RESUMO
Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression of Vibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression of tcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulate tcpA expression, the screen yielded ptsI and ptsH, which encode the enzyme I (EI) and Hpr components of the V. cholerae phosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIA(Glc) protein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression of tcpPH and aphAB, which themselves control expression of toxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that the V. cholerae PTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks.
Assuntos
Cólera/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/fisiologia , Vibrio cholerae/patogenicidade , Virulência/genética , Animais , Proteínas de Bactérias/biossíntese , Cólera/genética , Toxina da Cólera/biossíntese , AMP Cíclico , Modelos Animais de Doenças , Proteínas de Fímbrias/biossíntese , Citometria de Fluxo , Immunoblotting , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/biossínteseRESUMO
In an effort to initiate the development of a plant-based vaccination model against atherosclerosis, a cholera toxin B subunit (CTB)-based chimeric protein was designed to target both ApoB100 and CETP epitopes associated with immunotherapeutic effects in atherosclerosis. Epitopes were fused at the C-terminus of CTB to yield a protein called CTB:p210:CETPe. A synthetic gene coding for CTB:p210:CETPe was successfully transferred to tobacco plants with no phenotypic alterations. Plant-derived CTB:p210:CETPe was expressed and assembled in the pentameric form. This protein retained the target antigenic determinants, as revealed by GM1-ELISA and Western blot analyses. Higher expresser lines reached recombinant protein accumulation levels up to 10 µg/g fresh weight in leaf tissues and these lines carry a single insertion of the transgene as determined by qPCR. Moreover, when subcutaneously administered, the biomass from these CTB:p210:CETPe-producing plants was able to elicit humoral responses in mice against both ApoB100 and CETP epitopes and human serum proteins. These findings evidenced for the first time that atherosclerosis-related epitopes can be expressed in plants retaining immunogenicity, which opens a new path in the molecular farming field for the development of vaccines against atherosclerosis.
Assuntos
Apolipoproteína B-100/imunologia , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Proteínas de Transferência de Ésteres de Colesterol/imunologia , Nicotiana/genética , Animais , Aterosclerose/prevenção & controle , Toxina da Cólera/biossíntese , Epitopos/imunologia , Camundongos , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas/imunologiaRESUMO
Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2- to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.
Assuntos
Cólera/microbiologia , Vibrio cholerae O1/isolamento & purificação , Vibrio cholerae O1/patogenicidade , Fatores de Virulência/genética , Adolescente , Adulto , Idoso de 80 Anos ou mais , Animais , Proteínas de Bactérias/genética , Bangladesh , Criança , Pré-Escolar , Cólera/patologia , Toxina da Cólera/biossíntese , Toxina da Cólera/genética , Modelos Animais de Doenças , Feminino , Proteínas de Fímbrias/genética , Variação Genética , Haiti , Humanos , Masculino , Fatores de Transcrição/genética , Vibrio cholerae O1/classificação , Vibrio cholerae O1/genética , Virulência , Fatores de Virulência/biossíntese , Adulto JovemRESUMO
Genetic engineering revolutionized the concept of traditional vaccines since subunit vaccines became reality. Additionally, over the past two decades plant-derived antigens have been studied as potential vaccines with several advantages, including low cost and convenient administration. More specifically, genetic fusions allowed the expression of fusion proteins carrying two or more components with the aim to elicit immune responses against different targets, including antigens from distinct pathogens or strains. This review aims to provide an update in the field of the production of plant-based vaccine, focusing on those approaches based on the production of chimeric proteins comprising antigens from human pathogens, emphasizing the case of cholera toxin/E. coli enterotoxin fusions, chimeric viruses like particles approaches as well as the possible use of adjuvant-producing plants as expression hosts. Challenges for the near future in this field are also discussed.
Assuntos
Engenharia Genética/métodos , Plantas Geneticamente Modificadas/metabolismo , Vacinas/biossíntese , Adjuvantes Imunológicos/química , Toxina da Cólera/biossíntese , Toxina da Cólera/imunologia , Enterotoxinas/biossíntese , Enterotoxinas/imunologia , Engenharia Genética/tendências , Plantas Geneticamente Modificadas/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
BACKGROUND & AIMS: Dermatitis herpetiformis (DH) is characterized by variable degrees of enteropathy and increased intestinal permeability. Zonulin, a regulator of tight junctions, seems to play a key role in the altered intestinal permeability that characterizes the early phase of celiac disease. Our aim was to assess both intestinal permeability and serum zonulin levels in a group of patients with DH having variable grades of enteropathy. METHODS: We studied 18 DH patients diagnosed on the basis of characteristic immunoglobulin (Ig)A granular deposits in the dermal papillae of noninvolved skin. Results were compared with those of classic celiac patients, patients with linear IgA dermatosis, and healthy controls. RESULTS: According to Marsh's classification, 5 patients had no evidence of enteropathy (type 0), 4 patients had type II, 2 patients had type IIIb damage, and 7 patients had a more severe lesion (type IIIc). Intestinal permeability (lactulose/mannitol ratio [lac/man]) was abnormal in all patients with DH. Patients with more severe enteropathy had significantly greater permeability ( P < .05). The serum zonulin concentration (enzyme-linked immunosorbent assay) for patients with DH was 2.1 +/- .3 ng/mg with 14 of 16 (87.5%) patients having abnormally increased values. In contrast, patients with linear IgA dermatosis had normal histology, normal intestinal permeability, and negative celiac serology. CONCLUSIONS: Increased intestinal permeability and zonulin up-regulation are common and concomitant findings among patients with DH, likely involved in pathogenesis. Increased permeability can be observed even in patients with no evidence of histologic damage in biopsy specimens. Patients with linear IgA dermatosis appear to be a distinct population with no evidence of gluten sensitivity.
Assuntos
Doença Celíaca/diagnóstico , Doença Celíaca/epidemiologia , Toxina da Cólera/biossíntese , Dermatite Herpetiforme/diagnóstico , Dermatite Herpetiforme/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Idoso , Biomarcadores/análise , Estudos de Casos e Controles , Toxina da Cólera/sangue , Comorbidade , Feminino , Haptoglobinas , Humanos , Incidência , Absorção Intestinal/fisiologia , Masculino , Pessoa de Meia-Idade , Permeabilidade , Probabilidade , Prognóstico , Precursores de Proteínas , Valores de Referência , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Distribuição por SexoRESUMO
The hemagglutinin/protease (HA/P) seems to be an attractive locus for the insertion of heterologous tags in live cholera vaccine strains. A deltaCTXphi spontaneous mutant derived from a pathogenic strain of O139 Vibrio cholerae was sequentially manipulated to obtain hapA Colon, two colons celA derivatives which were later improved in their environmental safety by means of a thyA mutation. All the strains here obtained showed similar phenotypes in traits known to be remarkable for live cholera vaccines irrespective of their motility phenotypes, although the hapA mutants had a 10-fold decrease in their colonisation capacity compared with their parental strains in the infant mouse cholera model. However, the subsequent thyA mutation did not affect their colonisation properties in the same model. These preliminary results pave the way for further clinical assays to confirm the possibilities of these vaccine prototypes as safe and effective tools for the prevention of O139 cholera.
Assuntos
Vacinas contra Cólera/imunologia , Antígenos O/imunologia , Vibrio cholerae O139/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/biossíntese , Cápsulas Bacterianas/imunologia , Celulase/genética , Cólera/prevenção & controle , Toxina da Cólera/biossíntese , Toxina da Cólera/genética , Clostridium/genética , Resistência a Medicamentos , Farmacorresistência Bacteriana Múltipla , Genes Sintéticos , Testes de Hemaglutinação , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Metaloendopeptidases/genética , Mutagênese Insercional , Coelhos , Segurança , Estreptomicina/farmacologia , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Vibrio cholerae O139/efeitos dos fármacos , Vibrio cholerae O139/enzimologia , Vibrio cholerae O139/genéticaRESUMO
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.
Assuntos
Toxina da Cólera/genética , Toxina da Cólera/isolamento & purificação , Genes Bacterianos/genética , Histidina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Glândulas Suprarrenais/citologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Linhagem Celular , Toxina da Cólera/biossíntese , Toxina da Cólera/química , Códon/genética , DNA Recombinante/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Histidina/genética , Dados de Sequência Molecular , Subunidades Proteicas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Vibrio cholerae/químicaRESUMO
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.
Assuntos
Toxina da Cólera/biossíntese , Toxina da Cólera/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Toxina da Cólera/química , Vacinas contra Cólera/genética , Vacinas contra Cólera/toxicidade , Expressão Gênica , Genes Bacterianos , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/genética , Mutação Puntual , Conformação Proteica , Coelhos , Segurança , Vacinas Atenuadas/genética , Vacinas Atenuadas/toxicidade , Vibrio cholerae/imunologiaRESUMO
BACKGROUND: A specific probe was designed to identify part of the genetic sequence of the ctxB gene which encodes for the B subunit of the cholera toxin by polymerase chain reaction (PCR) which amplifies a 318 bp segment of the ctxB gene. Marked with P32, we used this probe for colony hybridization which is a technique for identifying the production capacity of subunit B of strains of Vibrio cholerae O1 from different outbreaks in South America (Perú 1992 and Ecuador 1993-1995) and from, collection strains. This probe was tested for the identification of the ctxB gene in Vibrio cholerae O139. METHOD: Thirty-eight phylogenetically related strains were studied: 24 V. cholerae O1, 4 V. cholerae non O1, 5 Aeromonas, 4 Plesiomonas and 1 Escherichia coli. RESULTS: The probe demonstrated to be useful for the identification of the ctxB gene (which codifies for the subunit B of the cholera toxin) in 24 strains of Vibrio cholerae O1 and in the Vibrio cholerae O139 strain. The ctxB gene was not detected in the remaining strains pertaining to the Vibrio cholerae non O1 species (non O139), Plesiomonas, Aeromonas spp. and E. coli. The specificity of this product was not demonstrated since no signal of unspecific hybridization appeared with phylogenetically related strains such as Escherichia coli K88 (LT+) and Aeromonas hydrophila ATCC (LT+), producers of the thermolabile LT toxin. It is important to indicate that the ctxB gene in V. cholerae O139 has been identified, for the first time, with our probe and thus it may be said that all the strains which have genetic codification for CT up to now may be identified. CONCLUSIONS: We conclude that the system herein described provides advantages over the immunologic and biologic methods for evaluating a large number of samples in a short time and with excellent specificity and sensitivity which are important in the diagnosis and the epidemiologic surveillance of the disease.
Assuntos
Toxina da Cólera/genética , Genes Bacterianos , Vibrio cholerae/genética , Aeromonas/genética , Cólera/epidemiologia , Cólera/microbiologia , Toxina da Cólera/biossíntese , Surtos de Doenças , Escherichia coli/genética , Humanos , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Plesiomonas/genética , Reação em Cadeia da Polimerase , América do Sul , Especificidade da Espécie , Vibrio cholerae/classificação , Vibrio cholerae/metabolismoRESUMO
Recientemente Vibrio cholerae ha llamado mucho la atención de los investigadores por ser un inmunógeno muy potente y, al mismo tiempo, un coadyuvante inmunomodulador de la respuesta inmunitaria en la mucosa intestinal, tanto para los antígenos que se administran mezclados como los ligados covalentemente a la toxina colérica. La inmunopatogenia del cólera es un fenómeno complejo. En este artículo se comunican los resultados preliminares de experimentos realizados con ratas de laboratorio para conocer la respuesta intestinal de la IgA en los roedores y los humanos