RESUMEN
Staphylococcus aureus colonizes the nose, throat, skin, and gastrointestinal (GI) tract of humans. GI carriage of S. aureus is difficult to eradicate and has been shown to facilitate the transmission of the bacterium among individuals. Although staphylococcal colonization of the GI tract is asymptomatic, it increases the likelihood of infection, particularly skin and soft tissue infections caused by USA300 isolates. We established a mouse model of persistent S. aureus GI colonization and characterized the impact of selected surface antigens on colonization. In competition experiments, an acapsular mutant colonized better than the parental strain Newman, whereas mutants defective in sortase A and clumping factor A showed impaired ability to colonize the GI tract. Mutants lacking protein A, clumping factor B, poly-N-acetyl glucosamine, or SdrCDE showed no defect in colonization. An S. aureus wall teichoic acid (WTA) mutant (ΔtagO) failed to colonize the mouse nose or GI tract, and the tagO and clfA mutants showed reduced adherence in vitro to intestinal epithelial cells. The tagO mutant was recovered in lower numbers than the wild type strain in the murine stomach and duodenum 1 h after inoculation. This reduced fitness correlated with the in vitro susceptibility of the tagO mutant to bile salts, proteases, and a gut-associated defensin. Newman ΔtagO showed enhanced susceptibility to autolysis, and an autolysin (atl) tagO double mutant abrogated this phenotype. However, the atl tagO mutant did not survive better in the mouse GI tract than the tagO mutant. Our results indicate that the failure of the tagO mutant to colonize the GI tract correlates with its poor adherence and susceptibility to bactericidal factors within the mouse gut, but not to enhanced activity of its major autolysin.
Asunto(s)
Antibacterianos/farmacología , Cápsulas Bacterianas/metabolismo , Enfermedades Gastrointestinales/microbiología , Proteínas de la Membrana/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Animales , Antibacterianos/metabolismo , Adhesión Bacteriana/efectos de los fármacos , Pared Celular/metabolismo , Enfermedades Gastrointestinales/metabolismo , Humanos , Ratones , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Staphylococcus aureus/genética , Ácidos Teicoicos/metabolismoRESUMEN
Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.
Asunto(s)
Ceramidas/metabolismo , Toxina del Cólera/metabolismo , Toxina del Cólera/farmacología , Gangliósido G(M1)/metabolismo , Transcitosis/efectos de los fármacos , Animales , Ceramidas/genética , Perros , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Endosomas/genética , Endosomas/metabolismo , Gangliósido G(M1)/genética , Células de Riñón Canino Madin DarbyRESUMEN
Sphingomyelinases (SMases) hydrolyze membrane sphingomyelin to ceramide and are expressed by diverse host and microbial cell types populating mucosal surfaces. Exogenous bacterial SMase acts on the basolateral membrane of polarized human intestinal epithelial cells to repress the cAMP-induced Cl(-) secretory response, but how this occurs is unknown. We show here that SMase acts by down-regulating a cAMP-gated basolateral membrane K(+) conductance. Neither phosphocholine, ceramide-1-phosphate, nor sphingosine-1-phosphate recapitulates this effect, indicating that ceramide production is the decisive factor. Basolaterally applied SMase induced the phosphorylation of c-Jun NH(2)-terminal kinase (JNK), and inhibition of JNK rescued the effect of SMase on cAMP-dependant secretion. SMase secreted by normal human fibroblasts specifically recapitulated the effect on cAMP-induced Cl(-) secretion, indicating that cell types inhabiting the subepithelial space can provide such an activity to the basolateral membrane of intestinal enterocytes in trans. Thus, conversion of sphingomyelin to ceramide in basolateral membranes of intestinal cells rapidly activates JNK to inhibit a cAMP-gated K(+) conductance and thereby attenuates Cl(-) secretion. These results define a novel lipid-mediated pathway for regulation of salt and water homeostasis at mucosal surfaces.
Asunto(s)
Cloruros/metabolismo , AMP Cíclico/metabolismo , Mucosa Intestinal/fisiología , MAP Quinasa Quinasa 4/metabolismo , Potasio/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Células Cultivadas , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Canales de Potasio con Entrada de Voltaje/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cholera toxin (CT) enters host cells by binding to ganglioside GM1 in the apical plasma membrane (PM). GM1 carries CT retrograde from the PM to the endoplasmic reticulum (ER), where a portion of the toxin, the A1-chain, retro-translocates to the cytosol, causing disease. Trafficking in this pathway appears to depend on the association of CT-GM1 complexes with sphingomyelin (SM)- and cholesterol-rich membrane microdomains termed lipid rafts. Here, we find that in polarized intestinal epithelia, the conversion of apical membrane SM to ceramide by bacterial sphingomyelinase attenuates CT toxicity, consistent with the lipid raft hypothesis. The effect is reversible, specific to toxin entry via the apical membrane, and recapitulated by the addition of exogenous long-chain ceramides. Conversion of apical membrane SM to ceramide inhibits the efficiency of toxin endocytosis, but retrograde trafficking from the apical PM to the Golgi and ER is not affected. This result suggests that the cause for toxin resistance occurs at steps required for retro-translocation of the CT A1-chain to the cytosol.
Asunto(s)
Membrana Celular/química , Ceramidas/metabolismo , Toxina del Cólera/toxicidad , Células Epiteliales/efectos de los fármacos , Esfingomielinas/metabolismo , Línea Celular , Humanos , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM(1). The homopentomeric B-subunit of the toxin can bind up to five GM(1) molecules at once. Here, we examined the role of polyvalent binding of GM(1) in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM(1). The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM(1) when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM(1) molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM(1) molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.
Asunto(s)
Toxina del Cólera/genética , Toxina del Cólera/metabolismo , Endocitosis/fisiología , Gangliósido G(M1)/metabolismo , Línea Celular , Gangliósido G(M1)/química , Humanos , Microdominios de Membrana/metabolismo , Mutación , Unión Proteica , Subunidades de Proteína , Transporte de ProteínasRESUMEN
Cholera toxin (CT), and members of the AB(5) family of toxins enter host cells and hijack the cell's endogenous pathways to induce toxicity. CT binds to a lipid receptor on the plasma membrane (PM), ganglioside GM1, which has the ability to associate with lipid rafts. The toxin can then enter the cell by various modes of receptor-mediated endocytosis and traffic in a retrograde manner from the PM to the Golgi and the endoplasmic reticulum (ER). Once in the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol so as to induce disease. GM1 is the vehicle that carries CT from PM to ER. Thus, the toxin pathway from PM to ER is a lipid-based sorting pathway, which is potentially meditated by the determinants of the GM1 ganglioside structure itself.
Asunto(s)
Membrana Celular/metabolismo , Toxina del Cólera/metabolismo , Endocitosis/fisiología , Retículo Endoplásmico/metabolismo , Animales , Humanos , Modelos Biológicos , Transporte de Proteínas/fisiologíaRESUMEN
Cholera and the related AB(5)-subunit toxins co-opt plasma membrane (PM) glycolipids to move retrograde into the endoplasmic reticulum (ER) of the host cell where a portion of the toxin is retro-translocated to the cytosol to induce disease. Only glycolipids that associate strongly with detergent insoluble membrane microdomains can sort the toxins backwards from PM to ER. The way certain lipids and proteins are clustered in the plane of the membrane to form lipid rafts likely explains how the glycolipids can function as sorting motifs for the toxins.
Asunto(s)
Toxinas Bacterianas/metabolismo , Endocitosis/fisiología , Microdominios de Membrana/metabolismo , Animales , Membrana Celular/metabolismo , Ceramidas/metabolismo , Toxina del Cólera/metabolismo , Retículo Endoplásmico/metabolismo , Glucolípidos/metabolismo , Aparato de Golgi/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Poliomavirus/metabolismo , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Virus 40 de los Simios/metabolismoRESUMEN
The apical brush border membrane (BBM) of intestinal epithelial cells forms a highly structured and dynamic environmental interface that serves to regulate cellular physiology and block invasion by intestinal microbes and their products. How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function. We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin. Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network. Accumulation of apical membrane ceramide is necessary and sufficient to induce the actin phenotype, and this coincides with altered membrane structure and augmented innate immune function as evidenced by resistance to invasion by Salmonella.
Asunto(s)
Citoesqueleto de Actina/microbiología , Mucosa Intestinal/microbiología , Microvellosidades/microbiología , Esfingomielina Fosfodiesterasa/metabolismo , Proteína de Unión al GTP rhoA/inmunología , Citoesqueleto de Actina/inmunología , Actinas , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Ceramidas/biosíntesis , Humanos , Inmunidad Innata , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Microvellosidades/inmunología , Microvellosidades/metabolismo , Salmonella typhimurium , Staphylococcus aureus/enzimologíaRESUMEN
Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl(-)/HCO(-) 3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.
RESUMEN
GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Despite the importance of GPR56 in brain development, where mutations cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), the signaling mechanism(s) remain largely unknown. Like many other adhesion GPCRs, GPR56 is cleaved via a GPCR autoproteolysis-inducing (GAIN) domain into N- and C-terminal fragments (GPR56N and GPR56C); however, the biological significance of this cleavage is elusive. Taking advantage of the recent identification of a GPR56 ligand and the presence of BFPP-associated mutations, we investigated the molecular mechanism of GPR56 signaling. We demonstrate that ligand binding releases GPR56N from the membrane-bound GPR56C and triggers the association of GPR56C with lipid rafts and RhoA activation. Furthermore, one of the BFPP-associated mutations, L640R, does not affect collagen III-induced lipid raft association of GPR56. Instead, it specifically abolishes collagen III-mediated RhoA activation. Together, these findings reveal a novel signaling mechanism that may apply to other members of the adhesion GPCR family.
Asunto(s)
Colágeno Tipo III/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Activación Enzimática/efectos de los fármacos , Evolución Molecular , Células HEK293 , Humanos , Ligandos , Malformaciones del Desarrollo Cortical/genética , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells. Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid sorting by ceramide structure and provide a molecular explanation for the diversity and specificity of retrograde trafficking by CT in host cells.
Asunto(s)
Membrana Celular/química , Ceramidas/química , Toxina del Cólera/química , Retículo Endoplásmico/química , Gangliósido G(M1)/química , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Ceramidas/metabolismo , Toxina del Cólera/metabolismo , Retículo Endoplásmico/metabolismo , Gangliósido G(M1)/síntesis química , Gangliósido G(M1)/metabolismo , Humanos , Isoformas de Proteínas/síntesis química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Vibrio cholerae, a Gram-negative facultative pathogen, is the etiologic agent for the diarrheal disease cholera. We previously characterized a clinical isolate, AM-19226, that translocates a type III secretion system (T3SS) effector protein with actin-nucleating activity, VopF, into the host cells. From comparative genomic studies, we identified a divergent T3SS island in additional isolates which possess a VopF homolog, VopN. Unlike the VopF-mediated protrusion formation, VopN localizes to stress fiber in host cells similarly to VopL, which is present in the pandemic strain of Vibrio parahaemolyticus. Chimera and yeast two-hybrid studies indicated that the amino-terminal regions of VopF and VopN proteins interact with distinct host cell factors. We determined that AM-19226-infected cells are arrested at S phase of the cell cycle and that VopF/VopN are antiapoptotic factors. To understand how VopF may contribute to the pathogenesis of AM-19226, we examined the effect of VopF in an in vitro polarized-epithelial model and an in vivo adult rabbit diarrheal model. Within the T3SS pathogenicity island is VopE, a homolog of YopE from Yersinia, which has been shown to loosen tight junctions. In polarized intestinal epithelia, VopF and VopE compromised the integrity of tight junctions by inducing cortical actin depolymerization and aberrant localization of the tight-junction protein ZO-1. An assay for pathogenicity in the adult rabbit diarrhea model suggested that these effectors are involved in eliciting the diarrheal response in infected rabbits.
Asunto(s)
Proteínas Bacterianas/metabolismo , Vibrio cholerae/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular , Diarrea/microbiología , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/microbiología , Unión Proteica , Mapeo de Interacción de Proteínas , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uniones Estrechas/fisiología , Técnicas del Sistema de Dos Híbridos , Factores de Virulencia/genéticaRESUMEN
Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro- translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endo-somal sorting of the toxin-GM1 complex.
Asunto(s)
Toxina del Cólera/toxicidad , Proteínas de la Membrana/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Secuencia de Bases , Transporte Biológico Activo , Células COS , Línea Celular , Chlorocebus aethiops , Toxina del Cólera/farmacocinética , Endosomas/metabolismo , Gangliósido G(M1)/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , ARN Interferente Pequeño/genética , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Flavonoids represent one of the oldest, largest, and most diverse families of plant secondary metabolites. These compounds serve a wide range of functions in plants, from pigmentation and UV protection to the regulation of hormone transport. Flavonoids also have interesting pharmacological activities in animals that are increasingly being characterized in terms of effects on specific proteins or other macromolecules. Although flavonoids are found in many different locations both inside and outside the cell, biosynthesis has long been believed to take place exclusively in the cytoplasm. Recent reports from a number of different plant species have documented the presence of flavonoids in nuclei, raising the possibility of novel mechanisms of action for these compounds. Here we present evidence that not only flavonoids, but also at least two of the biosynthetic enzymes, are located in the nucleus in several cell types in Arabidopsis. This is the first indication that differential targeting of the biosynthetic machinery may be used to regulate the deposition of plant secondary products at diverse sites of action within the cell.
Asunto(s)
Arabidopsis/enzimología , Núcleo Celular/enzimología , Flavonoides/biosíntesis , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Inmunoelectrónica , Modelos Moleculares , Raíces de Plantas/enzimología , Plantas Modificadas Genéticamente/enzimología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In the late 1990s, accumulated evidence led to the proposal that biological membranes are composed of microdomains of different lipids, which form functional "rafts." Recent work using atomic force microscopy has given us new insights into the factors influencing the formation and behavior of these physiological microenvironments
Asunto(s)
Microdominios de Membrana/fisiología , Microscopía de Fuerza Atómica/métodos , Microscopía de Fuerza Atómica/tendencias , Animales , HumanosRESUMEN
Cholesterol plays a crucial role in cell membranes, and has been implicated in the assembly and maintenance of sphingolipid-rich rafts. We have examined the cholesterol-dependence of model rafts (sphingomyelin-rich domains) in supported lipid monolayers and bilayers using atomic force microscopy. Sphingomyelin-rich domains were observed in lipid monolayers in the absence and presence of cholesterol, except at high cholesterol concentrations, when separate domains were suppressed. The effect of manipulating cholesterol levels on the behavior of these sphingomyelin-rich domains in bilayers was observed in real time. Depletion of cholesterol resulted in dissolution of the model lipid rafts, whereas cholesterol addition resulted in an increased size of the sphingomyelin-rich domains and eventually the formation of a single raftlike lipid phase. Cholesterol colocalization with sphingomyelin-rich domains was confirmed using the sterol binding agent filipin.
Asunto(s)
Colesterol/química , Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Microdominios de Membrana/química , Microscopía de Fuerza Atómica/métodos , Fosfatidilcolinas/química , Esfingomielinas/química , Filipina/química , Sustancias Macromoleculares , Conformación Molecular , Factores de TiempoRESUMEN
Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.