From endotoxin to exotoxin: De’s rich legacy to cholera

Abstract

Between 1951 and 1959, Sambhu Nath De made crucial discoveries on the pathogenesis of cholera that changed the course of our understanding of the disease. The discovery that cholera is caused by a potent exotoxin (cholera enterotoxin) affecting intestinal permeability, the demonstration that bacteria-free culture filtrates of Vibrio cholerae were enterotoxic, and the development of a reproducible animal model for the disease are considered milestones in the history of the fight against cholera. In this commentary, a classic article by De & Chatterje published in 1953 and its public health and research impact are highlighted.

Vibrio cholerae, the causative agent of the disease known as cholera, which causes watery diarrhoea, was first described by the Italian anatomist Filippo Pacini in 1854. That same year British physician John Snow demonstrated that the disease is water-borne. Thirty years later, Robert Koch found the characteristic comma-shaped bacterium in the intestinal tissue of Egyptian patients who died after developing the typical clinical symptoms of cholera. Later that year, Koch cultured the bacterium in Calcutta (now known as Kolkata), India, and is credited with the discovery of V. cholerae, which became known as “the comma bacillus”.

Having isolated the organism from cholera patients and grown it in culture, Koch had fulfilled two of his famous postulates for proving causality, but he had yet to fulfil the third, i.e. to show that pure cultures of the comma bacillus obtained from cholera victims could cause the disease in an animal model. This third postulate remained undemonstrated for the next 75 years, until the toxin that caused cholera was discovered by Sambhu Nath De in Kolkata in 1959.¹ De, in effect, also proved Koch’s third postulate by reproducing the disease in an animal model. The full significance of De’s discovery is highlighted by the fact that it took Koch just under 8 months to discover the more elusive and fastidious etiologic agent of tuberculosis, which he did in March 1882, including replicating the disease in a guinea pig model. It was the availability of an animal model for tuberculosis that enabled Koch to discover the pathogen.² However, in the case of cholera success eluded him because there was no animal model to provide proof that the comma bacillus could cause the disease. In 1959, when De reported the discovery of the cholera toxin,¹ another group in Bombay led by NK Dutta reported the development of an infant rabbit model for cholera and demonstrated that the symptoms of the disease were caused by a toxin.

Between 1951 and 1959, Sambhu Nath De, born in 1915 in Garibati near Calcutta, made critical discoveries on the pathogenesis of cholera that radically changed our understanding of the disease. The pioneering 1953 article of De & Chatterjee,³ reproduced in the original with this commentary, is a classic. It was the first in a series of papers that examined the action of V. cholerae on the intestinal mucous membrane and that culminated in the discovery of cholera toxin.¹ Prior to the above work, almost all research had consisted of administering the stools of cholera patients or various toxic preparations derived from V. cholerae to different animals by various routes using a multiplicity of techniques to check for potential systemic or lethal effects, and conflicting results had been obtained. De, however, contended that the primary site of activity of V. cholerae and/or its toxin was the intestinal mucosa.⁴ Few of the earlier studies had examined the effect of the toxic material on the intestinal mucosa because of the entrenched belief that an endotoxin was the main toxic principle in cholera. Thus, the 1953 article of De & Chatterje³ displayed a paradigm shift in thinking.

In the simple experiments that led to the article, living V. cholerae cultures were first introduced into the intraperitoneal cavity of a rabbit and later into the lumen of the rabbit’s ligated intestine. In this way, De & Chatterje demonstrated that V. cholerae alters the permeability of the intestinal mucosa and thereby causes fluid secretion. The intravenous injection of Evans blue dye, which combines firmly with plasma albumin, was an ingenious way to prove that the leakage of fluids in the intestinal lumen was from intestinal capillaries. De also had a rational explanation, based on experimental evidence, for why the intraperitoneal fluid was rich in protein, unlike cholera stools, and why the fluid that accumulated in the ligated intestine of rabbits was low in protein, like cholera stools.

The prodigious work of De & Chatterje³ was followed by the demonstration that the pathogenicity of some strains of Escherichia coli was very similar to that of V. cholerae, and such strains were what we know today as enterotoxigenic E. coli.⁵ The discovery of the cholera enterotoxin and its effect on intestinal permeability,³ the demonstration that bacteria-free culture filtrates of V. cholerae are enterotoxic¹ and the development of a reproducible animal model for cholera^1,3,4 are milestones in the history of the fight against the disease.

The work of De & Chatterje had a profound impact on public health. The realization that the cholera toxin impairs intestinal permeability without disrupting the intestinal mucosa, altering intestinal motility, or producing an inflammatory response set the stage for the impressive discovery in the late 1960s of oral rehydration therapy, a simple, cheap and effective treatment for the severe, rapid dehydration produced by cholera. Oral rehydration therapy dramatically brought down the cholera case fatality rate from 30% in 1980 to around 3.6% in 2000. The effectiveness of oral rehydration therapy became fully evident during a cholera epidemic that broke out during the Bangladesh Liberation war in 1971.⁶ Oral rehydration therapy was introduced globally by the World Health Organization in 1979 and rapidly became the cornerstone of programmes for the control of diarrhoeal diseases. Its use brought the annual number of deaths attributable to dehydration from diarrhoea among children aged less than 5 years from an estimated 4.6 million in 1980 to about 1.5 million in 2000.⁷ Recent trends suggest that diarrhoeal deaths among children continue to decline as a result of its use.

De’s work has made a mark in the history of efforts to understand cholera⁸ and in the history of cellular physiology and biochemistry⁹ because it marked the beginning of a new way of examining the complex process manifested as diarrhoea. The work of De also paved the way for the discovery of entire families of labile toxins from enterotoxigenic E. coli, and Shiga and Shiga-like toxins from Shigella spp. and diarrhoeagenic E. coli. To the immunologists, De’s work opened new vistas, particularly from the perspective of exploring the immune responses to the toxin and developing a vaccine containing antitoxin. A search done on 19 November 2009 in the PubMed database using the keyword “cholera toxin” yielded a phenomenal 11 168 publications that the work of De spawned.

The year 2009 heralded the 50th anniversary of the discovery of cholera toxin by De, and 128 years have elapsed since the first isolation of pure cultures of the comma bacillus by Koch. Despite the great wealth of knowledge accrued on V. cholerae over the past 128 years, including the sequencing of the entire genome of 24 isolates of V. cholerae, the problem of cholera continues unabated in many parts of the world. It has worsened since the 1990s, and Zimbabwe offers a striking recent example of how cholera can ravage a country. Good hygiene, sanitation and the provision of safe water can effectively reduce the burden of cholera, but implementing these measures realistically in low-resource settings is a complex matter with which we continue to grapple. Population growth and rising poverty, global climate change and rapid, unplanned urbanization are perfect ingredients in the recipe for cholera. The burden of this dangerous disease will continue to rise, for ultimately it is a question of “hygiene versus hunger” in the most impoverished areas, where the priorities are different from those in more prosperous parts of the world. We would need De’s pragmatic wisdom to solve the problem of cholera. Is there a simple solution? ■