RESUMEN
Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl(-) secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl(-) secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl(-) secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl(-) secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E. coli.
Asunto(s)
Toxinas Bacterianas/metabolismo , Polaridad Celular/fisiología , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Gangliósidos/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Superficie Celular/metabolismo , Línea Celular , Cloruros/metabolismo , Escherichia coli/patogenicidad , Gangliósido G(M1)/metabolismo , Gangliósidos/antagonistas & inhibidores , Humanos , Mucosa Intestinal/citología , Neuraminidasa/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Transducción de Señal , Toxina Tetánica/farmacologíaRESUMEN
Cholera toxin (CT) is the prototype for the Vibrio cholerae-Escherichia coli family of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68+V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10- and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12- to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.
Asunto(s)
Toxina del Cólera/genética , Toxina del Cólera/toxicidad , Proteínas de Escherichia coli , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/inmunología , Factores de Ribosilacion-ADP/toxicidad , Secuencia de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Sitios de Unión , Toxina del Cólera/inmunología , Secuencia Conservada , Enterotoxinas/genética , Enterotoxinas/toxicidad , Estabilidad de Enzimas , Epítopos , Variación Genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
The latent ADP-ribosyltransferase activity of cholera toxin (CT) that is activated after proteolytic nicking and reduction is associated with the CT A1 subunit (CTA1) polypeptide. This activity is stimulated in vitro by interaction with eukaryotic proteins termed ADP-ribosylation factors (ARFs). We analyzed this interaction in a modified bacterial two-hybrid system in which the T18 and T25 fragments of the catalytic domain of Bordetella pertussis adenylate cyclase were fused to CTA1 and human ARF6 polypeptides, respectively. Direct interaction between the CTA1 and ARF6 domains in these hybrid proteins reconstituted the adenylate cyclase activity and permitted cAMP-dependent signal transduction in an Escherichia coli reporter system. We constructed improved vectors and reporter strains for this system, and we isolated variants of CTA1 that showed greatly decreased ability to interact with ARF6. Amino acid substitutions in these CTA1 variants were widely separated in the primary sequence but were contiguous in the three-dimensional structure of CT. These residues, which begin to define the ARF interaction motif of CTA1, are partially buried in the crystal structure of CT holotoxin, suggesting that a change in the conformation of CTA1 enables it to bind to ARF. Variant CTA polypeptides containing these substitutions assembled into holotoxin as well as wild-type CTA, but the variant holotoxins showed greatly reduced enterotoxicity. These findings suggest functional interaction between CTA1 and ARF is required for maximal toxicity of CT in vivo.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Toxina del Cólera/metabolismo , Péptidos/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/genética , Adenilil Ciclasas/genética , Secuencias de Aminoácidos , Bordetella pertussis/enzimología , Bordetella pertussis/genética , Toxina del Cólera/química , Toxina del Cólera/genética , Expresión Génica , Prueba de Complementación Genética , Ingeniería Genética , Humanos , Modelos Moleculares , Péptidos/química , Péptidos/genética , Plásmidos , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.
Asunto(s)
Toxinas Bacterianas/farmacocinética , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Toxina del Cólera/farmacocinética , Enterotoxinas/farmacocinética , Células Epiteliales/fisiología , Proteínas de Escherichia coli , Gangliósido G(M1)/fisiología , Gangliósidos/fisiología , Mucosa Intestinal/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Glándulas Suprarrenales , Animales , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Polaridad Celular , Cloruros/metabolismo , Toxina del Cólera/biosíntesis , Toxina del Cólera/farmacología , Clonación Molecular , AMP Cíclico/metabolismo , Enterotoxinas/biosíntesis , Enterotoxinas/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Escherichia coli , Gangliósido G(M1)/química , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/ultraestructura , Cinética , Lípidos de la Membrana/fisiología , Ratones , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
One strategy for the induction of mucosal immune responses by oral immunization is to administer the antigen in conjunction with cholera toxin. Cholera toxin consists of one A polypeptide (CTA) which is noncovalently linked to five B subunits (CTB) via the A2 portion of the A subunit (CTA2). Coupling of antigens to the nontoxic B subunit of cholera toxin may improve the immunogenicity of antigens by targeting them to GM1 ganglioside on M cells and intestinal epithelial cells. Here, we describe the construction of a translational fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective amebic antigen, fused to a maltose binding protein (MBP) and to CTA2. When coexpressed in Escherichia coli with the CTB gene, these proteins assembled into a holotoxin-like chimera containing MBP-SREHP-CTA2 and CTB. This holotoxin-like chimera (SREHP-H) inhibited the binding of cholera toxin to GM1 ganglioside. Oral vaccination of mice with SREHP-H induced mucosal immunoglobulin A (IgA) and serum IgG antiamebic antibodies and low levels of mucosal anti-CTB antibodies. Our studies confirm that the genetic coupling of antigens to CTA2 and their coexpression in E. coli can produce holotoxin-like molecules that are mucosally immunogenic without the requirement for supplemental cholera toxin, and they establish the SREHP-H protein as a candidate for evaluation as a vaccine to prevent amebiasis.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Toxina del Cólera/inmunología , Entamoeba histolytica/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Femenino , Inmunidad Mucosa , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB CRESUMEN
The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.
Asunto(s)
Toxinas Bacterianas/química , Toxina del Cólera/química , Toxina del Cólera/genética , Enterotoxinas/química , Proteínas de Escherichia coli , Gangliósido G(M1)/química , Mutación , Receptores de Superficie Celular/química , Animales , Carbohidratos/química , Toxina del Cólera/metabolismo , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/patogenicidad , Gangliósido G(M1)/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Receptores de Superficie Celular/metabolismo , Propiedades de Superficie , PorcinosRESUMEN
Cholera and Escherichia coli heat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed "nicking"). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl- secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 microA/cm2). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the "basolateral" secretory responses to "apical" levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.
Asunto(s)
Toxinas Bacterianas/metabolismo , Toxina del Cólera/metabolismo , Endopeptidasas/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Transducción de Señal , Toxinas Bacterianas/genética , Transporte Biológico , Línea Celular , Toxina del Cólera/genética , Endosomas/metabolismo , Enterotoxinas/genética , Epitelio/metabolismo , Humanos , Hidrólisis , Receptores de Superficie Celular/metabolismoRESUMEN
Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones in Escherichia coli inhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic for E. coli, but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit of E. coli heat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield in E. coli. Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm of E. coli. We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, using phoA from E. coli, we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using the pspA gene that encodes pneumococcal surface protein A from Streptococcus pneumoniae, we produced a 299-residue amino-terminal fragment of PspA in E. coli in large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure-function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm in E. coli.
Asunto(s)
Fosfatasa Alcalina/biosíntesis , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Enterotoxinas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Vectores Genéticos , Señales de Clasificación de Proteína/genética , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Secuencia de Bases , Toxina del Cólera/biosíntesis , Toxina del Cólera/genética , Clonación Molecular , Citoplasma , ADN Bacteriano , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Streptococcus pneumoniae/genéticaRESUMEN
The Vibrio cholerae HA/protease gene (hap) promoter is inactive in Escherichia coli. We cloned and sequenced the 0.7kb hap promoter fragment from strain 3083-2 and showed that hap is located immediately 3' of ompW, encoding a minor outer membrane protein. A clone from a genomic library of strain 3083-2 was isolated, which was required for activation of the hap promoter in E. coli. Expression from the hap promoter only occurred late in the growth phase. A single complete open reading frame (ORF) designated HapR was identified on a 1.7 kb DNA fragment that was required for activation. Allelic replacements showed that hapR was also essential for hap expression in V. cholerae. In El Tor, but not in classical biotypes of V. cholerae, hapR mutations also produced a rugose colonial phenotype. HapR was shown to encode a 203-amino-acid polypeptide with 71% identity to LuxR of V. harveyi, an essential positive regulator of the lux operon that has no previously identified homologues. The amino-terminal domain (residues 21-68) showed significant homology to the TetR family of helix-turn-helix DNA-binding domains and was 95% identical to the same domain of LuxR. HapR and LuxR activated both the hap and the lux promoters at near wild-type levels, despite only limited homology in the promoter sequences (46% identity with 12 gaps over 420bp). DNA sequences and ORFs 5' (but not 3') of the hapR and luxR loci were homologous, suggesting a common origin for these loci, and hapR-hybridizing sequences were found in other vibrios. We conclude that HapR is absolutely required for hap expression and that HapR and LuxR form a new family of transcriptional activator proteins.
Asunto(s)
Genes Bacterianos , Genes Reguladores , Metaloendopeptidasas/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Transactivadores/genética , Vibrio cholerae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Escherichia coli/metabolismo , Genes Reporteros , Genoma Bacteriano , Operón Lac , Metaloendopeptidasas/biosíntesis , Datos de Secuencia Molecular , Mutagénesis , Operón , Plásmidos , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Vibrionaceae/genéticaRESUMEN
Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin-containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GM1 at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced Cl- secretion. At T1/2, dose dependencies for K(R)DEL-mutant toxins were increased > or = 10-fold. KDEL-mutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction. KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.
Asunto(s)
Toxinas Bacterianas/metabolismo , Toxina del Cólera/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/química , Oligopéptidos/fisiología , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Toxinas Bacterianas/análisis , Toxinas Bacterianas/genética , Secuencia de Bases , Compartimento Celular/fisiología , Línea Celular/metabolismo , Polaridad Celular/fisiología , Toxina del Cólera/análisis , Toxina del Cólera/genética , Endocitosis/fisiología , Enterotoxinas/análisis , Enterotoxinas/genética , Células Epiteliales , Epitelio/enzimología , Humanos , Datos de Secuencia Molecular , Mutación/fisiología , Oligopéptidos/química , Oligopéptidos/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica/fisiología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Factores de TiempoRESUMEN
The specificity of the pathway used by Vibrio cholerae for extracellular transport of cholera toxin (CT) and other proteins was examined in several different ways. First, V. cholerae was tested for the ability to secrete the B polypeptides of the type II heat-labile enterotoxins of Escherichia coli. Genes encoding the B polypeptide of LT-IIb in pBluescriptKS- phagemids were introduced into V. cholerae by electroporation. Culture supernatants and periplasmic extracts were collected from cultures of the V. cholerae transformants, and the enterotoxin B subunits were measured by an enzyme-linked immunosorbent assay. Results confirmed that the B polypeptides of both LT-IIa and LT-IIb were secreted by V. cholerae with efficiencies comparable to that measured for secretion of CT. Second, the plasmid clones were introduced into strain M14, an epsE mutant of V. cholerae. M14 failed to transport the B polypeptides of LT-IIa and LT-IIb to the extracellular medium, demonstrating that secretion of type II enterotoxins by V. cholerae proceeds by the same pathway used for extracellular transport of CT. These data suggest that an extracellular transport signal recognized by the secretory machinery of V. cholerae is present in LT-IIa and LT-IIb. Furthermore, since the B polypeptide of CT has little, if any, primary amino acid sequence homology with the B polypeptide of LT-IIa or LT-IIb, the transport signal is likely to be a conformation-dependent motif. Third, a mutant of the B subunit of CT (CT-B) with lysine substituted for glutamate at amino acid position 11 was shown to be secreted poorly by V. cholerae, although it exhibited immunoreactivity and ganglioside GM1-binding activity comparable to that of wild-type CT-B. These findings suggest that Glu-11 may be within or near the extracellular transport motif of CT-B. Finally, the genetic lesion in the epsE allele of V. cholerae M14 was determined by nucleotide sequence analysis.
Asunto(s)
Toxina del Cólera/farmacocinética , Proteínas de Escherichia coli , Proteínas de la Membrana , Vibrio cholerae/metabolismo , Alelos , Proteínas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Transporte Biológico , Toxina del Cólera/química , Enterotoxinas/metabolismo , Datos de Secuencia Molecular , Mutación , Relación Estructura-Actividad , Vibrio cholerae/genéticaRESUMEN
BACKGROUND: Because agents which inhibit the receptor binding of cholera toxin constitute possible lead compounds for the structure-based design of anti-cholera drugs, detailed investigation of the toxin's receptor-binding site is of key importance. The substitution Gly-->Asp at residue 33 of the cholera toxin B subunit (CTB) has been reported to abolish receptor-binding ability. The substitution Arg35-->Asp has been reported to result in deficient assembly of the AB5 holotoxin. The molecular basis for these effects was not readily apparent from analysis of an earlier crystal structure of the wild-type toxin B pentamer in a complex with the receptor pentasaccharide. RESULTS: We now report at a resolution of 2.0 A the crystal structure of a recombinant CTB pentamer containing the Gly33-->Asp substitution. The observed conformation of the Asp33 side chain suggests that the loss in binding affinity is due to a steric clash with atoms C9 and O9 of the sialic acid moiety of the receptor, ganglioside GM1. The crystal structure also reveals an unexpected mode of pentamer-pentamer interaction in which pairs of toxin pentamers are joined by reciprocal insertion of the imidazole ring of His13 from one subunit of each pentamer into one of the receptor-binding sites on the other. The surface of interaction at each pentamer-pentamer interface is on the order of 500 A2, and primarily involves contact of residues 10-14 with the receptor-binding site on the associated pentamer. This same pentamer-pentamer interaction is also present in the crystal structure of a second recombinant CTB containing an Arg-->Asp substitution at residue 35, which we have determined at 2.1 A resolution. CONCLUSIONS: These structures suggest that analogs to all or part of the pentapeptide Ala-Glu-Tyr-His-Asn, corresponding to residues 10-14 of CTB, may constitute lead compounds for the design of binding-site inhibitors.
Asunto(s)
Gangliósido G(M1)/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Arginina/genética , Arginina/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sitios de Unión , Unión Competitiva , Toxina del Cólera/genética , Cristalografía por Rayos X , Gangliósido G(M1)/antagonistas & inhibidores , Gangliósido G(M1)/química , Glicina/genética , Glicina/metabolismo , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaAsunto(s)
Secuencia de Aminoácidos , Toxinas Bacterianas/genética , Enterotoxinas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Vibrio cholerae/genética , Toxinas Bacterianas/química , Bases de Datos Factuales , Enterotoxinas/química , Escherichia coli/química , Genes Bacterianos/genética , Datos de Secuencia Molecular , Vibrio cholerae/químicaRESUMEN
The transpositional phage MudI 1734 lacZ was used to construct transcriptional fusions within the plasmid pMJ611, which contains the cloned tellurite resistance (TeR) determinant of the IncHI-2 plasmid pMER610. A series of 70 MudI insertions, in both orientations, causing loss of tellurite resistance in pMJ611, mapped within a 4.3 kb region which included the genes terA-terD and a 0.4 kb region upstream of the site previously reported as the 5' limit of the TeR determinant. Expression of beta-galactosidase from these transcriptional fusions, including those involving the 5' upstream region, occurred only from inserts transcribed in the direction terA-terD, confirming the transcriptional orientation of the TeR determinant deduced from DNA sequence analysis. Sixteen of the tellurite-sensitive MudI fusions, distributed over the entire determinant and in both orientations, showed the same pattern of expression when transferred by conjugation and homologous recombination to pMER610, except that the beta-galactosidase levels were consistently 2- to 3-fold higher in the parent plasmid. Northern analysis with a DNA probe spanning the TeR determinant identified five transcripts of 4.8, 4.0, 2.7, 1.5 and 1.0 kb synthesised by pMER610. Further hybridisations with DNA probes defining sub-sections of the TeR determinant, together with DNA sequence analysis, suggested the presence of three transcriptional start sites, at approximately 0.9 and 0.1 kb upstream of terA, and near the junction between terC and terD. Three transcriptional termination sites, located within terA, near the terC-terD junction and at the 3' end of terE are also indicated. Both the expression of beta-galactosidase from the MudI fusions and the synthesis of ter gene transcripts are constitutive and were not affected by prior exposure of cultures to sub-toxic levels of tellurite. Further DNA sequence analysis reveals that the extensive homology between terD and terE extends to a section of terA.
Asunto(s)
Plásmidos , Telurio/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Elementos Transponibles de ADN , ADN Bacteriano , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , beta-Galactosidasa/genéticaRESUMEN
Cholera enterotoxin (CT) is produced by Vibrio cholerae and excreted into the culture medium as an extracellular protein. CT consists of one A polypeptide and five B polypeptides associated by noncovalent bonds, and CT-B interacts with CT-A primarily via the A2 domain. Treatment of CT with trypsin cleaves CT-A into A1 and A2 fragments that are linked by a disulfide bond. CT-B binds to ganglioside GM1, which functions as the plasma membrane receptor for CT, and the enzymatic activity of A1 causes the toxic effects of CT on target cells. We constructed translational fusions that joined foreign proteins via their carboxyl termini to the A2 domain of CT-A, and we studied the interactions of the fusion proteins with CT-B. The A2 domain was necessary and sufficient to enable bacterial alkaline phosphatase (BAP), maltose-binding protein (MBP) or beta-lactamase (BLA) to associate with CT-B to form stable, immunoreactive, holotoxin-like chimeras. Each holotoxin-like chimera was able to bind to ganglioside GM1. Holotoxin-like chimeras containing the BAP-A2 and BLA-A2 fusion proteins had BAP activity and BLA activity, respectively. We constructed BAP-A2 mutants with altered carboxyl-terminal sequences and tested their ability to assemble into holotoxin-like chimeras. Although the carboxyl-terminal QDEL sequence of the BAP-A2 fusion protein was not required for interaction with CT-B, most BAP-A2 mutants with altered carboxyl termini did not form holotoxin-like chimeras. When holotoxin-like chimeras containing BAP-A2, MBP-A2, or BLA-A2 were synthesized in V. cholerae, they were found predominantly in the periplasm. The toxin secretory apparatus of V. cholerae was not able, therefore, to translocate these holotoxin-like chimeras across the outer membrane.
Asunto(s)
Toxina del Cólera/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Toxina del Cólera/inmunología , Toxina del Cólera/metabolismo , Clonación Molecular , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutación , Oligonucleótidos/química , Proteínas Recombinantes de Fusión/inmunología , Relación Estructura-ActividadRESUMEN
Although the physiologic function of gangliosides is unknown, evidence suggests they play a role in the regulation of cell growth. The binding of ganglioside GM1 by recombinant B subunit of cholera toxin (rCT-B) inhibited mitogen-stimulated B cell proliferation without elevating intracellular cAMP. CT-B paradoxically enhanced the expression of MHC class II (Ia) molecules and minor lymphocyte-stimulating determinants without altering the expression of some other immunologically relevant B cell surface Ag. Increased expression of Ia was not detected until 4 h after stimulation, kinetics similar to those seen when B cells are stimulated with anti-Ig antibody or IL-4, suggesting that the enhancement was not the result of redistribution of existing cell surface markers but rather the result of a new metabolic event. Both the inhibitory and stimulatory effects of CT-B could be blocked by incubation of CT-B with ganglioside GM1. Furthermore, enhancement of the CT-B-mediated effect was seen when additional ganglioside GM1 was incorporated into the B cell membrane. rCT-B with a mutation that interfered with its binding to ganglioside GM1 did not enhance Ia expression. Taken together, these results indicate that the observed effects of CT-B were most likely mediated through the binding of cell surface ganglioside GM1. CT-B-mediated stimulation of Ia expression provides a potential explanation for the previously described ability of CT-B to act as an immunoadjuvant. These results suggest that the binding of ganglioside GM1 has multiple B cell growth-regulating effects.
Asunto(s)
Linfocitos B/efectos de los fármacos , Toxina del Cólera/farmacología , AMP Cíclico/fisiología , Gangliósido G(M1)/metabolismo , Activación de Linfocitos/efectos de los fármacos , Animales , Linfocitos B/inmunología , Femenino , Gangliósido G(M1)/farmacología , Antígenos de Histocompatibilidad Clase II/análisis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Antígenos Estimulantes de Linfocito Menor/inmunologíaRESUMEN
Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.
Asunto(s)
Enterotoxinas/química , Gangliósidos/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , Análisis Mutacional de ADN , Enterotoxinas/genética , Expresión Génica , Hemólisis/inmunología , Inmunoensayo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Biosíntesis de Proteínas , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.