Lipid rafts have been implicated in several important cellular functions, such as internalization of receptors and their ligands, signal transduction, and cholesterol transport. The relationship between membrane receptors and lipid rafts is highly variable. For example, epidermal growth factor and ␤ 2 adrenergic receptors move away from lipid rafts following receptor activation. In contrast, insulin and angiotensin II receptors move into lipid rafts f...

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“…It has also been shown that invasion requires bacterial interaction with host cell targets such as lipid rafts (39,66,67), which are themselves associated with specific targets for P. aeruginosa invasion, for example, the cystic fibrosis transmembrane conductance regulator (38,49) and asialo-GM1 (10). These targets interact with bacterial invasion ligands, including lipopolysaccharide (LPS), flagellin, and pili (10,22,69).…”

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confidence: 99%

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

et al. 2008

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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.

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Internalization ofPseudomonas aeruginosaIs Mediated by Lipid Rafts in Contact Lens–Wearing Rabbit and Cultured Human Corneal Epithelial Cells

Abstract: These findings demonstrate that contact-lens-mediated PA internalization involves lipid raft formation. Also, hTCEpi cells may be used as an experimental model for studying further the molecular mechanism(s) of PA infection in the corneal epithelium.

Cited by 62 publications

References 39 publications

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia byPseudomonas aeruginosa

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia byPseudomonas aeruginosa

Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and Clathrin-Dependent Manner

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

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