After De΄s pivotal demonstration in 1959 of a diarrhoeogenic exo-enterotoxin in cell-free culture filtrates from Vibrio cholerae (of classical biotype), much insight has been gained about cholera toxin (CT), which is arguably now the best known of all microbial toxins. The subunit structure and function of CT, its receptor (the GM1 ganglioside), and its effects on the cyclic AMP system and on intestinal secretion were defined in the 1970s, and the essential aspects of the genetic organization in the 1980s. Recent findings have generated additional perspectives. The 3D-crystal structure of CT has been established, the CT-encoding operon has been shown to be carried by a non-lytic bacteriophage, and in depth knowledge has been gained on how the bacterium controls CT gene expression in response to cell density and various environmental signals. The mode of entry into target cells and the intracellular transport of CT are becoming clearer. CT has become the prototype enterotoxin and a widely used tool for elucidating important aspects of cell biology and physiology, e.g., cell membrane receptors, the cyclic AMP system, G proteins, as well as normal and pathological ion transport mechanisms. In immunology, CT has emerged as a potent, widely used experimental adjuvant, and the strong oral-mucosal immunogenicity of the non-toxic B-subunit (CTB) has led to the use of CTB as a protective antigen together with killed vibrios in a widely licensed oral cholera vaccine. CTB has also been shown to promote immunological tolerance against certain types of mucosally co-administered antigens, preferably tissue antigens linked to the CTB molecule; this has stimulated research and development to use CTB in this context for treatment of autoimmune and allergic diseases. In summary, in the 50 years after De΄s discovery of CT, this molecule has emerged from being the cholera patient΄s "foe" to also becoming a highly useful scientist΄s "friend".

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Cholera toxin (CT) was discovered exactly half a century ago by S.N. De. We have come a long way since this epoch-making discovery. Retrospectively, science had to wait a long time since Koch's prediction of the existence of a toxin, and its actual discovery by De. CT is not just another enterotoxin that causes the signs and symptoms of the dreaded disease, cholera. It is unique in many respects, starting from its structure to its functions. CT is a multifunctional protein that is capable of influencing the immune system in many ways. It not only has remarkable adjuvant properties, but also acts as an anti-inflammatory agent, by modulating specific signal transduction pathways. Its immunomodulatory properties can be harnessed for treatment of various autoimmune disorders, and have shown great promise in the area of immunotherapeutics. CT can truly be considered as a paradigm of a multifunctional protein.

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It was shown earlier that immune responses against cholera toxin (CT) as well as Vibrio cholerae lipopolysaccharide (LPS) or whole bacterial cells (WC) were protective and that these different antibody specificities co-operated synergistically for protection against experimental cholera. Similarly, antibodies against the heat-labile toxin (LT) and major colonization factors (CFs) of enterotoxingenic Escherichia coli (ETEC) co-operated synergistically for protection against LT-producing ETEC expressing homologous CFs. Studies in humans revealed that repeated oral antigen administration was optimal in inducing intestinal immune responses. Based on these findings oral inactivated vaccines consisting of toxin antigen and whole cells, i.e. the licensed recombinant cholera B subunit (rCTB)-WC cholera vaccine Dukoral®, and candidate ETEC vaccines have been developed. In different trials the rCTB-WC cholera vaccine has provided very high (85-100%) short term protection, which was significantly higher than that induced by the WC component alone, whereas rCTB-WC and WC alone provided comparable (50-60%), long term protection. An oral ETEC vaccine consisting of rCTB and formalin-inactivated E. coli bacteria expressing major CFs was shown to be safe and immunogenic in adults and children in different countries. The vaccine also induced significant protection against non-mild ETEC diarrhoea, i.e. diarrhoea interfering with daily activity in American travellers but not against ETEC diarrhoea in young children in Egypt. Against this background, a modified ETEC vaccine consisting of recombinant E. coli strains overexpressing the major CFs and a more LT like hybrid toxoid (LCTBA) has been developed. This vaccine will be tested soon alone and together with a mucosal adjuvant, i.e. dmLT, in clinical trials.

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Non-vibrio cholera has been recognized as a clinical entity for as long as cholera was known to be caused by Vibrio cholerae. Until 1968, the aetiologic agent of this syndrome was not known. Following a series of studies in patients with non-vibrio cholera it was found that these patients had large concentrations of Escherichia coli in the small bowel and stools which produced cholera toxin-like enterotoxins, and had fluid and electrolyte transport abnormalities in the small bowel similar to patients with documented cholera. Furthermore, these patients developed antibodies to the cholera-like enterotoxin. Later studies showed that these strains, when fed to volunteers produced a cholera-like disease and that two enterotoxins were found to be produced by these organisms: a heat-labile enterotoxin (LT) which is nearly identical to cholera toxin, and a heat-stable enterotoxin (ST), a small molecular weight polypeptide. E. coli that produced one or both of these enterotoxins were designated enterotoxigenic E. coli (ETEC). ETEC are now known not only to cause a severe cholera-like illness, but to be the most common bacterial cause of acute diarrhoea in children in the developing world, and to be the most common cause of travellers' diarrhoea in persons who visit the developing world.

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Nutritional stress elicits stringent response in bacteria involving modulation of expression of several genes. This is mainly triggered by the intracellular accumulation of two small molecules, namely, guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bis(diphosphate), collectively called (p)ppGpp. Like in other Gram-negative bacteria, the cellular level of (p)ppGpp is maintained in Vibrio cholerae, the causative bacterial pathogen of the disease cholera, by the products of two genes relA and spoT. However, apart from relA and spoT, a novel gene relV has recently been identified in V. cholerae, the product of which has been shown to be involved in (p)ppGpp synthesis under glucose or fatty acid starvation in a ∆relA ∆spoT mutant background. Furthermore, the GTP binding essential protein CgtA and a non-DNA binding transcription factor DksA also seem to play several important roles in modulating stringent response and regulation of other genes in this pathogen. The present review briefly discusses about the role of all these genes mainly in the management of stringent response in V. cholerae.

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The rise in multi-drug resistant Vibrio cholerae strains is a big problem in treatment of patients suffering from severe cholera. Only a few studies have evaluated the potential of natural compounds against V. cholerae. Extracts from plants like 'neem', 'guazuma', 'daio', apple, hop, green tea and elephant garlic have been shown to inhibit bacterial growth or the secreted cholera toxin (CT). However, inhibiting bacterial growth like common antimicrobial agents may also impose selective pressure facilitating development of resistant strains. A natural compound that can inhibit virulence in V. cholerae is an alternative choice for remedy. Recently, some common spices were examined to check their inhibitory capacity against virulence expression of V. cholerae. Among them methanol extracts of red chili, sweet fennel and white pepper could substantially inhibit CT production. Fractionation of red chili methanol extracts indicated a hydrophobic nature of the inhibitory compound(s), and the n-hexane and 90 per cent methanol fractions could inhibit >90 per cent of CT production. Purification and further fractionation revealed that capsaicin is one of the major components among these red chili fractions. Indeed, capsaicin inhibited the production of CT in various V. cholerae strains regardless of serogroups and biotypes. The quantitative reverse transcription real-time PCR assay revealed that capsaicin dramatically reduced the expression of major virulence-related genes such as ctxA, tcpA and toxT but enhanced the expression of hns gene that transcribes a global prokaryotic gene regulator (H-NS). This indicates that the repression of CT production by capsaicin or red chili might be due to the repression of virulence genes transcription by H-NS. Regular intake of spices like red chili might be a good approach to fight against devastating cholera.

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Vibrio cholerae is the causative agent of the disease cholera, characterized by profuse watery diarrhoea. Two of the main virulence factors associated with the disease are cholera toxin (CT) and toxin-coregulated pilus (TCP). Expression of CT and TCP is regulated via a complex cascade of factors that respond to environmental signals, but ultimately ToxT is the direct transcriptional activator of the genes encoding CT and TCP. Recent studies have begun to unveil the mechanisms behind ToxT-dependent transcription. We review current knowledge of transcriptional activation by ToxT and the environmental stimuli that allow ToxT to regulate virulence gene expression, resulting in cholera pathogenesis.

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Antimicrobial resistance poses a major threat in the treatment of infectious diseases. Though significant progress in the management of diarrhoeal diseases has been achieved by improved hygiene, development of new antimicrobials and vaccines, the burden remains the same, especially in children below 5 yr of age. In the case of cholera, though oral rehydration treatment is the mainstay, antimicrobial therapy is mandatory at times to reduce the volume of stool and shorten the duration of the disease. Though for many pathogens, antimicrobial resistance emerged soon after the introduction of antibiotics, Vibrio cholerae remained sensitive to most of the antibiotics for quite a long period. However, the scenario changed over the years and today, V. cholerae strains isolated world over are resistant to multiple antibiotics. A myriad number of mechanisms underlie this phenomenon. These include production of extended-spectrum beta-lactamases, enhanced multi-drug efflux pump activity, plasmid-mediated quinolone and fluoroquinolone resistance, and chromosomal mutations. Horizontal transfer of resistance determinants with mobile genetic elements like integrons and the integrating conjugative elements (ICEs), SXTs help in the dissemination of drug resistance. Though all strains isolated are not resistant to all antibiotics and we are not as yet "stranded", expanding spectrum of drug resistance is a definite cause for concern. Pipelines of discovery of new antibiotics are drying up as major pharmaceutical companies are losing interest in investing money in this endeavour, mainly due to the short shelf-life of the antibiotics and also due to the fast emergence of drug resistance. To address this issue, attempts are now being made to discover drugs which are pathogen specific and target their "virulence mechanisms". It is expected that development of resistance against such antibiotics would take much longer. This review briefly focuses on all these issues.

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Cholera is an acute form of diarrhoeal disease that plagued human civilization over the centuries. The sudden and explosive onset of the disease in the form of an outbreak or epidemic, coupled with high mortality and morbidity rates, had a tragic impact on the personal as well as social life of people living in the affected areas. The enormity of human sufferings led clinicians and scientists to carry out extensive research on cholera and Vibrio cholerae (the causative bacterium of the disease) leading to major discoveries that opened up novel areas of research or new disciplines in biomedical sciences. An attempt is made here to summarize some of these breakthroughs and outline their significance in broader perspectives. Finally, the possible impact of the global socio-political scenario on the spread of cholera epidemics (pandemicity of cholera) is briefly discussed.

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One of the major pathogenic determinants of Vibrio cholerae, the cholera toxin, is encoded in the genome of a filamentous phage, CTXφ. CTXφ makes use of the chromosome dimer resolution system of V. cholerae to integrate its single stranded genome into one, the other, or both V. cholerae chromosomes. Here, we review current knowledge about this smart integration process .

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Background & objectives : Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that β1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus β-prism lectin domain contributed to pore formation in erthrocytes. Methods : VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of β-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. Results : Proteolytic truncation of the C-terminus β-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. Interpretation & conclusions : Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

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The 50-year commemoration of S.N. De's seminal 1959 publication in Nature provides an opportunity to reflect on scientific discovery, recognition, and public health. De's paper marked the first major conceptual advance in cholera research since 1884, when Robert Koch definitively identified Der Kommabazillus as the aetiological agent of cholera. Unfortunately, Koch reported that systemic toxinosis and multi-organ failure led to severe dehydrating diarrhoea, thereby mistaking cause for effect. As a consequence, while work on other microbial pathogens advanced into the development of vaccines and therapeutics, cholera research languished as scientists injected animals parenterally in decades of futile effort to develop an animal model of diarrhoea. This fundamental misconception in cholera pathogenesis was swept away when S.N. De used ligated loops of rabbit ileum to demonstrate lumenal fluid accumulation in the presence of Vibrio cholerae culture filtrates. After some delay, De's observation of a diarrhoeagenic exotoxin became the founding principle of modern cholera research, vaccination, and treatment; and a burst of discovery saw V. cholerae transformed into the enteric pathogen best understood at the molecular level. The scientific basis for orally administering vaccines to induce mucosal immunity was established, and the success of oral rehydration, what has been described as one of the 20 ^th century's most important medical advances, was explained. Nobel laureate Joshua Lederberg wrote of De's iconoclastic creativity, experimental skill, and observational mastery, and many other leaders in the field concurred. De was nominated for the Nobel Prize in Physiology or Medicine more than once. But despite the passage of half a century from De's work, cholera remains a frustrating problem: we are clearly missing something. In reviewing the scientific and programmatic impact of S.N. De on cholera, it is clear that a defining victory against the disease is achievable, but only if basic scientific discoveries are relentlessly driven towards progress in public health.

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Background & objectives : Factor causing the elimination of the classical biotype of Vibrio cholerae O1, and its replacement by the El Tor biotype causing the 7 ^th cholera pandemic are unclear. Possible ability of the El Tor strains to adapt better than the classical strains to undefined environmental forces have been largely implicated for the change. Here we describe an environmental bacteriophage designated JSF9 which might have contributed to the range of factors. Methods : Competition assays were conducted in the infant mice model and in microcosms between representative El Tor and classical biotype strains in the absence or in the presence of JSF9 phage. Results : The JSF9 phage was found to kill classical strains and favour enrichment of El Tor strains, when mixtures containing strains of the two biotypes and JSF9 phage were subjected to alternate passage in infant mice and in samples of environmental water. Spontaneous derivatives of the classical biotype strains, as well as transposon mutants which developed resistance to JSF9 phage were found to be defective in colonization in the infant mouse model. Interpretation & conclusions : These results suggest that in addition to other factors, the inherent ability of El Tor biotype strains to evade predation by JSF9 or similar phages which kill classical biotype strains, might have enhanced the emergence of El Tor strains as the predominant pandemic biotype.

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Background & objectives : Spread of cholera in West Bengal is known to be related to its ecosystem which favours Vibrio cholerae. Incidence of cholera has not been correlated with temperature, relative humidity and rainfall, which may act as favourable factors. The aim of this study was to investigate the relational impact of climate changes on cholera. Methods : Monthly V. cholerae infection data for of the past 13 years (1996-2008), average relative humidity (RH), temperature and rainfall in Kolkata were considered for the time series analysis of Seasonal Auto-Regressive Integrated Moving Average (SARIMA) model to investigate relational impact of climatic association of V. cholerae infection and General Linear Model (GLM) for point estimation. Results : The SARIMA (1,0,0)(0,1,1) model revealed that monthly average RH was consistently linear related to V. cholerae infection during monsoon season as well as temperature and rainfall were non-stationary, AR(1), SMA(1) and SI(1) (P<0.001) were highly significant with seasonal difference. The GLM has identified that consistent (<10%) range of RH (86.78 ± 4.13, CV=5.0, P <0.001) with moderate to highest (>7 cm) rainfall (10.1 ± 5.1, CV=50.1, P <0.001) and wide (>5-10ºC) range of temperature (29.00 ± 1.64, CV=5.6, P <0.001) collectively acted as an ideal climatic condition for V. cholerae infection. Increase of RH to 21 per cent influenced an unusual V. cholerae infection in December 2008 compared to previous years. Interpretation & conclusions : V. cholerae infection was associated higher RH (>80%) with 29°C temperature with intermittent average (10 cm) rainfall. This model also identified periodicity and seasonal patterns of cholera in Kolkata. Heavy rainfall indirectly influenced the V. cholerae infection, whereas no correlation was found with high temperature.

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Campylobacter jejuni is a foodborne pathogen and a leading cause of diarrhoea worldwide. It is believed that a cholera toxin-like toxin (CTLT) produced by C. jejuni may mediate watery diarrhoea. However, the production of a CTLT by C. jejuni is controversial. A cholera toxin gene (ctx) homologue has not been identified in Campylobacter species. We investigated the identity of the CT cross-reactive antigen from Campylobacter species previously and the results are reviewed here. Filtrates of C. jejuni grown in four different liquid media, reported to promote CTLT production, were tested by Chinese hamster ovary (CHO) cell elongation assay for functional toxin and for reactivity with CT antibody using GM1 ganglioside ELISA (GM1 ELISA) and immunoblotting. Protein sequence of the CT antibody-reactive band was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Non-jejuni species (C. coli, C. lari, C. foetus, C. hyointestinalis and C. upsaliensis) were investigated by CHO cell assay and immunoblotting. Filtrates from seven C. jejuni reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay. However, filtrates from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains irrespective of GM1 ELISA reactivity, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein (PorA) of C. jejuni. CT antibody reacted with a C. jejuni recombinant PorA on immunoblotting. All non-C. jejuni strains were negative by CHO cell assay, but the common 53-kDa proteins reacted with CT antibody on immunoblots. The cross-reactivity of PorAs of Campylobacter species with CT may lead to the erroneous conclusion that Campylobacter species produce a functional CTLT.

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