In-vitro study of the effect of Centella asiatica on cholera toxin production and the gene expression level of ctxA gene in Vibrio cholerae isolates.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urb or Indian pennywort is a plant of ethnopharmacological relevance, commonly called as Brahmi in South India known for its antimicrobial property in gut and for the treatment of other gut ailments. Natural anti-virulence drugs that disarm pathogens by directly targeting virulence factors or the cell viability and are thus preferred over antibiotics as these drugs impose limited selection pressure for resistance development. In this regard, an in-vitro experimental study was conducted to know the effect of extract of Centella asiatica(L.) Urb. on cholera toxin, gene expression and its vibriocidal effect on five standard strains of Vibrio cholerae; IDH03097 (El Tor variant), N16961 (El Tor), O395 (Classical) as well as five clinical strains (Haitian variant). AIM OF THE STUDY: To study the effect of extract of Centella asiatica on Vibrio cholerae. MATERIALS AND METHODS: Crude extract was prepared from the leaves and stem part of the plant. The vibriocidal concentration was tested at different concentrations of the extract. The amount of cholera toxin released from the strains before and after exposure to the extract of Centella asiatica to Vibrio cholerae was measured using Bead ELISA. ctxA gene expression in the strains before and after exposure to extract of Centella asiatica was measured using quantitative real time PCR. All the above assays were performed with commercially obtained asiaticoside as well. RESULTS: The vibriocidal activity was tested at the different concentration of the extract, where 1g/mL of crude extract and 12.5mg/mL of asiaticoside was found to be vibriocidal. The amount of cholera toxin released before and after the exposure to extract of C. asiatica was measured using Bead ELISA, showing a reduction of 70%, 89% and 93% toxin produced by classical, El Tor and variant respectively. ctxA gene expression before and after exposure to extract of Centella asiatica as well as asiaticoside was measured using qRT-PCR. We found a decrease in expression of ctxA gene transcription by 6.19 fold in classical strain, 4.29 fold in El Tor, 1.133 fold in variant strains and about 10.13-10.20 fold for the clinical strains of V. cholerae using the extract of C.asiatica while, the reduction with the exposure to the asiaticoside were 2.762 fold in classical strain, 4.809 in El Tor, 24.1 in variant strain and 34.77 - 34.8 for the clinical strains. CONCLUSION: Centella asiatica extract inhibited the CT production in Vibrio cholerae as well as decreased the transcription of ctxA gene expression.

In Vitro Inhibition of Cholera Toxin Production in Vibrio cholerae by Methanol Extract of Sweet Fennel Seeds and Its Components.

A newly emerged Vibrio cholerae O1 El Tor variant strain with multidrug resistance is considered a threat to public health. Recent strategies to suppress virulence factors production instead of bacterial growth may lead to less selective pressure for the emergence of resistant strains. The use of spices and their active constituents as the inhibitory agents against cholera toxin (CT) production in V. cholerae may be an alternative approach to treat cholera. In this study, we examined the potential of sweet fennel seed (Foeniculum vulgare Miller var. dulce) methanol extract to inhibit CT production in V. cholerae without affecting viability. The methanol extract of sweet fennel seeds significantly inhibited CT production in various V. cholerae strains, regardless of serogroup or biotype. Interestingly, trans-anethole and 4-allylanisole, essential oil components of sweet fennel seeds, also demonstrated similar effects. Here, we report that sub-bactericidal concentrations of sweet fennel seed methanol extract and its major components can drastically inhibit CT production in various V. cholerae strains.

Conjugated Linoleic Acid Reduces Cholera Toxin Production In Vitro and In Vivo by Inhibiting Vibrio cholerae ToxT Activity.

The severe diarrheal disease cholera is endemic in over 50 countries. Current therapies for cholera patients involve oral and/or intravenous rehydration, often combined with the use of antibiotics to shorten the duration and intensity of the disease. However, as antibiotic resistance increases, treatment options will become limited. Linoleic acid has been shown to be a potent negative effector of V. cholerae virulence that acts on the major virulence transcription regulator protein, ToxT, to inhibit virulence gene expression. ToxT activates transcription of the two major virulence factors required for disease, cholera toxin (CT) and toxin-coregulated pilus (TCP). A conjugated form of linoleic acid (CLA) is currently sold over the counter as a dietary supplement and is generally recognized as safe by the U.S. Food and Drug Administration. This study examined whether CLA could be used as a new therapy to reduce CT production, which, in turn, would decrease disease duration and intensity in cholera patients. CLA could be used in place of traditional antibiotics and would be very unlikely to generate resistance, as it affects only virulence factor production and not bacterial growth or survival.

Inhibition of the sodium-translocating NADH-ubiquinone oxidoreductase [Na+-NQR] decreases cholera toxin production in Vibrio cholerae O1 at the late exponential growth phase.

Two virulence factors produced by Vibrio cholerae, cholera toxin (CT) and toxin-corregulated pilus (TCP), are indispensable for cholera infection. ToxT is the central regulatory protein involved in activation of CT and TCP expression. We previously reported that lack of a respiration-linked sodium-translocating NADH-ubiquinone oxidoreductase (Na(+)-NQR) significantly increases toxT transcription. In this study, we further characterized this link and found that Na(+)-NQR affects toxT expression only at the early-log growth phase, whereas lack of Na(+)-NQR decreases CT production after the mid-log growth phase. Such decreased CT production was independent of toxT and ctxB transcription. Supplementing a respiratory substrate, l-lactate, into the growth media restored CT production in the nqrA-F mutant, suggesting that decreased CT production in the Na(+)-NQR mutant is dependent on electron transport chain (ETC) activity. This notion was supported by the observations that two chemical inhibitors, a Na(+)-NQR specific inhibitor 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO) and a succinate dehydrogenase (SDH) inhibitor, thenoyltrifluoroacetone (TTFA), strongly inhibited CT production in both classical and El Tor biotype strains of V. cholerae. Accordingly, we propose the main respiratory enzyme of V. cholerae, as a potential drug target to treat cholera because human mitochondria do not contain Na(+)-NQR orthologs.

Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea.

The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The highest expression level recorded in T(1) generation seeds was 0.0014% of total aqueous soluble protein (TASP). Expression level of the same event in the T(2) generation was significantly increased to 0.0197% of TASP. Immunogenicity of maize derived CT-B was evaluated in mice with an oral immunization trial. Anti-CTB IgG and anti-CTB IgA were detected in the sera and fecal samples of the orally immunized mice, respectively. The mice were protected against holotoxin challenge with CT. An additional group of mice was administrated with an equal amount (5 µg per dose each) of mixed maize-derived CT-B and LT-B (B subunit of E. coli heat labile toxin). In the sera and fecal samples obtained from this group, the specific antibody levels were enhanced compared to either the same or a higher amount of CT-B alone. These results suggest that a synergistic action may be achieved using a CT-B and LT-B mixture that can lead to a more efficacious combined vaccine to target diarrhea induced by both cholera and enterotoxigenic strains of Escherichia coli.

Newer insights into the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea.

BACKGROUND: Psidium guajava L., Myrtaceae, is used widely in traditional medicine for the treatment of diarrhoea, dysentery, gastroenteritis, stomachaches, and indigestion. However, the effect of the leaf extract of P. guajava on the pathogenesis of infectious diarrhoea has not been studied. The present study evaluates the effect of a hot aqueous extract (decoction) of dried leaves of P. guajava on parameters associated with pathogenicity of infectious diarrhoea. The aim was to understand its possible mechanism(s) of action in controlling infectious diarrhoea and compare it with quercetin, one of the most reported active constituents of P. guajava with antidiarrhoeal activity. METHODS: The crude decoction and quercetin were studied for their antibacterial activity and effect on virulence features of common diarrhoeal pathogens viz. colonization of epithelial cells and production and action of enterotoxins. Colonization as measured by adherence of enteropathogenic Escherichia coli (EPEC) and invasion of enteroinvasive E. coli (EIEC) and Shigella flexneri was assessed using HEp-2 cell line. The production of E. coli heat labile toxin (LT) and cholera toxin (CT) and their binding to ganglioside monosialic acid (GM1) were studied by GM1-ELISA whereas the production and action of E. coli heat stable toxin (ST) was assessed by suckling mouse assay. RESULTS: The decoction of P. guajava showed antibacterial activity towards S. flexneri and Vibrio cholerae. It decreased production of both LT and CT and their binding to GM1. However, it had no effect on production and action of ST. The decoction also inhibited the adherence of EPEC and invasion by both EIEC and S. flexneri to HEp-2 cells. Quercetin, on the other hand, had no antibacterial activity at the concentrations used nor did it affect any of the enterotoxins. Although it did not affect adherence of EPEC, it inhibited the invasion of both EIEC and S. flexneri to HEp-2 cells. CONCLUSION: Collectively, the results indicate that the decoction of P. guajava leaves is an effective antidiarrhoeal agent and that the entire spectrum of its antidiarrhoeal activity is not due to quercetin alone.

Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato.

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P2 promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.

Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato.

A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5' to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016-0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.

A plant-based multicomponent vaccine protects mice from enteric diseases.

Cholera toxin (CT) B and A2 subunit complementary DNAs (cDNAs) were fused to a rotavirus enterotoxin and enterotoxigenic Escherichia coli fimbrial antigen genes and transferred into potato. Immunoblot and enzyme-linked immunosorbent assay (ELISA) results indicated that the fusion antigens were synthesized in transformed tuber tissues and assembled into cholera holotoxin-like structures that retained enterocyte-binding affinity. Orally immunized mice generated detectable levels of serum and intestinal antibodies against the pathogen antigens. Elevated levels of interleukin 2 (IL2) and interferon gamma (INFgamma) detected in immunogen-challenged spleen cells from the immunized mice indicated the presence of a strong Th1 immune response to the three plant-synthesized antigens. This result was supported by flow cytometry analysis of immunized mouse spleen cells that showed a significant increase in CD4+ lymphocyte numbers. Diarrhea symptoms were reduced in severity and duration in passively immunized mouse neonates following rotavirus challenge. The results suggest that food plants can function as vaccines for simultaneous protection against infectious virus and bacterial diseases.

Expression of cholera toxin B subunit oligomers in transgenic potato plants.

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M(r) approximately 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M(r) approximately 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.

Expression of the cholera toxin B subunit in the Golgi apparatus of Swiss 3T3 cells inhibits DNA synthesis induced by basic fibroblast growth factor.

We attempted to express the cholera toxin B subunit (CTXB) in the Golgi apparatus of cultured mammalian cells by means of gene transfection. Complementary DNA of CTXB was ligated with the Golgi-retention signal sequence of human beta 1,4 galactosyltransferase cDNA, and the chimeric gene yielded was inserted into a mammalian expression vector. The resultant construct was transfected into COS-1 cells for transient expression and into Swiss 3T3 cells for stable expression. The expression of a fusion protein encoded by the chimeric gene was demonstrated according to the following criteria: first, detection of a protein exhibiting the expected molecular mass on Western blot analysis using an anti-CTXB antibody; second, detection of the protein located in the Golgi area by indirect immunofluoresence microscopy; and third, detection of GM1 binding activity in cell lysates. Stable transformants satisfying the above criteria were subjected to an assay for mitogen-induced DNA synthesis. These transformants exhibited significantly lower DNA synthesis than mock transfection cells on stimulation with basic fibroblast growth factor (bFGF), whereas the two types of cells exhibited similar responses to 10% fetal calf serum and other mitogens, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and platelet-derived growth factor. Analysis of the binding of radio-iodinated bFGF to the cells revealed that the transformants did not exhibit a significant decrease in the binding affinity or the number of high affinity sites. These results suggest that the fusion protein specifically inhibits the bFGF signaling not at the binding step but rather at a later step(s) triggered by the binding.

Isolation and characterization of melanin-producing (mel) mutants of Vibrio cholerae.

Vibrio cholerae strain Htx-3, a hypertoxinogenic mutant of V. cholerae 569B Inaba, produces a dark brown pigment under certain growth conditions, whereas strain 569B does not. We investigated the biochemical basis for this pigment production and the possible relationships between pigmentation and other phenotypic properties in V. cholerae. After mutagenesis of V. cholerae 569B with N-methyl-N'-nitro-N-nitrosoguanidine, 28 independently derived pigment-forming (mel) mutants were isolated and characterized. The mel mutants frequently differed from wild type in toxinogenicity or motility and occasionally differed in other phenotypic traits. Individual mel mutants differed from wild type both in the amount of toxin produced and in the growth conditions optimal for toxin production. It has not yet been established whether multiple phenotypic changes in individual mel mutants represent pleiotropic effects of single mutations or induction of multiple mutations by N-methyl-N'-nitro-N-nitrosoguanidine or both. Production of pigment by mel mutants occurred at temperatures from 22 to 40 degrees C, was inhibited by anaerobiosis, and was stimulated by supplementation of growth media with the amino acid precursors of melanin (l-phenylalanine, l-tyrosine, or l-tyrosine plus l-cysteine). The pigment possessed several other properties reported for microbial melanins. We conclude that a biochemical pathway for melanin production is present in V. cholerae, that this pathway cannot be fully expressed in wild-type strain 569B, and that mutations in the gene(s) which we have designated mel can permit hyperproduction of melanin under appropriate conditions.

[Viability and properties of cholera vibrios cultivated in mineral water]. / Vyzhivaemost' i svoistva kholernykh vibrionov pri kul'tivirovanii v mineral'noi vode.

Samples of mineral water from thermal spas of "Matsesta" health resorts were infected with cholera vibrio strains. In nonsterile mineral water diluted with distilled water 1 : 2, 1 : 4, and 1 : 8 and stored at 37 degrees C survival periods of cholera vibrio constituted 289 days for classic biotype, and 413 days for El Tor biotype. In boiled mineral water stored at 20-24 degrees C this period was 1429 days for both biotypes. Prolonged cultivation of cholera vibrios in mineral water had no effect on biochemical and serological properties, as well as on cholera phage and antibiotic sensitivity, but there occurred dissociation of the strains into S- and R-forms.