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1.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33430070

RESUMEN

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.

2.
Front Microbiol ; 11: 1069, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32523583

RESUMEN

Mitochondria play crucial roles in cellular metabolism, signaling, longevity, and immune defense. Recent evidences have revealed that the host microbiota, including bacterial pathogens, impact mitochondrial behaviors and activities in the host. The pathogenicity of Pseudomonas aeruginosa requires quorum sensing (QS) cell-cell communication allowing the bacteria to sense population density and collectively control biofilm development, virulence traits, adaptation and interactions with the host. QS molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL), can also modulate the behavior of host cells, e.g., epithelial barrier properties and innate immune responses. Here, in two types of cells, fibroblasts and intestinal epithelial cells, we investigated whether and how P. aeruginosa 3O-C12-HSL impacts the morphology of mitochondrial networks and their energetic characteristics, using high-resolution transmission electron microscopy, fluorescence live-cell imaging, assay for mitochondrial bioenergetics, and quantitative mass spectrometry for mitoproteomics and bioinformatics. We found that 3O-C12-HSL induced fragmentation of mitochondria, disruption of cristae and inner membrane ultrastructure, altered major characteristics of respiration and energetics, and decreased mitochondrial membrane potential, and that there are distinct cell-type specific details of these effects. Moreover, this was mechanistically accompanied by differential expression of both common and cell-type specific arrays of components in the mitochondrial proteome involved in their structural organization, electron transport chain complexes and response to stress. We suggest that this effect of 3O-C12-HSL on mitochondria may represent one of the events in the interaction between P. aeruginosa and host mitochondria and may have an impact on the pathogens strategy to hijack host cell activities to support their own survival and spreading.

3.
J Innate Immun ; 11(3): 263-279, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30428481

RESUMEN

Cell-to-cell signaling via small molecules is an essential process to coordinate behavior in single species within a community, and also across kingdoms. In this review, we discuss the quorum sensing (QS) systems used by the opportunistic pathogen Pseudomonas aeruginosa to sense bacterial population density and fitness, and regulate virulence, biofilm development, metabolite acquisition, and mammalian host defense. We also focus on the role of N-acylhomoserine lactone-dependent QS signaling in the modulation of innate immune responses connected together via calcium signaling, homeostasis, mitochondrial and cytoskeletal dynamics, and governing transcriptional and proteomic responses of host cells. A future perspective emphasizes the need for multidisciplinary efforts to bring current knowledge of QS into a more detailed understanding of the communication between bacteria and host, as well as into strategies to prevent and treat P. aeruginosa infections and reduce the rate of antibiotic resistance.


Asunto(s)
Interacciones Microbiota-Huesped , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/fisiología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/fisiología , Adhesión Bacteriana , Biopelículas , Señalización del Calcio , Movimiento Celular , Homoserina/análogos & derivados , Homoserina/fisiología , Humanos , Hierro/metabolismo , Lipopolisacáridos/fisiología , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/patogenicidad
4.
Methods Mol Biol ; 1673: 213-225, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29130176

RESUMEN

The human mucosal environment in the gut is rich with interactions between microbiota and mammalian epithelia. Microbes such as the Gram-negative bacterium Pseudomonas aeruginosa may use quorum sensing to communicate with other microorganisms and mammalian cells to alter gene expression. Here, we present methodologies to evaluate the effects of P. aeruginosa N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C12-HSL) on Caco-2 cell monolayers. First, we describe a method for assessing barrier function and permeability of epithelial cells when exposed to 3O-C12-HSL by measuring transepithelial electrical resistance (TER) and paracellular flow using fluorescently labeled dextran. Secondly, we detail methods to investigate the effect of 3O-C12-HSL on protein-protein interactions of epithelial junction proteins. Lastly, we will detail imaging techniques to visualize Caco-2 barrier disruption following exposure to 3O-C12-HSL through the use of confocal laser scanning microscopy (CLSM) and a super resolution technique, stimulated emission depletion (STED) microscopy, to achieve nanoscale visualization of Caco-2 monolayers.


Asunto(s)
Células Epiteliales/efectos de los fármacos , Homoserina/análogos & derivados , Lactonas/farmacología , Mamíferos/metabolismo , Animales , Células CACO-2 , Extractos Celulares , Impedancia Eléctrica , Electroforesis en Gel de Poliacrilamida , Células Epiteliales/metabolismo , Fluorescencia , Homoserina/farmacología , Humanos , Immunoblotting , Inmunoprecipitación , Microscopía Confocal , Uniones Estrechas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo
5.
Front Cell Dev Biol ; 5: 98, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29209610

RESUMEN

Normal epithelial and endothelial renewal and healing after bacterial and viral challenges are essential for homeostasis along the intestine and the blood and lymphatic vessels. We thus investigated whether and how virus affects migration of human epithelial cells and specifically how the nucleocapsid protein (N) modulates the cellular proteome and interactome using human Caco-2 cells in a wound-healing assay with Hazara virus as a model. Here, Hazara virus blocked cell migration in a dose- and time-dependent manner, disrupted the actin cytoskeleton and specifically reduced the expression of the IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and water channel aquaporin 6 (AQP6) that regulate cytoskeletal organization, water homeostasis and vesicle communication. Moreover, in the Caco-2 cell proteome, we identified several distinct groups of molecules associating with N upon Hazara virus infection, being involved in the ensemble of important cellular processes, e.g., chaperone activity, metabolism, cellular defense against infections, cell morphology, and migration. These events do not only facilitate the virus life cycle, but they are also crucial for membrane and cytoskeleton dynamics, cellular self-renewal and wound healing, being so essential for body integrity and homeostasis.

6.
Artículo en Inglés | MEDLINE | ID: mdl-27047801

RESUMEN

Quorum sensing (QS) communication allows Pseudomonas aeruginosa to collectively control its population density and the production of biofilms and virulence factors. QS signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL), can also affect the behavior of host cells, e.g., by modulating the chemotaxis, migration, and phagocytosis of human leukocytes. Moreover, host water homeostasis and water channels aquaporins (AQP) are critical for cell morphology and functions as AQP interact indirectly with the cell cytoskeleton and signaling cascades. Here, we investigated how P. aeruginosa 3O-C12-HSL affects cell morphology, area, volume and AQP9 expression and distribution in human primary macrophages, using quantitative PCR, immunoblotting, two- and three-dimensional live imaging, confocal and nanoscale imaging. Thus, 3O-C12-HSL enhanced cell volume and area and induced cell shape and protrusion fluctuations in macrophages, processes tentatively driven by fluxes of water across cell membrane through AQP9, the predominant AQP in macrophages. Moreover, 3O-C12-HSL upregulated the expression of AQP9 at both the protein and mRNA levels. This was accompanied with enhanced whole cell AQP9 fluorescent intensity and redistribution of AQP9 to the leading and trailing regions, in parallel with increased cell area in the macrophages. Finally, nanoscopy imaging provided details on AQP9 dynamics and architecture within the lamellipodial area of 3O-C12-HSL-stimulated cells. We suggest that these novel events in the interaction between P. aeruginosa and macrophage may have an impact on the effectiveness of innate immune cells to fight bacteria, and thereby resolve the early stages of infections and inflammations.


Asunto(s)
4-Butirolactona/análogos & derivados , Acuaporinas/metabolismo , Forma de la Célula/efectos de los fármacos , Homoserina/análogos & derivados , Macrófagos/metabolismo , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum/efectos de los fármacos , 4-Butirolactona/farmacología , Acuaporinas/genética , Biopelículas , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Homoserina/farmacología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/inmunología , Infecciones por Pseudomonas , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , ARN Mensajero/biosíntesis , Transducción de Señal/efectos de los fármacos , Factores de Virulencia/metabolismo , Agua/metabolismo
7.
FEMS Microbiol Lett ; 363(8)2016 04.
Artículo en Inglés | MEDLINE | ID: mdl-26976854

RESUMEN

Crimean-Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne pathogen that causes infectious disease with severe hemorrhagic manifestations in vascular system in humans. The proper function of the cells in the vascular system is critically regulated by aquaporins (AQP), water channels that facilitate fluxes of water and small solutes across membranes. With Hazara virus as a model for CCHFV, we investigated the effects of viruses on AQP6 and the impact of AQP6 on virus infectivity in host cells, using transiently expressed GFP-AQP6 cells, immunofluorescent assay for virus detection, epifluorescent imaging of living cells and confocal microscopy. In GFP-AQP6 expressing cells, Hazara virus reduced both the cellular and perinuclear AQP6 distribution and changed the cell area. Infection of human cell with CCHFV strain IbAR 10200 downregulated AQP6 expression at mRNA level. Interestingly, the overexpression of AQP6 in host cells decreased the infectivity of Hazara virus, speaking for a protective role of AQP6. We suggest the possibility for AQP6 being a novel player in the virus-host interactions, which may lead to less severe outcomes of an infection.


Asunto(s)
Acuaporina 6/biosíntesis , Virus de la Fiebre Hemorrágica de Crimea-Congo/patogenicidad , Fiebre Hemorrágica de Crimea/patología , Interacciones Huésped-Patógeno/fisiología , Replicación Viral/fisiología , Animales , Acuaporina 6/genética , Línea Celular , Proteínas Fluorescentes Verdes , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Fiebre Hemorrágica de Crimea/virología , Humanos , Ratones , Ratones Endogámicos C3H , ARN Mensajero/biosíntesis
8.
Front Microbiol ; 6: 915, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26388857

RESUMEN

Pseudomonas aeruginosa controls production of its multiple virulence factors and biofilm development via the quorum sensing (QS) system. QS signals also interact with and affect the behavior of eukaryotic cells. Host water homeostasis and aquaporins (AQP) are essential during pathological conditions since they interfere with the cell cytoskeleton and signaling, and hereby affect cell morphology and functions. We investigated the contribution of P. aeruginosa QS genes lasI/rhlI to phagocytosis, cell morphology, AQP9 expression, and distribution in human macrophages, using immunoblotting, confocal, and nanoscale imaging. Wild type P. aeruginosa with a functional QS system was a more attractive prey for macrophages than the lasI/rhlI mutant lacking the production of QS molecules, 3O-C12-HSL, and C4-HSL, and associated virulence factors. The P. aeruginosa infections resulted in elevated AQP9 expression and relocalization to the leading and trailing regions in macrophages, increased cell area and length; bacteria with a functional QS system lasI/rhlI achieved stronger responses. We present evidence for a new role of water fluxes via AQP9 during bacteria-macrophage interaction and for the QS system as an important stimulus in this process. These novel events in the interplay between P. aeruginosa and macrophages may influence on the outcome of infection, inflammation, and development of disease.

9.
FEMS Microbiol Lett ; 362(11)2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25956174

RESUMEN

Helicobacter pylori produces outer membrane vesicles (OMV), delivering bacterial substances including the oncogenic cytotoxin-associated CagA protein to their surroundings. We investigated the effects of H. pylori OMV carrying CagA (OMV-CagA) on cell junctions and ATP-binding proteome of epithelial monolayers, using proteomics, mass spectrometry and imaging. OMV-CagA localized in close vicinity of ZO-1 tight junction protein and induced histone H1 binding to ATP. We suggest the expression of novel events in the interactions between H. pylori OMV and epithelia, which may have an influence on host gene transcription and lead to different outcomes of an infection and development of cancer.


Asunto(s)
Adenosina Trifosfato/metabolismo , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Helicobacter pylori/metabolismo , Histonas/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Células CACO-2 , Helicobacter pylori/genética , Interacciones Huésped-Patógeno , Humanos , Microscopía Confocal , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructura , Proteína de la Zonula Occludens-1/metabolismo
10.
Front Plant Sci ; 5: 309, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25018766

RESUMEN

Both direct and long-range interactions between pathogenic Pseudomonas aeruginosa bacteria and their eukaryotic hosts are important in the outcome of infections. For cell-to-cell communication, these bacteria employ the quorum sensing (QS) system to pass on information of the density of the bacterial population and collectively switch on virulence factor production, biofilm formation, and resistance development. Thus, QS allows bacteria to behave as a community to perform tasks which would be impossible for individual cells, e.g., to overcome defense and immune systems and establish infections in higher organisms. This review highlights these aspects of QS and our own recent research on how P. aeruginosa communicates with human cells using the small QS signal molecules N-acyl homoserine lactones (AHL). We focus on how this conversation changes the behavior and function of neutrophils, macrophages, and epithelial cells and on how the signaling machinery in human cells responsible for the recognition of AHL. Understanding the bacteria-host relationships at both cellular and molecular levels is essential for the identification of new targets and for the development of novel strategies to fight bacterial infections in the future.

11.
J Virol ; 88(6): 3161-9, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24371070

RESUMEN

UNLABELLED: The disease mechanisms associated with onset and secondary effects of rotavirus (RV) diarrhea remain to be determined and may not be identical. In this study, we investigated whether onset of RV diarrhea is associated with increased intestinal permeability and/or motility. To study the transit time, fluorescent fluorescein isothiocyanate (FITC)-dextran was given to RV-infected adult and infant mice. Intestinal motility was also studied with an opioid receptor agonist (loperamide) and a muscarinic receptor antagonist (atropine). To investigate whether RV increases permeability at the onset of diarrhea, fluorescent 4- and 10-kDa dextran doses were given to infected and noninfected mice, and fluorescence intensity was measured subsequently in serum. RV increased transit time in infant mice. Increased motility was detected at 24 h postinfection (h p.i.) and persisted up to 72 h p.i in pups. Both loperamide and atropine decreased intestinal motility and attenuated diarrhea. Analysis of passage of fluorescent dextran from the intestine into serum indicated unaffected intestinal permeability at the onset of diarrhea (24 to 48 h p.i.). We show that RV-induced diarrhea is associated with increased intestinal motility via an activation of the myenteric nerve plexus, which in turn stimulates muscarinic receptors on intestinal smooth muscles. IMPORTANCE: We show that RV-infected mice have increased intestinal motility at the onset of diarrhea, and that this is not associated with increased intestinal permeability. These new observations will contribute to a better understanding of the mechanisms involved in RV diarrhea.


Asunto(s)
Diarrea/fisiopatología , Mucosa Intestinal/metabolismo , Infecciones por Rotavirus/fisiopatología , Rotavirus/fisiología , Animales , Dextranos/metabolismo , Diarrea/metabolismo , Diarrea/virología , Femenino , Motilidad Gastrointestinal , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Permeabilidad , Infecciones por Rotavirus/metabolismo , Infecciones por Rotavirus/virología
12.
Biochem Biophys Res Commun ; 430(3): 993-8, 2013 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-23261438

RESUMEN

Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.


Asunto(s)
Acuaporinas/metabolismo , Células Epiteliales/fisiología , Agua/metabolismo , Cicatrización de Heridas , Animales , Movimiento Celular , Perros , Células Epiteliales/metabolismo , Células de Riñón Canino Madin Darby
13.
PLoS Pathog ; 8(10): e1002953, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23071436

RESUMEN

Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxo-dodecanoyl-L-homoserine lactone 3O-C(12)-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C(12)-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C(12)-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C(12)-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C(12)-HSL with a sensor bioassay. Moreover, 3O-C(12)-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P. aeruginosa 3O-C(12)-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C(12)-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial--human cell communication.


Asunto(s)
Acil-Butirolactonas/metabolismo , Movimiento Celular , Pseudomonas aeruginosa/metabolismo , Percepción de Quorum , Proteínas Activadoras de ras GTPasa/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Biopelículas/crecimiento & desarrollo , Células CACO-2 , Línea Celular , Células Epiteliales/metabolismo , Homoserina/análogos & derivados , Homoserina/metabolismo , Humanos , Fosforilación , Pseudomonas aeruginosa/patogenicidad , Transducción de Señal , Factores de Virulencia/biosíntesis , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo
14.
Infect Immun ; 80(5): 1670-80, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22354024

RESUMEN

Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can impair the intestinal epithelial barrier and thereby facilitate the translocation of luminal bacteria into the blood, which may in turn aggravate a septic condition. Here, we showed that staphylococcal alpha-toxin disrupts the barrier integrity of human intestinal epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER) and reduced cellular levels of junctional proteins, such as ZO-1, ZO-3, and E-cadherin. The Caco-2 cells also responded to alpha-toxin with an elevated cytosolic calcium ion concentration ([Ca(2+)](i)), elicited primarily by calcium influx from the extracellular environment, as well as with a significant reduction in TER, which was modulated by intracellular calcium chelation. Moreover, a significantly larger reduction in TER and amounts of the junctional proteins, viz., ZO-3 and occludin, was achieved by basolateral than by apical application of the alpha-toxin. These experimental findings thus support the hypothesis that free staphylococcal alpha-toxin in the bloodstream may cause intestinal epithelial barrier dysfunction and further aggravate the septic condition by promoting the release of intestinal bacteria into the underlying tissues and the blood.


Asunto(s)
Toxinas Bacterianas/toxicidad , Células Epiteliales/efectos de los fármacos , Proteínas Hemolisinas/toxicidad , Uniones Intercelulares/efectos de los fármacos , Staphylococcus aureus/metabolismo , Células CACO-2 , Cadherinas/genética , Cadherinas/metabolismo , Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Citosol/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ocludina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Proteínas de la Zonula Occludens , Proteína de la Zonula Occludens-1 , beta Catenina/genética , beta Catenina/metabolismo
15.
J Leukoc Biol ; 91(1): 15-26, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21807742

RESUMEN

In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C(12)-HSL and -C(10)-HSL but not C(4)-HSL in a concentration-dependent manner. Moreover, 3O-C(12)-HSL and -C(10)-HSL activate PLCγ1 but not -γ2, mobilize intracellular calcium, and up-regulate IP(3)R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C(12)-HSL- and 3O-C(10)-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C(4)-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C(12)-HSL and -C(10)-HSL. These findings show that the bacterial QS molecules 3O-C(12)-HSL and -C(10)-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.


Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Acil-Butirolactonas/farmacología , Señalización del Calcio/fisiología , Calcio/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Neutrófilos/citología , Citoesqueleto de Actina/inmunología , Señalización del Calcio/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Bacterias Gramnegativas/inmunología , Bacterias Gramnegativas/metabolismo , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/metabolismo , Humanos , Neutrófilos/inmunología , Neutrófilos/microbiología
16.
Eur J Cell Biol ; 89(8): 584-97, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20434232

RESUMEN

In Pseudomonas aeruginosa, cell-cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We demonstrate here that 3O-C(12)-HSL can induce changes in calcium signalling through influx and release of calcium from thapsigargin-sensitive stores and delocalization of inositol 1,4,5-trisphosphate receptors (IP(3)R), but not of ryanodine receptors (RyR). In parallel, P. aeruginosa 3O-C(12)-HSL disrupts junctions in human Caco-2 cells as evidenced by a reduction of the expression and distribution of ZO-3 and JAM-A. Using co-immunoprecipitation we also found an alteration in the binding of ZO-3 to JAM-A in protein complexes. Moreover, 3O-C(12)-HSL-treatment resulted in tyrosine hyperphosphorylation of ZO-3 and JAM-A. On the contrary, serine and threonine residues of ZO-1 and JAM-A became less phosphorylated after exposition of 3O-C(12)-HSL. The 3O-C(12)-HSL-induced intracellular calcium signalling and alteration in the phosphorylation status of junction proteins furthermore correlated with changes in the association between JAM-A-ZO-3. The calcium inhibitors thapsigargin, xestospongin C, and dantrolene partly prevented the 3O-C(12)-HSL-induced decreases in TER and increases in the paracellular flux of 10kDa dextran. These findings clearly suggest that P. aeruginosa 3O-C(12)-HSL can cause the loss of epithelial barrier function via calcium signalling and further alteration in the phosphorylation status of junction proteins; and that bacterial quorum sensing signals represent inter-kingdom signalling.


Asunto(s)
Proteínas Bacterianas/fisiología , Señalización del Calcio/fisiología , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/fisiología , Uniones Estrechas/patología , Acil-Butirolactonas , Western Blotting , Células CACO-2 , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Inmunoglobulinas/metabolismo , Inmunoprecipitación , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Fosforilación , Receptores de Superficie Celular , Uniones Estrechas/metabolismo , Proteínas de la Zonula Occludens
17.
Exp Cell Res ; 315(2): 313-26, 2009 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-19038248

RESUMEN

In Pseudomonas aeruginosa, cell-cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We report that 3O-C(12)-HSL can disrupt adherens junctions in human epithelial Caco-2 cells as evidenced by a reduction of the expression and distribution of E-cadherin and beta-catenin. Using co-immunoprecipitation we also found that P. aeruginosa 3O-C(12)-HSL-treatment resulted in tyrosine hyperphosphorylation of E-cadherin, beta-catenin, occludin and ZO-1. Similarly, serine and threonine residues of E-cadherin and ZO-1 became more phosphorylated after 3O-C(12)-HSL treatment. On the contrary, occludin and beta-catenin underwent dephosphorylation on serine and threonine residues after exposition of 3O-C(12)-HSL. These changes in the phosphorylation state were paralleled by alteration in the structure of junction complexes and increased paracellular permeability. Moreover, pre-treatment of the Caco-2 cells with protein phosphatase and kinase inhibitors prevented 3O-C(12)-HSL-induced changes in paracellular permeability and interactions between occludin-ZO-1 and the E-cadherin-beta-catenin. These findings clearly suggest that an alteration in the phosphorylation status of junction proteins are involved in the changes in cell junction associations and enhanced paracellular permeability, and that bacterial signals are indeed sensed by the host cells.


Asunto(s)
Células Epiteliales/efectos de los fármacos , Uniones Intercelulares/efectos de los fármacos , Pseudomonas aeruginosa/química , Percepción de Quorum , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Células CACO-2 , Cadherinas/metabolismo , Citoplasma/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Genisteína/farmacología , Homoserina/análogos & derivados , Homoserina/farmacología , Humanos , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Ocludina , Ácido Ocadaico/farmacología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Unión Proteica/efectos de los fármacos , Estaurosporina/farmacología , Proteína de la Zonula Occludens-1 , beta Catenina/metabolismo
18.
FEBS Lett ; 580(30): 6921-8, 2006 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-17157842

RESUMEN

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.


Asunto(s)
4-Butirolactona/análogos & derivados , Células Epiteliales/efectos de los fármacos , Homoserina/análogos & derivados , Pseudomonas aeruginosa/química , Percepción de Quorum , 4-Butirolactona/farmacología , Actinas/metabolismo , Células CACO-2 , Adhesión Celular , Proliferación Celular , Dextranos/farmacología , Impedancia Eléctrica , Activación Enzimática/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Homoserina/farmacología , Humanos , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Rastreo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ocludina , Fosfoproteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteína de la Zonula Occludens-1
19.
Microbes Infect ; 7(15): 1512-8, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16039899

RESUMEN

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.


Asunto(s)
4-Butirolactona/análogos & derivados , Homoserina/análogos & derivados , Factores Inmunológicos/farmacología , Macrófagos/inmunología , Fagocitosis/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , 4-Butirolactona/farmacología , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Homoserina/farmacología , Humanos , Immunoblotting , Macrófagos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo
20.
Immunol Lett ; 85(1): 29-33, 2003 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-12505193

RESUMEN

The ability of culture filtrate (CF) of two laboratory Pseudomonas aeruginosa strains to inhibit delayed type hypersensitivity (DTH) to non-bacterial antigen in CBA mice has been studied. It was shown that intraperitoneal injection of native CF of the strains did not affect the level of DTH. However, redox treated CF expressed the immunosuppressive activity. Gel filtration of redox activated CF through Sephadex G-200 showed that CF contains three immunosuppressive components differing by their molecular weight and specificity. All components contained lipid group and O-polysaccharide chains that indicated their lipopolysaccharide (LPS) nature. These experiments show that laboratory P. aeruginosa strains have three LPS components but not all of them display the immunosuppressive activity.


Asunto(s)
Inmunidad/efectos de los fármacos , Inmunosupresores/farmacología , Lipopolisacáridos/farmacología , Pseudomonas aeruginosa/química , Animales , Hipersensibilidad Retardada/inmunología , Inmunosupresores/química , Lipopolisacáridos/química , Ratones , Ratones Endogámicos CBA , Peso Molecular , Oxidación-Reducción
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