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1.
Front Cell Infect Microbiol ; 13: 1196581, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37680748

RESUMEN

Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.


Asunto(s)
Fibrosis Quística , Gliotoxina , Micosis , Aspergillus fumigatus , Fibrosis Quística/complicaciones , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Cloruros , Células Epiteliales
2.
Cell Rep ; 37(1): 109795, 2021 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-34610318

RESUMEN

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.


Asunto(s)
AMP Cíclico/metabolismo , Canales Epiteliales de Sodio/metabolismo , Epitelio/metabolismo , Sodio/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Amilorida/farmacología , Animales , Animales Modificados Genéticamente/metabolismo , Colforsina/farmacología , Fibrosis Quística , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Bloqueadores del Canal de Sodio Epitelial/farmacología , Canales Epiteliales de Sodio/química , Transporte Iónico/efectos de los fármacos , Masculino , Porcinos
3.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L931-L942, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32130033

RESUMEN

The human airway is protected by an efficient innate defense mechanism that requires healthy secretion of airway surface liquid (ASL) to clear pathogens from the lungs. Most of the ASL in the upper airway is secreted by submucosal glands. In cystic fibrosis (CF), the function of airway submucosal glands is abnormal, and these abnormalities are attributed to anomalies in ion transport across the epithelia lining the different sections of the glands that function coordinately to produce the ASL. However, the ion transport properties of most of the anatomical regions of the gland have never been measured, and there is controversy regarding which segments express CFTR. This makes it difficult to determine the glandular abnormalities that may contribute to CF lung disease. Using a noninvasive, extracellular self-referencing ion-selective electrode technique, we characterized ion transport properties in all four segments of submucosal glands from wild-type and CFTR-/- swine. In wild-type airways, the serous acini, mucus tubules, and collecting ducts secrete Cl- and Na+ into the lumen in response to carbachol and forskolin stimulation. The ciliated duct also transports Cl- and Na+ but in the opposite direction, i.e., reabsorption from the ASL, which may contribute to lowering Na+ and Cl- activities in the secreted fluid. In CFTR-/- airways, the serous acini, collecting ducts, and ciliated ducts fail to transport ions after forskolin stimulation, resulting in the production of smaller volumes of ASL with normal Cl-, Na+, and K+ concentration.


Asunto(s)
Células Acinares/metabolismo , Cilios/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Pulmón/metabolismo , Células Acinares/efectos de los fármacos , Células Acinares/patología , Animales , Carbacol/farmacología , Cationes Monovalentes , Cloruros/metabolismo , Cilios/efectos de los fármacos , Cilios/patología , Colforsina/farmacología , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Modelos Animales de Enfermedad , Técnicas Electroquímicas , Electrodos , Eliminación de Gen , Expresión Génica , Humanos , Transporte Iónico , Pulmón/efectos de los fármacos , Pulmón/patología , Potasio/metabolismo , Sodio/metabolismo , Porcinos
4.
Sci Rep ; 9(1): 540, 2019 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-30679487

RESUMEN

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR-/- and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR-/- swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.


Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Quiste Mediastínico/tratamiento farmacológico , Quiste Mediastínico/metabolismo , Solución Salina Hipertónica/uso terapéutico , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Administración por Inhalación , Animales , Animales Modificados Genéticamente , Secreciones Corporales/efectos de los fármacos , Secreciones Corporales/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Femenino , Técnicas de Inactivación de Genes , Transporte Iónico/efectos de los fármacos , Masculino , Ósmosis/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Solución Salina Hipertónica/administración & dosificación , Solución Salina Hipertónica/farmacología , Porcinos
5.
Cell Microbiol ; 20(1)2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-28876505

RESUMEN

N-(3-Oxododecanoyl)-l-homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum-sensing molecule for bacteria-bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12-triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in "initiator" caspases or "effector" caspases. Our data indicate that C12 selectively induces the mitochondria-dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both "initiator" and "effector" caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.


Asunto(s)
4-Butirolactona/análogos & derivados , Apoptosis/fisiología , Homoserina/análogos & derivados , Membranas Mitocondriales/metabolismo , Percepción de Quorum/fisiología , 4-Butirolactona/metabolismo , Animales , Caspasa 3/genética , Caspasa 7/genética , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Fibroblastos/metabolismo , Células HCT116 , Homoserina/metabolismo , Humanos , Ratones , Ratones Noqueados , Interacciones Microbianas/fisiología , Mitocondrias/metabolismo , Pseudomonas aeruginosa/metabolismo
6.
Nat Commun ; 8(1): 786, 2017 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-28983075

RESUMEN

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel, which can result in chronic lung disease. The sequence of events leading to lung disease is not fully understood but recent data show that the critical pathogenic event is the loss of the ability to clear bacteria due to abnormal airway surface liquid secretion (ASL). However, whether the inhalation of bacteria triggers ASL secretion and whether this is abnormal in cystic fibrosis has never been tested. Here we show, using a novel synchrotron-based in vivo imaging technique, that wild-type pigs display both a basal and a Toll-like receptor-mediated ASL secretory response to the inhalation of cystic fibrosis relevant bacteria. Both mechanisms fail in CFTR-/- swine, suggesting that cystic fibrosis airways do not respond to inhaled pathogens, thus favoring infection and inflammation that may eventually lead to tissue remodeling and respiratory disease.Cystic fibrosis is caused by mutations in the CFTR chloride channel, leading to reduced airway surface liquid secretion. Here the authors show that exposure to bacteria triggers secretion in wild-type but not in pig models of cystic fibrosis, suggesting an impaired response to pathogens contributes to infection.


Asunto(s)
Fibrosis Quística/metabolismo , Pulmón/metabolismo , Pseudomonas aeruginosa , Mucosa Respiratoria/metabolismo , Animales , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Exposición por Inhalación , Pulmón/diagnóstico por imagen , Masculino , Radiografía , Porcinos
7.
PLoS One ; 11(3): e0150109, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27031335

RESUMEN

Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10-80 fold increase, termed "swarming"), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds.


Asunto(s)
Adhesión Bacteriana/fisiología , Fibrosis Quística/patología , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Quimiotaxis , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Microscopía Fluorescente , Microscopía por Video , Pseudomonas aeruginosa/genética , Receptores de Aminoácidos/deficiencia , Receptores de Aminoácidos/genética , Mucosa Respiratoria/citología
8.
Am J Physiol Gastrointest Liver Physiol ; 310(9): G671-81, 2016 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-26847387

RESUMEN

A computer model, constructed for evaluation of integrated functioning of cellular components involved in acid secretion by the gastric parietal cell, has provided new interpretations of older experimental evidence, showing the functional significance of a canalicular space separated from a mucosal bath by a gland lumen and also shedding light on basolateral Cl(-) transport. The model shows 1) changes in levels of parietal cell secretion (with stimulation or H-K-ATPase inhibitors) result mainly from changes in electrochemical driving forces for apical K(+) and Cl(-) efflux, as canalicular [K(+)] ([K(+)]can) increases or decreases with changes in apical H(+)/K(+) exchange rate; 2) H-K-ATPase inhibition in frog gastric mucosa would increase [K(+)]can similarly with low or high mucosal [K(+)], depolarizing apical membrane voltage similarly, so electrogenic H(+) pumping is not indicated by inhibition causing similar increase in transepithelial potential difference (Vt) with 4 and 80 mM mucosal K(+); 3) decreased H(+) secretion during strongly mucosal-positive voltage clamping is consistent with an electroneutral H-K-ATPase being inhibited by greatly decreased [K(+)]can (Michaelis-Menten mechanism); 4) slow initial change ("long time-constant transient") in current or Vt with clamping of Vt or current involves slow change in [K(+)]can; 5) the Na(+)-K(+)-2Cl(-) symporter (NKCC) is likely to have a significant role in Cl(-) influx, despite evidence that it is not necessary for acid secretion; and 6) relative contributions of Cl(-)/HCO3 (-) exchanger (AE2) and NKCC to Cl(-) influx would differ greatly between resting and stimulated states, possibly explaining reported differences in physiological characteristics of stimulated open-circuit Cl(-) secretion (≈H(+)) and resting short-circuit Cl(-) secretion (>>H(+)).


Asunto(s)
Simulación por Computador , Células Parietales Gástricas/metabolismo , Potasio/metabolismo , Animales , Bicarbonatos/metabolismo , Cloruros/metabolismo , Humanos , Bombas Iónicas/metabolismo
9.
Oncotarget ; 7(5): 5924-42, 2016 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-26758417

RESUMEN

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells.


Asunto(s)
4-Butirolactona/análogos & derivados , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Homoserina/análogos & derivados , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , 4-Butirolactona/farmacología , Animales , Apoptosis/efectos de los fármacos , Arildialquilfosfatasa/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Homoserina/farmacología , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
10.
J Biol Chem ; 290(11): 7247-58, 2015 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-25627690

RESUMEN

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca(2+) was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca(2+)] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca(2+) release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12.


Asunto(s)
4-Butirolactona/análogos & derivados , Apoptosis , Arildialquilfosfatasa/metabolismo , Homoserina/análogos & derivados , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiología , Percepción de Quorum , 4-Butirolactona/metabolismo , Animales , Células Cultivadas , Células HEK293 , Homoserina/metabolismo , Interacciones Huésped-Patógeno , Humanos , Ratones , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología
11.
mBio ; 5(6): e01924, 2014 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-25352618

RESUMEN

UNLABELLED: A defining characteristic of Chlamydia spp. is their developmental cycle characterized by outer membrane transformations of cysteine bonds among cysteine-rich outer membrane proteins. The reduction-oxidation states of host cell compartments were monitored during the developmental cycle using live fluorescence microscopy. Organelle redox states were studied using redox-sensitive green fluorescent protein (roGFP1) expressed in CF15 epithelial cells and targeted to the cytosol, mitochondria, and endoplasmic reticulum (ER). The redox properties of chlamydiae and the inclusion were monitored using roGFP expressed by Chlamydia trachomatis following transformation. Despite the large morphological changes associated with chlamydial infection, redox potentials of the cytosol (Ψ(cyto) [average, -320 mV]), mitochondria (Ψ(mito) [average, -345 mV]), and the ER (ΨER [average, -258 mV]) and their characteristic redox regulatory abilities remained unchanged until the cells died, at which point Ψ(cyto) and Ψ(mito) became more oxidized and Ψ(ER) became more reduced. The redox status of the chlamydial cytoplasm was measured following transformation and expression of the roGFP biosensor in C. trachomatis throughout the developmental cycle. The periplasmic and outer membrane redox states were assessed by the level of cysteine cross-linking of cysteine-rich envelope proteins. In both cases, the chlamydiae were highly reduced early in the developmental cycle and became oxidized late in the developmental cycle. The production of a late-developmental-stage oxidoreductase/isomerase, DsbJ, may play a key role in the regulation of the oxidoreductive developmental-stage-specific process. IMPORTANCE: Infectious Chlamydia organisms have highly oxidized and cysteine cross-linked membrane proteins that confer environmental stability when outside their host cells. Once these organisms infect a new host cell, the proteins become reduced and remain reduced during the active growth stage. These proteins become oxidized at the end of their growth cycle, wherein infectious organisms are produced and released to the environment. How chlamydiae mediate and regulate this key step in their pathogenesis is unknown. Using biosensors specifically targeted to different compartments within the infected host cell and for the chlamydial organisms themselves, the oxidoreductive states of these compartments were measured during the course of infection. We found that the host cell redox states are not changed by infection with C. trachomatis, whereas the state of the chlamydial organisms remains reduced during infection until the late developmental stages, wherein the organisms' cytosol and periplasm become oxidized and they acquire environmental resistance and infectivity.


Asunto(s)
Chlamydia trachomatis/química , Chlamydia trachomatis/crecimiento & desarrollo , Citoplasma/química , Células Epiteliales/química , Células Epiteliales/microbiología , Orgánulos/química , Oxidación-Reducción , Línea Celular , Membrana Celular/química , Genes Reporteros , Humanos , Periplasma/química
12.
Proc Natl Acad Sci U S A ; 111(35): 12930-5, 2014 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-25136096

RESUMEN

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Tráquea/metabolismo , Tráquea/microbiología , Animales , Fibrosis Quística/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Femenino , Haemophilus influenzae/metabolismo , Inmunidad Innata/fisiología , Masculino , Mucinas/metabolismo , Infecciones por Pseudomonas/inmunología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Staphylococcus aureus/metabolismo , Porcinos , Sincrotrones , Tráquea/inmunología
13.
J Immunol ; 193(3): 1459-67, 2014 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-24990083

RESUMEN

Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (HSL-C12) as a quorum-sensing molecule to regulate bacterial gene expression. Because HSL-C12 is membrane permeant, multiple cell types in P. aeruginosa-infected airways may be exposed to HSL-C12, especially adjacent to biofilms where local (HSL-C12) may be high. Previous reports showed that HSL-C12 causes both pro- and anti-inflammatory effects. To characterize HSL-C12's pro- and anti-inflammatory effects in host cells, we measured protein synthesis, NF-κB activation, and KC (mouse IL-8) and IL-6 mRNA and protein secretion in wild-type mouse embryonic fibroblasts (MEF). To test the role of the endoplasmic reticulum stress inducer, PERK we compared these responses in PERK(-/-) and PERK-corrected PERK(-/-) MEF. During 4-h treatments of wild-type MEF, HSL-C12 potentially activated NF-κB p65 by preventing the resynthesis of IκB and increased transcription of KC and IL-6 genes (quantitative PCR). HSL-C12 also inhibited secretion of KC and/or IL-6 into the media (ELISA) both in control conditions and also during stimulation by TNF-α. HSL-C12 also activated PERK (as shown by increased phosphorylation of eI-F2α) and inhibited protein synthesis (as measured by incorporation of [(35)S]methionine by MEF). Comparisons of PERK(-/-) and PERK-corrected MEF showed that HSL-C12's effects were explained in part by activation of PERK→phosphorylation of eI-F2α→inhibition of protein synthesis→reduced IκBα production→activation of NF-κB→increased transcription of the KC gene but reduced translation and secretion of KC. HSL-C12 may be an important modulator of early (up to 4 h) inflammatory signaling in P. aeruginosa infections.


Asunto(s)
4-Butirolactona/análogos & derivados , Factor 2 Eucariótico de Iniciación/fisiología , Mediadores de Inflamación/fisiología , Pseudomonas aeruginosa/inmunología , Percepción de Quorum/inmunología , Transducción de Señal/inmunología , eIF-2 Quinasa/fisiología , 4-Butirolactona/fisiología , Animales , Línea Celular , Estrés del Retículo Endoplásmico/inmunología , Ratones , eIF-2 Quinasa/deficiencia
14.
Am J Physiol Gastrointest Liver Physiol ; 306(8): G699-710, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24578340

RESUMEN

Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle.


Asunto(s)
Actinas/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Células Parietales Gástricas , Vesículas Transportadoras , Animales , Azepinas/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Naftalenos/farmacología , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/patología , Conejos , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/fisiología
15.
Am J Physiol Cell Physiol ; 306(9): C844-55, 2014 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-24598360

RESUMEN

Pseudomonas aeruginosa secretes N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression. Micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a loss of transepithelial resistance within 1 h that was accompanied by disassembly of tight junctions, as indicated by relocation of the tight junction protein zonula occludens 1 from the apical to the basolateral pole and into the cytosol of polarized human airway epithelial cell cultures (Calu-3 and primary tracheal epithelial cells). These effects were blocked by carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone, a pan-caspase blocker, indicating that tight junction disassembly was an early event in C12-triggered apoptosis. Short-duration (10 min) pretreatment of airway epithelial (Calu-3 and JME) cells with 1 µM thapsigargin (Tg), an inhibitor of Ca(2+) uptake into the endoplasmic reticulum (ER), was found to be protective against the C12-induced airway epithelial barrier breakdown and also against other apoptosis-related effects, including shrinkage and fragmentation of nuclei, activation of caspase 3/7 (the executioner caspase in apoptosis), release of ER-targeted redox-sensitive green fluorescent protein into the cytosol, and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA-AM [to buffer cytosolic Ca(2+) concentration (Cacyto)] or Ca(2+)-free solution + BAPTA-AM reduced C12 activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca(2+). Because C12 and Tg reduced Ca(2+) concentration in the ER and increased Cacyto, while Tg increased mitochondrial Ca(2+) concentration (Camito) and C12 reduced Camito, it is proposed that Tg may reduce C12-induced apoptosis in host cells not by raising Cacyto, but by preventing C12-induced decreases in Camito.


Asunto(s)
4-Butirolactona/análogos & derivados , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Pulmón/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Tapsigargina/farmacología , Tráquea/efectos de los fármacos , 4-Butirolactona/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular , Quelantes/farmacología , Citoprotección , Impedancia Eléctrica , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Activación Enzimática , Humanos , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Cultivo Primario de Células , Transporte de Proteínas , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Factores de Tiempo , Tráquea/metabolismo , Tráquea/microbiología , Tráquea/patología , Proteína de la Zonula Occludens-1/metabolismo
16.
Cell Microbiol ; 16(7): 1094-104, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24438098

RESUMEN

Pseudomonas aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12-triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild-type (WT) and Bax(-/-) /Bak(-/-) (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψmito ), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax-dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψmito and release of cytochrome C and cell death. Re-expression of Bax or Bak in DKO MEF did not alter the WT-like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax-independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψmito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.


Asunto(s)
4-Butirolactona/análogos & derivados , Apoptosis , Citocromos c/metabolismo , Fibroblastos/fisiología , Mitocondrias/metabolismo , Pseudomonas aeruginosa/fisiología , 4-Butirolactona/fisiología , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Forma de la Célula , Fibroblastos/microbiología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Células 3T3 NIH , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo
17.
J Pharmacol Sci ; 122(3): 213-22, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23877017

RESUMEN

The proposed mechanism for proton pump inhibitors (PPIs) is that PPIs are activated at low pH to the sulfenamide form, which reacts with the sulfhydryl group of cysteine(s) at the active site of the proton pump, to produce reducible disulfide-bonded PPI-proton pump conjugates. However, this mechanism cannot explain the observations that some PPI-protein conjugates are irreducible. This study was designed to investigate the chemistry of the irreducible conjugates by mass spectrometry, using three PPIs and 17 cysteine-containing peptides. While some peptides favored the formation of reducible PPI-peptide adduct, the other peptides mainly produced irreducible adducts. Characterization of the irreducible adduct revealed that the irreducible bonding required the participation of both a sulfhydryl group and a nearby primary amino group. High resolution mass spectrometry suggested a molecular structure of the irreducible adduct. These results suggested a reaction mechanism in which the PPI pyridone form reacted with an amino group and a sulfhydryl group to form an irreducible adduct. The irreducible adduct becomes the dominant product over time because of the irreversible nature of the pyridone-mediated reaction. These findings may explain the irreducible inhibition of H/K-ATPase by PPIs and their relatively slow biological turnover in vivo. [Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.13058FP].


Asunto(s)
Cisteína/química , Péptidos/química , Inhibidores de la Bomba de Protones/química , Inhibidores de la Bomba de Protones/farmacología , 2-Piridinilmetilsulfinilbencimidazoles/química , 2-Piridinilmetilsulfinilbencimidazoles/farmacología , Dominio Catalítico , Inhibidores Enzimáticos , ATPasa Intercambiadora de Hidrógeno-Potásio , Concentración de Iones de Hidrógeno , Lansoprazol/química , Lansoprazol/farmacología , Espectrometría de Masas , Estructura Molecular , Omeprazol/química , Omeprazol/farmacología , Pantoprazol , Piridonas , Sulfamerazina
18.
Am J Physiol Cell Physiol ; 303(12): C1301-11, 2012 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-23099641

RESUMEN

In primary culture, the gastric parietal cell's deeply invaginated apical membrane, seen in microscopy by phalloidin binding to F-actin (concentrated in microvilli and a subapical web), is engulfed into the cell, separated from the basolateral membrane (which then becomes the complete plasma membrane), and converted, from a lacy interconnected system of canaliculi, into several separate vacuoles. In this study, vacuolar morphology was achieved by 71% of parietal cells 8 h after typical collagenase digestion of rabbit gastric mucosa, but the tight-junctional protein zonula occludens-1 (ZO-1) was completely delocalized after ∼2 h, when cells were ready for culturing. Use of low-Ca(2+) medium (4 mM EGTA) to release cells quickly from gastric glands yielded parietal cells in which ZO-1 was seen in a small spot or ring, a localization quickly lost if these cells were then cultured in normal Ca(2+) but remaining up to 20 h if they were cultured in low Ca(2+). The cells in low Ca(2+) mostly retained, at 20 h, an intermediate morphology of many bulbous canalicular expansions ("prevacuoles"), seemingly with narrow interconnections. Histamine stimulation of 20-h cells with intermediate morphology caused colocalization of proton-pumping H-K-ATPase with canaliculi and prevacuoles but little swelling of those structures, consistent with a remaining apical pore through which secreted acid could escape. Apparent canalicular interconnections, lack of stimulated swelling, and lingering ZO-1 staining indicate inhibition of membrane fission processes that separate apical from basolateral membrane and vacuoles from each other, suggesting an important role for extracellular Ca(2+) in these, and possibly other, endocytotic processes.


Asunto(s)
Calcio/farmacología , Células Parietales Gástricas/citología , Vacuolas/metabolismo , Animales , Células Cultivadas , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Histamina/farmacología , Microvellosidades/metabolismo , Células Parietales Gástricas/efectos de los fármacos , Células Parietales Gástricas/metabolismo , Conejos , Vacuolas/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismo
19.
Am J Physiol Lung Cell Mol Physiol ; 303(4): L327-33, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22683572

RESUMEN

The airway is kept sterile by an efficient innate defense mechanism. The cornerstone of airway defense is mucus containing diverse antimicrobial factors that kill or inactivate pathogens. Most of the mucus in the upper airways is secreted by airway submucosal glands. In patients with cystic fibrosis (CF), airway defense fails and the lungs are colonized by bacteria, usually Pseudomonas aeruginosa. Accumulating evidence suggests that airway submucosal glands contribute to CF pathogenesis by failing to respond appropriately to inhalation of bacteria. However, the regulation of submucosal glands by the innate immune system remains poorly understood. We studied the response of submucosal glands to the proinflammatory cytokines interleukin-1ß and tumor necrosis factor-α. These are released into the airway submucosa in response to infection with the bacterium P. aeruginosa and are elevated in CF airways. Stimulation with IL-1ß and TNF-α increased submucosal gland secretion in a concentration-dependent manner with a maximal secretion rate of 240 ± 20 and 190 ± 40 pl/min, respectively. The half maximal effective concentrations were 11 and 20 ng/ml, respectively. The cytokine effect was dependent on cAMP but was independent of cGMP, nitric oxide, Ca(2+), or p38 MAP kinase. Most importantly, IL-1ß- and TNF-α-stimulated secretion was blocked by the CF transmembrane conductance regulator (CFTR) blocker, CFTRinh172 (100 µmol/l) but was not affected by the Ca(2+)-activated Cl(-) channel blocker, niflumic acid (1 µmol/l). The data suggest, that during bacterial infections and resulting release of proinflammatory cytokines, the glands are stimulated to secrete fluid, and this response is mediated by cAMP-activated CFTR, a process that would fail in patients with CF.


Asunto(s)
Líquidos Corporales/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Interleucina-1beta/farmacología , Moco/metabolismo , Sistema Respiratorio/anatomía & histología , Sistema Respiratorio/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Líquidos Corporales/efectos de los fármacos , Calcio/metabolismo , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Femenino , Técnicas In Vitro , Masculino , Moco/efectos de los fármacos , Ácido Niflúmico/farmacología , Óxido Nítrico/metabolismo , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/enzimología , Sistemas de Mensajero Secundario/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Porcinos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
20.
Cell Microbiol ; 14(5): 698-709, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22233488

RESUMEN

Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis (CF) patients, a process regulated by quorum-sensing molecules including N-(3-oxododecanoyl)-l-homoserine lactone (C12). C12 (10-100 µM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CF ΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψ(mito)) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca(2+) and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 min and were complete in 1-2 h. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψ(mito) and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψ(mito) and increases in Ca(cyto) like 10-50 µM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed.


Asunto(s)
4-Butirolactona/análogos & derivados , Apoptosis , Biopelículas/crecimiento & desarrollo , Células Epiteliales/efectos de los fármacos , Homoserina/análogos & derivados , Pseudomonas aeruginosa/fisiología , 4-Butirolactona/metabolismo , 4-Butirolactona/toxicidad , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Homoserina/metabolismo , Homoserina/toxicidad , Humanos , Membranas Mitocondriales/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Factores de Tiempo
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