RESUMEN
Cholera continues to be an important public health concern in developing countries where proper hygiene and sanitation are compromised. This severe diarrheal disease is caused by the Gram-negative pathogen Vibrio cholerae belonging to serogroups O1 and O139. Cholera toxin (CT) is the prime virulence factor and is directly responsible for the disease manifestation. The ctxB gene encodes cholera toxin B subunit (CTB) whereas the A subunit (CTA) is the product of ctxA gene. Enzymatic action of CT depends on binding of B pentamers to the lipid-based receptor ganglioside GM1. In recent years, emergence of V. cholerae Haitian variant strains with ctxB7 allele and their rapid spread throughout the globe has been linked to various cholera outbreaks in Africa and Asia. These strains produce classical type (WT) CTB except for an additional mutation in the signal sequence region where an asparagine (N) residue replaces a histidine (H) at the 20th amino acid position (H20N) of CTB precursor (pre-CTB). Here we report that Haitian variant V. cholerae O1 strains isolated in Kolkata produced higher amount of CT compared to contemporary O1 El Tor variant strains under in vitro virulence inducing conditions. We observed that the ctxB7 allele, itself plays a pivotal role in higher CT production. Based on our in silico analysis, we hypothesized that higher accumulation of toxin subunits from ctxB7 allele might be attributed to the structural alteration at the CTB signal peptide region of pre-H20N CTB. Overall, this study provides plausible explanation regarding the hypertoxigenic phenotype of the Haitian variant strains which have spread globally, possibly through positive selection for increased pathogenic traits.
Asunto(s)
Alelos , Toxina del Cólera/genética , Cólera/microbiología , Genes Bacterianos/genética , Vibrio cholerae O1/genética , Técnicas de Tipificación Bacteriana , Cólera/epidemiología , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Brotes de Enfermedades , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Haití/epidemiología , Humanos , ARN Bacteriano , Serogrupo , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: The antimicrobial susceptibility patterns and genetic characteristics of Vibrio cholerae O1, which is responsible for several cholera epidemics in Nigeria, are not reported in detail since 2007. In this study, we screened V. cholerae O1 El Tor biotype isolates from cholera cases and water samples from different states to investigate their phenotypic and genetic attributes with special reference to their clonality. RESULTS: All the V. cholerae O1 biotype El Tor isolates isolated during 2007-2013 were susceptible to fluoroquinolones and tetracycline, the drugs currently used in the treatment of cholera cases in Nigeria. Emergence of CT genotype 7 (Haitian type of ctxB allele) was predominantly seen among Ogawa serotype and the CT genotype 1 (classical ctxB allele) was mostly found in Inaba serotype. Overall, V. cholerae O1 from clinical and water samples were found to be closely related as determined by the pulsed-field gel electrophoresis. V. cholerae isolates from Abia, Kano and Bauchi were found to be genetically distinct from the other states of Nigeria. CONCLUSION: Fecal contamination of the water sources may be the possible source of the cholera infection. Combined prevalence of Haitian and classical ctxB alleles were detected in Ogawa and Inaba serotypes, respectively. This study further demonstrated that V. cholerae O1 with the ctxB has been emerged similar to the isolates reported in Haiti. Our findings suggest that the use of fluoroquinolones or tetracycline/doxycycline may help in the effective management of acute cholera in the affected Nigerian states. In addition, strengthening the existing surveillance in the hospitals of all the states and supply of clean drinking water may control cholera outbreaks in the future.
Asunto(s)
Toxina del Cólera/genética , Cólera/diagnóstico , Vibrio cholerae O1/genética , Alelos , Antibacterianos/farmacología , Cólera/epidemiología , Cólera/microbiología , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Estudios Transversales , Electroforesis en Gel de Campo Pulsado , Fluoroquinolonas/farmacología , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Nigeria/epidemiología , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Serogrupo , Tetraciclina/farmacología , Vibrio cholerae O1/efectos de los fármacos , Vibrio cholerae O1/aislamiento & purificación , Virulencia/genéticaRESUMEN
Retinal ischemic injury is an important cause of visual impairment. The loss of retinal ganglion cells (RGCs) is a key sign of retinal ischemic damage. A subset of RGCs expressing the photopigment melanopsin (mRGCs) regulates non-image-forming visual functions such as the pupillary light reflex (PLR), and circadian rhythms. We studied the effect of retinal ischemia on mRGCs and the non-image-forming visual system function. For this purpose, transient ischemia was induced by raising intraocular pressure to 120 mm Hg for 40 min followed by retinal reperfusion by restoring normal pressure. At 4 weeks post-treatment, animals were subjected to electroretinography and histological analysis. Ischemia induced a significant retinal dysfunction and histological alterations. At this time point, a significant decrease in the number of Brn3a(+) RGCs and in the anterograde transport from the retina to the superior colliculus and lateral geniculate nucleus was observed, whereas no differences in the number of mRGCs, melanopsin levels, and retinal projections to the suprachiasmatic nuclei and the olivary pretectal nucleus were detected. At low light intensity, a decrease in pupil constriction was observed in intact eyes contralateral to ischemic eyes, whereas at high light intensity, retinal ischemia did not affect the consensual PLR. Animals with ischemia in both eyes showed a conserved locomotor activity rhythm and a photoentrainment rate which did not differ from control animals. These results suggest that the non-image forming visual system was protected against retinal ischemic damage.
Asunto(s)
Isquemia/patología , Retina/fisiopatología , Animales , Toxina del Cólera/química , Ritmo Circadiano/fisiología , Electrorretinografía , Cuerpos Geniculados/patología , Luz , Masculino , Movimiento , Ratas , Ratas Wistar , Retina/patología , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones/metabolismo , Núcleo Supraquiasmático/metabolismo , Factores de Tiempo , Visión OcularRESUMEN
The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.
Asunto(s)
Toxina del Cólera/química , Toxina del Cólera/metabolismo , Replegamiento Proteico , Vibrio cholerae/genética , Toxina del Cólera/genética , Dicroismo Circular , Escherichia coli/metabolismo , Presión Hidrostática/efectos adversos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vibrio cholerae/metabolismoRESUMEN
Cationic supported bilayers on latex are useful to isolate and immobilize oppositely charged proteins as a monomolecular layer over a range of low protein concentrations and particle number densities. Cholera toxin (CT) from Vibrio cholerae, an 87 kDa AB5 hexameric protein and bovine serum albumin (BSA) self-assembled on dioctadecyldimethylammonium bromide (DODAB) supported bilayers with high affinity yielding highly organized and monodisperse particulates at 5 x 10(9) particles/mL, over a range of low protein concentrations (0-0.025 mg/mL BSA or CT). Protein association onto the bilayer-covered polystyrene sulfate (PSS) was determined from adsorption isotherms, dynamic light scattering for size distributions and zeta-potential analysis revealing a monomolecular, thin and highly organized protein layer surrounding each particle with potential for biospecific recognition such as antigen-antibody, receptor-ligand, hybridization of oligonucleotide sequences, all of them important in immunodiagnosis, selective biomolecular chromatographic separations, microarrays design and others.
Asunto(s)
Cationes , Membrana Dobles de Lípidos , Proteínas/química , Adsorción , Toxina del Cólera/química , Imitación Molecular , Compuestos de Amonio Cuaternario/química , Albúmina Sérica Bovina/química , TermodinámicaRESUMEN
Many notions regarding the function, structure and regulation of cholera toxin expression have remained essentially unaltered in the last 15 years. At the same time, recent findings have generated additional perspectives. For example, the cholera toxin genes are now known to be carried by a non-lytic bacteriophage, a previously unsuspected condition. Understanding of how the expression of cholera toxin genes is controlled by the bacterium at the molecular level has advanced significantly and relationships with cell-density-associated (quorum-sensing) responses have recently been discovered. Regarding the cell intoxication process, the mode of entry and intracellular transport of cholera toxin are becoming clearer. In the immunological field, the strong oral immunogenicity of the non-toxic B subunit of cholera toxin (CTB) has been exploited in the development of a now widely licensed oral cholera vaccine. Additionally, CTB has been shown to induce tolerance against co-administered (linked) foreign antigens in some autoimmune and allergic diseases.
Asunto(s)
Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/farmacología , Toxina del Cólera/genética , Toxina del Cólera/farmacología , Adyuvantes Inmunológicos/química , Enfermedades Autoinmunes/tratamiento farmacológico , Toxinas Bacterianas/farmacología , Cólera/microbiología , Toxina del Cólera/química , Vacunas contra el Cólera , Enterotoxinas/farmacología , Proteínas de Escherichia coli/farmacología , Regulación Bacteriana de la Expresión Génica , Humanos , Hipersensibilidad/tratamiento farmacológico , Inmunidad MucosaRESUMEN
Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB5 hexameric protein, and its receptor, the monosialoganglioside GM1, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM1 molar ratio in perfect agreement with literature. This receptor-ligand recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM(1) showed that GM(1) incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2-0.3 mM PC and then decreased as a function of PC concentration. At 5 microM GM(1), 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m2 silica, which corresponded to the 5 GM(1)/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.
Asunto(s)
Toxina del Cólera/química , Gangliósido G(M1)/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Receptores de Superficie Celular/fisiología , Adsorción , Animales , Biomimética/métodos , Bovinos , Fenómenos Químicos , Química Física , Calor , Control de Infecciones , Liposomas/química , Lípidos de la Membrana , Tamaño de la Partícula , Fosfatidilcolinas/química , Pirenos/química , Receptores de Superficie Celular/aislamiento & purificación , Dióxido de Silicio/química , Vibrio cholerae/enzimologíaRESUMEN
In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Fibrinolisina/fisiología , Proteínas Inmediatas-Precoces/biosíntesis , Sistema de Señalización de MAP Quinasas , Plasminógeno/fisiología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Sitios de Unión , Northern Blotting , Western Blotting , Línea Celular , Toxina del Cólera/química , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Proteína 1 de la Respuesta de Crecimiento Precoz , Activación Enzimática , Flavonoides/farmacología , Genes Dominantes , Humanos , Leupeptinas/química , Lisina/química , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Unión Proteica , ARN/metabolismo , ARN Mensajero/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.
Asunto(s)
Toxina del Cólera/genética , Toxina del Cólera/aislamiento & purificación , Genes Bacterianos/genética , Histidina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Glándulas Suprarrenales/citología , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Línea Celular , Toxina del Cólera/biosíntesis , Toxina del Cólera/química , Codón/genética , ADN Recombinante/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Expresión Génica , Vectores Genéticos , Histidina/genética , Datos de Secuencia Molecular , Subunidades de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Vibrio cholerae/químicaRESUMEN
A mutant cholera toxin B subunit containing a G33E substitution was constructed and expressed in V. cholerae. The G33E amino acid substitution did not affect the amount of recombinant CTB secreted to the culture medium. The overexpression of the mutant B subunits in wild-type toxigenic cholera vibrios led to an 80% decrease in production of active cholera toxin in vitro and in vivo. Overexpression of BG33E subunits could be instrumental in the increase of the biosafety of live attenuated cholera candidate vaccine strains.
Asunto(s)
Toxina del Cólera/biosíntesis , Toxina del Cólera/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Toxina del Cólera/química , Vacunas contra el Cólera/genética , Vacunas contra el Cólera/toxicidad , Expresión Génica , Genes Bacterianos , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/genética , Mutación Puntual , Conformación Proteica , Conejos , Seguridad , Vacunas Atenuadas/genética , Vacunas Atenuadas/toxicidad , Vibrio cholerae/inmunologíaRESUMEN
The cholera toxin A-subunit (CTA) was genetically engineered at its amino end and tested for carriage of epitopes by fusion of the STa heat-stable enterotoxin analogue CAELCCNPAC. Efficient holotoxin formation by complementation in trans with cholera toxin B-subunit (CTB) indicated no decrease in affinity for CTB but evidence of reduced toxicity suggests steric interference by the decapeptide with the active site. The holotoxin was stable, able to bind to GM1 and was recognized by anti-STa and anti-CTA antibodies. The use of a full-length CTA might have been a key step for successful genetic fusions. Based on these findings, it seems worthwhile pursue the development of CTA for construction of recombinant mucosal immunoadjuvants.
Asunto(s)
Toxina del Cólera/química , Epítopos/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Recombinante , Escherichia coli/química , Datos de Secuencia MolecularRESUMEN
Vibrio cholerae has recently called the attention of researchers due to its strong immunogenicity and also because it serves as coadjunct immunomodulator of the immune response of the intestinal mucosae for the mixed added antigens as well as for those covalently linked to the toxin. The immunopathogeny of cholera is a complex phenomenon. This article presents the preliminary results of experiments conducted with laboratory rats in order to find the IgA intestinal response of rodents and humans.