Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Here, we ectopically inserted exogenous PtdIns(3,4,5)P3 into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface.
We previously found that water transport across hepatocyte plasma membranes occurs mainly via a nonchannel mediated pathway. Recently, it has been reported that mRNA for the water channel, aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected an N-glycosylated band of ϳ34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt 2 cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt 2 cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt 2 cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.Bile is formed primarily by hepatocytes and subsequently delivered to the bile ducts where it is modified by cholangiocytes (i.e. the epithelial cells that line the bile ducts). Bile secretion by hepatocytes involves the active transport of solutes followed by the passive movement of water into the bile canaliculus in response to osmotic gradients created by these solutes (1, 2). Although a substantial amount of data have been published about the molecular identification of solute transporters and the mechanisms regulating solute transport by hepatocytes (3), little attention has been focused on the mechanistic and regulatory aspects involved in hepatocyte water transport.Water can cross cellular plasma membranes through the lipid portion of the bilayer by a diffusion mechanism or through aquaporin water channels. Aquaporins, a family of recently identified integral membrane proteins, increase cell membrane water permeability facilitating rapid movement of water in response to osmotic gradients (4, 5).We previously found based on biophysical and molecular biology studies that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway (6). As this observation seems to be in contradiction with the recent identification of transcript for the water channel aquaporin-8 (AQP8) 1 in hepatocytes (7-9), we further explored this issue by studying the protein expression, subcellular localization, and possible regulation of AQP8 water channels in isolated rat hepatocytes. MATERIALS AND METHODSIsolation and Incubation of ...
Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P 3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells. INTRODUCTIONPseudomonas aeruginosa is one of the leading causes of nosocomial infections in humans (reviewed in Engel, 2003). This Gram negative opportunistic pathogen causes acute infections of the respiratory and urinary tract, skin, and eye in the setting of preexisting epithelial tissue damage and/or host immunocompromise. P. aeruginosa is also a cause of chronic lung infections and ultimately death in patients with cystic fibrosis.Although usually considered an extracellular pathogen, ϳ50% of clinical, laboratory, and environmental P. aeruginosa isolates demonstrate measurable internalization in vivo as well as in vitro (Chi et al., 1991;Fleiszig et al., 1994Fleiszig et al., , 1995Fleiszig et al., , 1997bFleiszig et al., , 1998Hirakata et al., 1998;Grassmé et al., 2000). These two different phenotypes correlate with the differences in type III secreted effectors (reviewed in Engel, 2003). Both classes of strains are virulent in animal models of P. aeruginosa infection. The noninvasive, cytotoxic strains secrete ExoU (Hauser et al., 1998), a potent phospholipase (Sato et al., 2003), and ExoT, a bifunctional enzyme with N-terminal GAP activity toward Rho family GTPases (Krall et al., 2000;Kazmierczak and Engel, 2002) and C-terminal ADP ribosyltransferase (ADPRT) activity toward Crk (Sun and Barbieri, 2003). Both domains of ExoT contribute to its anti-internalization activity (Garrity-Ryan et al., 2000;Garrity-Ryan et al., 2004). The invasive strains are much less cytotoxic due to the loss of the ExoU gene (Allewelt et al., 2000). Interestingly, these strains secrete ExoT and a closely related protein ExoS that also possesses an N-terminal GAP domain whose substrates include Rho family GTPases (Goehring et al., 1999...
Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577–2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963–970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell–cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier.
Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.
Summary The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to the organisms’ exterior. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells leads to the striking formation of an actin-rich membrane protrusion with ‘inverted’ polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a Type III secretion system apparatus. Host protrusions form de novo underneath bacterial aggregates and involve the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NFκB response. Our data reveal an unanticipated role for spatio-temporal epithelial polarity changes in the activation of innate immune responses.
Summary Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Transition from planktonic to host cell-associated multicellular structures is an essential infection step that has not been described for the opportunistic human pathogen Pseudomonas aeruginosa. In this study we show that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell–cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)- enriched host cell membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates both PIP3- enriched protrusion formation and aggregate internalization. Our results establish the first framework of principles that define P. aeruginosa transition to multicellular structures during interaction with host cells.
Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.