Reversible adhesion by type IV pili leads to formation of permanent localized clusters.

The formation of long-lived, multicellular clusters is a fundamental step in the physiopathology of many disease-causing bacteria. Experiments on abiotic surfaces suggest that bacterial colonization, including initial cluster formation, requires (1) irreversible adhesion, (2) cell proliferation, and (3) a phenotypic transition. However, here we show that on infection of a polarized MDCK epithelium, Pseudomonas aeruginosa (PA) forms long-lived - i.e., permanent - bacterial clusters without requiring irreversible adhesion, cell proliferation, or a phenotypic transition. By combining experiments and a mathematical model, we reveal that the cluster formation process is mediated by type IV pili (T4P). Furthermore, we unveil how T4P quantitatively operate during adhesion, finding that it is a stochastic process that involves an activation time, requires the retraction of pili, and results in reversible attachment. We explain how such reversible attachment process leads to the formation of permanent bacterial clusters and quantify the cluster growth dynamics.

The inflammatory response induced by Pseudomonas aeruginosa in macrophages enhances apoptotic cell removal.

Pathogens phagocytosis and the uptake of apoptotic cells (efferocytosis) are essential macrophages tasks, classically considered as mutually exclusive. Macrophages have been observed to polarize into either pro-inflammatory/microbicidal or anti-inflammatory/efferocytic phenotypes. However, macrophage functions have shown to be more complex. Furthermore, little is known about the regulation of efferocytosis under inflammatory conditions. In this study, we elucidate the modulation of the macrophage efferocytic function during an inflammatory stimulus. We find that bone marrow-derived macrophages (BMDM) are very efficient in engulfing both the bacterial pathogen Pseudomonas aeruginosa and apoptotic cells. BMDM showed a high bactericidal capacity unaffected by the concomitant presence of apoptotic material. Plasticity in macrophage programming, in response to changing environmental cues, may modulate efferocytic capability. In this work, we further show that, after phagocyting and processing Pseudomonas aeruginosa, macrophages highly increase their efferocytic capacity without affecting their phagocytic function. Moreover, we demonstrate that Pseudomonas aeruginosa enhances efferocytosis of these phagocytes through the IL-6 signaling pathway. Our results show that the inflammatory response generated by the bacterial processing enhances these macrophages' capacity to control inflammation through an increased efferocytosis.

Akap350 Recruits Eb1 to The Spindle Poles, Ensuring Proper Spindle Orientation and Lumen Formation in 3d Epithelial Cell Cultures.

The organization of epithelial cells to form hollow organs with a single lumen requires the accurate three-dimensional arrangement of cell divisions. Mitotic spindle orientation is defined by signaling pathways that provide molecular links between specific spots at the cell cortex and astral microtubules, which have not been fully elucidated. AKAP350 is a centrosomal/Golgi scaffold protein, implicated in the regulation of microtubule dynamics. Using 3D epithelial cell cultures, we found that cells with decreased AKAP350 expression (AKAP350KD) formed polarized cysts with abnormal lumen morphology. Analysis of mitotic cells in AKAP350KD cysts indicated defective spindle alignment. We established that AKAP350 interacts with EB1, a microtubule associated protein that regulates spindle orientation, at the spindle poles. Decrease of AKAP350 expression lead to a significant reduction of EB1 levels at spindle poles and astral microtubules. Conversely, overexpression of EB1 rescued the defective spindle orientation induced by deficient AKAP350 expression. The specific delocalization of the AKAP350/EB1complex from the centrosome decreased EB1 levels at astral microtubules and lead to the formation of 3D-organotypic structures which resembled AKAP350KD cysts. We conclude that AKAP350 recruits EB1 to the spindle poles, ensuring EB1 presence at astral microtubules and proper spindle orientation during epithelial morphogenesis.

The EAL-domain protein FcsR regulates flagella, chemotaxis and type III secretion system in Pseudomonas aeruginosa by a phosphodiesterase independent mechanism.

The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.

Elimination of Pseudomonas aeruginosa through Efferocytosis upon Binding to Apoptotic Cells.

For opportunistic pathogens such as Pseudomonas aeruginosa, the mucosal barrier represents a formidable challenge. Infections develop only in patients with altered epithelial barriers. Here, we showed that P. aeruginosa interacts with a polarized epithelium, adhering almost exclusively at sites of multi-cellular junctions. In these sites, numerous bacteria attach to an extruded apoptotic cell or apoptotic body. This dead cell tropism is independent of the type of cell death, as P. aeruginosa also binds to necrotic cells. We further showed that P. aeruginosa is internalized through efferocytosis, a process in which surrounding epithelial cells engulf and dispose of extruded apoptotic cells. Intracellularly, along with apoptotic cell debris, P. aeruginosa inhabits an efferocytic phagosome that acquires lysosomal features, and is finally killed. We propose that elimination of P. aeruginosa through efferocytosis is part of a host defense mechanism. Our findings could be relevant for the study of cystic fibrosis, which is characterized by an exacerbated number of apoptotic cells and ineffective efferocytosis.

Centrosomal AKAP350 and CIP4 act in concert to define the polarized localization of the centrosome and Golgi in migratory cells.

The acquisition of a migratory phenotype is central in processes as diverse as embryo differentiation and tumor metastasis. An early event in this phenomenon is the generation of a nucleus-centrosome-Golgi back-to-front axis. AKAP350 (also known as AKAP9) is a Golgi and centrosome scaffold protein that is involved in microtubule nucleation. AKAP350 interacts with CIP4 (also known as TRIP10), a cdc42 effector that regulates actin dynamics. The present study aimed to characterize the participation of centrosomal AKAP350 in the acquisition of migratory polarity, and the involvement of CIP4 in the pathway. The decrease in total or in centrosomal AKAP350 led to decreased formation of the nucleus-centrosome-Golgi axis and defective cell migration. CIP4 localized at the centrosome, which was enhanced in migratory cells, but inhibited in cells with decreased centrosomal AKAP350. A decrease in the CIP4 expression or inhibition of the CIP4-AKAP350 interaction also led to defective cell polarization. Centrosome positioning, but not nuclear movement, was affected by loss of CIP4 or AKAP350 function. Our results support a model in which AKAP350 recruits CIP4 to the centrosome, providing a centrosomal scaffold to integrate microtubule and actin dynamics, thus enabling centrosome polarization and ensuring cell migration directionality.

The Pseudomonas aeruginosa type III translocon is required for biofilm formation at the epithelial barrier.

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.

Host cell polarity proteins participate in innate immunity to Pseudomonas aeruginosa infection.

The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. 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 led 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 formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses.

An image analysis method to quantify CFTR subcellular localization.

Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering "apical CFTR ratio". We obtained an apical CFTR ratio of 0.67 ± 0.05 for wtCFTR and 0.11 ± 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 °C rendered an apical CFTR ratio value of 0.23 ± 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 ± 0.03 for wild type and 0.05 ± 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify mutations that completely or mildly affect CFTR processing. Moreover, it could be extended to other studies on the biology underlying protein subcellular localization in health and disease.

A confocal microscopy image analysis method to measure adhesion and internalization of Pseudomonas aeruginosa multicellular structures into epithelial cells.

Formation of multicellular structures such as biofilms is an important feature in the physiopathology of many disease-causing bacteria. We recently reported that Pseudomonas aeruginosa adheres to epithelial cells rapidly forming early biofilm-like aggregates, which can then be internalized into cells. Conventional methods to measure adhesion/internalization, such as dilution plating for total cell-associated or antibiotic protected bacteria, do not distinguish between single and aggregated bacteria. We report a procedure that combining double bacteria labeling, confocal microscopy and image analysis allows identification and quantification of the number of adhered and internalized bacteria distinguishing between single and aggregated bacterial cells. A plugin for Fiji to automatically perform these procedures has been generated.

Pseudomonas aeruginosa interacts with epithelial cells rapidly forming aggregates that are internalized by a Lyn-dependent mechanism.

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.

Species-specific interactions of Src family tyrosine kinases regulate Chlamydia intracellular growth and trafficking.

Src family kinases (SFKs) regulate key cellular processes and are emerging as important targets for intracellular pathogens. In this commentary, we briefly review the role of SFKs in bacterial pathogenesis and highlight new work on the role of SFKs during the intracellular cycle of Chlamydia species.

Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane.

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.

Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells.

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.

Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytes.

Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes (e.g., 102% with 1 micromol/L glucagon) and a simultaneous decrease in intracellular membranes (e.g., 38% with 1 micromol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion.

Water permeability properties of the human small intestine in vitro: effects of Escherichia coli heat-stable enterotoxin

The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive). 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (deltaOsm) in both intestinal segments. The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 ñ 0.049 cm/s vs. 0.156 ñ 0.022 cm/s and 0.0012 ñ 0.0001 cm/s vs. 0.0019 ñ O.0003, respectively. 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt). 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum. 5) 2 mug/ml of E. coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc. 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum. In the ileum, STa action caused a 48 per cent decrease in the absorptive Jwt values.

Water permeability properties of the human small intestine in vitro: effects of Escherichia coli heat-stable enterotoxin

The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive). 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (deltaOsm) in both intestinal segments. The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 ñ 0.049 cm/s vs. 0.156 ñ 0.022 cm/s and 0.0012 ñ 0.0001 cm/s vs. 0.0019 ñ O.0003, respectively. 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt). 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum. 5) 2 mug/ml of E. coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc. 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum. In the ileum, STa action caused a 48 per cent decrease in the absorptive Jwt values. (AU)