Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.     

Clonal relationship among Vibrio cholerae O1 El Tor strains isolated in Somalia

One hundred and three Vibrio cholerae O1 strains, selected to represent the cholera outbreaks which occurred in Somalia in 1998-1999, were characterized by random amplified polymorphic DNA patterns, ribotyping, and antimicrobial susceptibility. All strains showed a unique amplified DNA pattern and 2 closely related ribotypes (B5a and B8a), among which B5a was the more frequently identified. Ninety-one strains were resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim, conferred, except for spectinomycin, by a conjugative plasmid IncC. These findings indicated that the group of strains active in Somalia in the late 1990s had a clonal origin.     

Prolonged colonization of mice by Vibrio cholerae El Tor O1 depends on accessory toxins

Cholera epidemics caused by Vibrio cholerae El Tor O1 strains are typified by a large number of asymptomatic carriers who excrete vibrios but do not develop diarrhea. This carriage state was important for the spread of the seventh cholera pandemic as the bacterium was mobilized geographically, allowing the global dispersion of this less virulent strain. Virulence factors associated with the development of the carriage state have not been previously identified. We have developed an animal model of cholera in adult C57BL/6 mice wherein V. cholerae colonizes the mucus layer and forms microcolonies in the crypts of the distal small bowel. Colonization occurred 1 to 3 h after oral inoculation and peaked at 10 to 12 h, when bacterial loads exceeded the inoculum by 10- to 200-fold, indicating bacterial growth within the small intestine. After a clearance phase, the number of bacteria within the small intestine, but not those in the cecum or colon, stabilized and persisted for at least 72 h. The ability of V. cholerae to prevent clearance and establish this prolonged colonization was associated with the accessory toxins hemolysin, the multifunctional autoprocessing RTX toxin, and hemagglutinin/protease and did not require cholera toxin or toxin-coregulated pili. The defect in colonization attributed to the loss of the accessory toxins may be extracellularly complemented by inoculation of the defective strain with an isogenic colonization-proficient V. cholerae strain. This work thus demonstrates that secreted accessory toxins modify the host environment to enable prolonged colonization of the small intestine in the absence of overt disease symptoms and thereby contribute to disease dissemination via asymptomatic carriers.     

Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh

We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.     

The role of toxin-coregulated pili in the pathogenesis of Vibrio cholerae O1 El Tor

Studies in the infant mouse cholera model have evaluated the significance of toxin-coregulated pili (TCP) in the pathogenesis of Vibrio cholerae strains of El Tor biotype. Four El Tor strains—two which produce TCP during in vitro growth and two which do not—were mutated by the insertion of an antibiotic-resistance cartridge into the tcpA gene (encoding the pilin monomer). The resulting mutants were otherwise indistinguishable from wild-type and in particular were unaltered in their sensitivity to antibody-dependent, complement-mediated bacteriolysis. All were dramatically attenuated and showed a marked impairment in terms of in vivo persistence in mixed-infection competition experiments. Virulence was restored by provision of a functional tcp operon in trans, confirming that the pathogenic potential of El Tor strains is critically dependent upon product(s) of this operon.     

Epitope differences in toxin-coregulated pili produced by classical and El Tor Vibrio cholerae O1.

A toxin-coregulated pilus (TCP), that is important for intestinal colonization of Vibrio cholerae O1, may be produced by vibrios of both classical and EI Tor biotypes. By comparing TCP produced by various strains of the two biotypes in immunoblotting and enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies (mAbs) and polyclonal antisera against TCP from classical vibrios, we have found biotype-related epitope differences in TCP. Our results indicate that TCP of classical strains has an epitope in the TcpA-subunit (20.5 kDa) that is missing in EI Tor TcpA, and an additional epitope that is more strongly expressed in classical TcpA. A polyclonal antiserum reacted strongly with TcpA from strains of both biotypes in immunoblotting suggesting both the presence of major shared TcpA epitopes and that the low or absent reactivity of EI Tor TcpA with the mAbs was not due to lower production of TcpA by EI Tor strains. Whereas all the TcpA-positive classical strains inhibited the binding of polyclonal antiserum and mAbs to solid phase-bound TCP-positive bacteria in an inhibition ELISA, practically no inhibition was observed with TcpA-positive EI Tor strains. This together with findings in immunoelectron microscopy studies that TCP 'bundles' were only detected on classical strains, suggest that TCP is poorly expressed on EI Tor vibrios.     

Cholera Toxin Production by the El Tor Variant of Vibrio cholerae O1 Compared to Prototype El Tor and Classical Biotypes

Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.Vibrio cholerae O1 is classified into classical and El Tor biotypes. Among other genetic, biochemical, and physiological differences, each biotype has unique gene sequences encoding cholera toxin B subunit (CTB), that is, classical ctxB and El Tor ctxB. Besides these two prototype biotypes of V. cholerae O1, Nair et al. (9) in 2002 in Bangladesh isolated strains that possess phenotypic properties of both classical and El Tor biotypes carrying classical ctxB. The same group also isolated El Tor strains that had classical ctxB (10). For these new types of strains of V. cholerae O1, we have recently proposed the designations of hybrid and El Tor variants, respectively (13). Subsequent to the isolation of the El Tor variant in Bangladesh by Nair et al. (10), El Tor variant strains were isolated from several countries and areas in Asia and Africa (1, 11, 15-18). In Kolkata, India, we showed that El Tor variant strains appeared in 1990 and that a complete replacement of prototype El Tor strains by El Tor variant strains has occurred since 1995 (14).In this study, we investigated the amount of cholera toxin (CT) produced both in vitro and in vivo by V. cholerae O1 El Tor variant strains isolated in Kolkata during a period from 1996 to 2007. It was found that El Tor variant strains produced a much larger amount of CT than did prototype El Tor strains and that the amount of CT produced by El Tor variant strains was more or less equivalent to that produced by classical strains.V. cholerae O1 strains used in this study are listed in Table ​Table1.1. AKI (3) and Syncase medium (2) were used for culturing the test strains. The rationale for selecting these media was that AKI preferentially supports the production of El Tor CT (3) while Syncase medium is reported to be the best medium supporting the production of CT by the classical biotype (2). Measurement of CT concentration produced by V. cholerae O1 strains was carried out as follows. Each strain was cultured either in AKI medium at 37°C for 20 h without shaking or in Syncase medium at 37°C for 20 h with shaking, and the optical density of the culture was measured at 600 nm (OD₆₀₀). After centrifugation, the supernatants were collected and the concentration of CT (ng/ml/OD₆₀₀) in the samples was measured by bead enzyme-linked immunosorbent assay (ELISA). The method of the bead ELISA employed was essentially that described by Oku et al. (12). In brief, a polystyrene bead (6.5 mm in diameter) was coated with anti-CT IgG and used as a solid phase. The coated bead was first incubated with the sample and then incubated with anti-CT IgG [F(ab′)]-horseradish peroxidase conjugate. Peroxidase activity was determined colorimetrically with 3,3′,5,5′-tetramethylbenzidine as the substrate. The absorbance at 450 nm (OD₄₅₀) was linear between 0 and 0.5, representing CT concentrations of 0 to 20 ng/ml. The sample prepared as described above (the supernatant of the culture of the strain) was appropriately diluted so that the OD₄₅₀ fell in the range of 0.1 to 0.5, and the amount of CT produced by the strain was expressed as ng/ml/OD₆₀₀.

TABLE 1.

V. cholerae O1 strains used

Identity of hemolysins produced by Vibrio cholerae non-O1 and V. cholerae O1, biotype El Tor.

Hemolysins purified from non-O1 Vibrio cholerae (non-O1 hemolysin) and a Vibrio cholerae O1, biotype El Tor (El Tor hemolysin) were investigated for their homology. The hemolysins were isolated from the culture supernatant fluids by ammonium sulfate precipitation and gel filtration on Sephadex G-100 columns. The purified hemolysins gave single bands with an identical mobility on conventional polyacrylamide gel disc electrophoresis. The molecular weights of the non-O1 and El Tor hemolysins were estimated to be about 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the amino acid compositions of the hemolysins were very similar. The specific activities of the hemolysins were identical, and both hemolysins were neutralized to the same extent with antisera against the homologous and heterologous hemolysins. Ouchterlony double immunodiffusion tests with both hemolysins and antihemolysin serum gave a common (fused) precipitin line. These data indicate that the non-O1 hemolysin is biologically, physicochemically, and immunologically indistinguishable from the El Tor hemolysin.     

New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor.

A new medium that stimulates in vitro production of cholera toxin by Vibrio cholerae O1 El Tor (El Tor vibrios) was developed. The medium contains 0.5% NaCl, 0.3% NaHCO3, 0.4% yeast extract, and 1.5% Bacto-Peptone. El Tor vibrios were cultured in a stationary test tube at 37 degrees C for 20 h. The culture supernatant was assayed for cholera toxin by a reversed passive latex agglutination method. Most vibrios grown in this medium produced 10 to 20 times more toxin than in traditional syncase medium. The number of live vibrios at the end of culture was about 10(8)/ml in the new medium (AKI medium) and about 10(10)/ml in the syncase medium. As a result, each individual organism in the new medium should have produced as much as 1,000 times more toxin than in syncase medium. Sodium bicarbonate was found to be the most important factor in toxin production by El Tor vibrios in the new medium. We recommend this new medium because of its high yield of cholera toxin and its technical simplicity.     

ICEVchInd5 is prevalent in epidemic Vibrio cholerae O1 El Tor strains isolated in India

Integrative conjugative elements (ICEs) of the SXT/R391 family are self-transmissible mobile elements mainly involved in antibiotic resistance spread among γ-Proteobacteria, including Vibrio cholerae. We demonstrated that the recently described ICEVchInd5 is prevailing in V. cholerae O1 clinical strains isolated in Wardha province (Maharashtra, India) from 1994 to 2005. Genetic characterization by ribotyping and multiple-locus SSR analysis proved the same clonal origin for V. cholerae O1 isolates in Wardha province over an 11-year period and was used to assess the correlation between strain and ICE content among ours and different Indian reference strains. In silico analysis showed the existence of at least 3 sibling ICEs of ICEVchInd5 in V. cholerae O1 El Tor reference strains, isolated in the Indian subcontinent after 1992.     

The Seventh Pandemic Vibrio cholerae O1 El Tor Isolate in China Has Undergone Genetic Shifts

A total of 330 clinical Vibrio cholerae O1 serogroups from China dating between 1961 and 2010 were investigated. By phenotypic biotyping and genetic analysis, during the seventh pandemic of V. cholerae O1 in China, the isolates of hybrid biotype (mixed classical phenotypes) were present during the entire1961-2010 period, while El Tor genetic shifts appeared in 1992 and replaced the prototype El Tor from 2002 to 2010.     

Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba from the prevailing O1 Ogawa serotype strains in India

The toxigenic Inaba serotype of Vibrio cholerae O1 biotype El Tor reappeared in India in 1998 and 1999, almost 10 years after its last dominance in Calcutta in 1989. Extensive molecular characterization by ribotyping, restriction fragment length polymorphism, and pulsed-field gel electrophoresis indicated that recent Inaba strains are remarkably different from the earlier Inaba strains but are very similar to the prevailing V. cholerae O1 Ogawa El Tor biotype strains. The antibiograms of the Inaba strains were also similar to those of the recent V. cholerae Ogawa strains. These V. cholerae O1 Inaba strains appear to have evolved from the currently prevailing Ogawa strains and are likely to dominate in the coming years.     

Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V. cholerae O139 Bengal in Bangladesh.

The emergence of Vibrio cholerae O139 Bengal in 1993, its rapid spread in an epidemic form, in which it replaced existing strains of V. cholerae O1 during 1992 and 1993, and the subsequent reemergence of V. cholerae O1 of the El Tor biotype in Bangladesh since 1994 have raised questions regarding the origin of the reemerged El Tor vibrios. We studied 50 El Tor vibrio strains isolated in Bangladesh and four other countries in Asia and Africa before the emergence of V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 to determine whether the reemerged El Tor vibrios were genetically different from the El Tor vibrios which existed before the emergence of V. cholerae O139. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA, cholera toxin (ctxA), and zonula occludens toxin (zot) or in DNA sequences flanking the genes showed that the El Tor strains isolated before the emergence of V. cholerae O139 belonged to four different ribotypes and four different ctx genotypes. Of 32 El Tor strains isolated after the emergence of O139 vibrios, 30 strains (93.7%) including all the clinical isolates belonged to a single new ribotype and a distinctly different ctx genotype. These results provide evidence that the reemerged El Tor strains represent a new clone of El Tor vibrios distinctly different from the earlier clones of El Tor vibrios which were replaced by the O139 vibrios. Further analysis showed that all the strains carried the structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR). All strains of the new clone produced cholera toxin (CT) in vitro, as assayed by the GM1-dependent enzyme-linked immunosorbent assay, and the level of CT production was comparable to that of previous epidemic isolates of El Tor vibrios. Further studies are required to assess the epidemic potential of the newly emerged clone of V. cholerae O1 and to understand the mechanism of emergence of new clones of toxigenic V. cholerae.     

Cholera toxin expression by El Tor Vibrio cholerae in shallow culture growth conditions

Vibrio cholerae O1 classical, El Tor and O139 are the primary biotypes that cause epidemic cholera, and they also express cholera toxin (CT). Although classical V. cholerae produces CT in various settings, the El Tor and O139 strains require specific growth conditions for CT induction, such as the so-called AKI conditions, which consist of growth in static conditions followed by growth under aerobic shaking conditions. However, our group has demonstrated that CT production may also take place in shallow static cultures. How these type of cultures induce CT production has been unclear, but we now report that in shallow culture growth conditions, there is virtual depletion of dissolved oxygen after 2.5 h of growth. Concurrently, during the first three to 4 h, endogenous CO₂ accumulates in the media and the pH decreases. These findings may explain CT expression at the molecular level because CT production relies on a regulatory cascade, in which the key regulator AphB may be activated by anaerobiosis and by low pH. AphB activation stimulates TcpP synthesis, which induces ToxT production, and ToxT directly stimulates ctxAB expression, which encodes CT. Importantly, ToxT activity is enhanced by bicarbonate. Therefore, we suggest that in shallow cultures, AphB is activated by initial decreases in oxygen and pH, and subsequently, ToxT is activated by intracellular bicarbonate that has been generated from endogenous CO₂. This working model would explain CT production in shallow cultures and, possibly, also in other growth conditions.     

Suppressed induction of proinflammatory cytokines by a unique metabolite produced by Vibrio cholerae O1 El Tor biotype in cultured host cells

Vibrio cholerae O1 has two biotypes, El Tor and Classical, and the latter is now presumed to be extinct in nature. Under carbohydrate-rich growth conditions, El Tor biotype strains produce the neutral fermentation end product 2,3-butanediol (2,3-BD), which prevents accumulation of organic acids from mixed acid fermentation and thus avoids a lethal decrease in the medium pH, while the Classical biotype strains fail to do the same. In this study, we investigated the inhibitory effect of 2,3-BD on the production of two proinflammatory biomarkers, intreleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), in human intestinal epithelial HT29 and alveolar epithelial A549 cells. Cell-free culture supernatants of El Tor strain N16961 grown in LB supplemented with 1% glucose induced a negligible amount of IL-8 or TNF-α, while the Classical O395 strain induced much higher levels of these proinflammatory cytokines. On the other hand, three mutant strains constructed from the N16961 strain with defects in the constitutive 2,3-BD pathway were also able to induce high levels of cytokines. When HT29 and A549 cells were treated with bacterial flagella, known proinflammatory cytokine inducers, and chemically synthesized 2,3-BD at various concentrations, a dose-dependent decrease in IL-8 and TNF-α production was observed, demonstrating the suppressive effect of 2,3-BD on the production of proinflammatory cytokines in epithelial cells. Upon cotreatment with extraneous 2,3-BD, elevated levels of IκBα, the inhibitor of the NF-κB pathway, were detected in both HT29 and A549 cells. Furthermore, treatments containing 2,3-BD elicited lower levels of NF-κB-responsive luciferase activity, demonstrating that the reduced cytokine production is likely through the inhibition of the NF-κB pathway. These results reveal a novel and potential role of 2,3-BD as an immune modulator that might have conferred a superior pathogenic potential of the El Tor over the Classical biotype.